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Sample records for homologous recombination events

  1. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  2. High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast

    PubMed Central

    Dominska, Margaret; Moriel-Carretero, María; Herrera-Moyano, Emilia; Aguilera, Andrés; Petes, Thomas D.

    2016-01-01

    The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102) have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister

  3. A conditional mouse model for measuring the frequency of homologous recombination events in vivo in the absence of essential genes.

    PubMed

    Brown, Adam D; Claybon, Alison B; Bishop, Alexander J R

    2011-09-01

    The ability to detect and repair DNA damage is crucial to the prevention of various diseases. Loss of function of genes involved in these processes is known to result in significant developmental defects and/or predisposition to cancer. One such DNA repair mechanism, homologous recombination, has the capacity to repair a wide variety of lesions. Knockout mouse models of genes thought to be involved in DNA repair processes are frequently lethal, making in vivo studies very difficult, if not impossible. Therefore, we set out to develop an in vivo conditional mouse model system to facilitate investigations into the involvement of essential genes in homologous recombination. To test our model, we measured the frequency of spontaneous homologous recombination using the pink-eyed unstable mouse model, in which we conditionally excised either Blm or full-length Brca1 (breast cancer 1, early onset). These two genes are hypothesized to have opposing roles in homologous recombination. In summary, our in vivo data supports in vitro studies suggesting that BLM suppresses homologous recombination, while full-length BRCA1 promotes this process. PMID:21709021

  4. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  5. Biochemistry of homologous recombination in Escherichia coli.

    PubMed Central

    Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M

    1994-01-01

    Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921

  6. DNA Sequence Alignment during Homologous Recombination.

    PubMed

    Greene, Eric C

    2016-05-27

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. PMID:27129270

  7. [Homologous recombination among bacterial genomes: the measurement and identification].

    PubMed

    Xianwei, Yang; Ruifu, Yang; Yujun, Cui

    2016-02-01

    Homologous recombination is one of important sources in shaping the bacterial population diversity, which disrupts the clonal relationship among different lineages through horizontal transferring of DNA-segments. As consequence of blurring the vertical inheritance signals, the homologous recombination raises difficulties in phylogenetic analysis and reconstruction of population structure. Here we discuss the impacts of homologous recombination in inferring phylogenetic relationship among bacterial isolates, and summarize the tools and models separately used in recombination measurement and identification. We also highlight the merits and drawbacks of various approaches, aiming to assist in the practical application for the analysis of homologous recombination in bacterial evolution research. PMID:26907777

  8. Homologous recombination deficiency and ovarian cancer.

    PubMed

    Ledermann, Jonathan A; Drew, Yvette; Kristeleit, Rebecca S

    2016-06-01

    The discovery that PARP inhibitors block an essential pathway of DNA repair in cells harbouring a BRCA mutation has opened up a new therapeutic avenue for high-grade ovarian cancers. BRCA1 and BRCA2 proteins are essential for high-fidelity repair of double-strand breaks of DNA through the homologous recombination repair (HRR) pathway. Deficiency in HRR (HRD) is a target for PARP inhibitors. The first PARP inhibitor, olaparib, has now been licensed for BRCA-mutated ovarian cancers. While mutated BRCA genes are individually most commonly associated with HRD other essential HRR proteins may be mutated or functionally deficient potentially widening the therapeutic opportunities for PARP inhibitors. HRD is the first phenotypically defined predictive marker for therapy with PARP inhibitors in ovarian cancer. Several different PARP inhibitors are being trialled in ovarian cancer and this class of drugs has been shown to be a new selective therapy for high-grade ovarian cancer. Around 20% of high-grade serous ovarian cancers harbour germline or somatic BRCA mutations and testing for BRCA mutations should be incorporated into routine clinical practice. The expanded use of PARP inhibitors in HRD deficient (non-BRCA mutant) tumours using a signature of HRD in clinical practice requires validation. PMID:27065456

  9. TALEN-mediated homologous recombination in Daphnia magna

    PubMed Central

    Nakanishi, Takashi; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2015-01-01

    Transcription Activator-Like Effector Nucleases (TALENs) offer versatile tools to engineer endogenous genomic loci in various organisms. We established a homologous recombination (HR)-based knock-in using TALEN in the crustacean Daphnia magna, a model for ecological and toxicological genomics. We constructed TALENs and designed the 67 bp donor insert targeting a point deletion in the eyeless mutant that shows eye deformities. Co-injection of the TALEN mRNA with donor DNA into eggs led to the precise integration of the donor insert in the germ line, which recovered eye deformities in offspring. The frequency of HR events in the germ line was 2% by using both plasmid and single strand oligo DNA with 1.5 kb and 80 nt homology to the target. Deficiency of ligase 4 involved in non-homologous end joining repair did not increase the HR efficiency. Our data represent efficient HR-based knock-in by TALENs in D. magna, which is a promising tool to understand Daphnia gene functions. PMID:26674741

  10. Ku86 deficiency leads to reduced intrachromosomal homologous recombination in vivo in mice.

    PubMed

    Reliene, Ramune; Bishop, Alexander J R; Li, Gloria; Schiestl, Robert H

    2004-02-01

    Ku70 and Ku86 together with DNA-PKcs form the DNA-dependent protein kinase (DNA-PK) complex that is involved in DNA double-strand break repair by nonhomologous end joining. We investigated the effect of Ku86 mutation on intrachromosomal homologous recombination (HR) resulting in deletions in vivo in mice. We quantified such deletion events using a phenotypic pigmentation assay. Deletion of one copy of a 70 kb DNA duplication in the pink-eyed unstable (pun) allele results in reversion to the wildtype pink-eyed dilution (p) gene, allowing black pigment accumulation in cells of the retinal pigment epithelium (RPE). We found that the frequency of homologous recombination was significantly reduced in Ku86 deficient mice. Furthermore, the proliferation of cells in which recombination events occurred was reduced and developmentally delayed in the Ku86 deficient mice. These data indicate a role for Ku86 directly or indirectly in homologous recombination in vivo. PMID:14706343

  11. Enhancing radiotherapy through a greater understanding of homologous recombination

    PubMed Central

    Barker, Christopher A.; Powell, Simon N.

    2016-01-01

    Radiotherapy for the treatment of cancer can cause a wide range of cellular effects, the most biologically potent of which is the double strand break in DNA. The process of repairing DNA double strand breaks involves one of two major mechanisms: non-homologous end-joining or homologous recombination. In this review, we review the molecular mechanisms of homologous recombination, in particular as it relates to the repair of DNA damage from ionizing radiation. We also present specific situations where homologous recombination may be dysfunctional in human cancers, and how this functional abnormality can be recognized. We also discuss the therapeutic opportunities that can be exploited based on deficiencies in homologous recombination at various steps in the DNA repair pathway. Side-by-side with these potential therapeutic opportunities, we review the contemporary clinical trials in which strategies to exploit these defects in homologous recombination can be enhanced by the use of radiotherapy in conjunction with biologically-targeted agents. We conclude that the field of radiation oncology has only scratched the surface of a potentially highly efficacious therapeutic strategy. PMID:20832019

  12. RPA homologs and ssDNA processing during meiotic recombination.

    PubMed

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair. PMID:26520106

  13. Intrachromosomal recombination between well-separated, homologous sequences in mammalian cells.

    PubMed Central

    Baker, M D; Read, L R; Ng, P; Beatty, B G

    1999-01-01

    In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin mu-gene bearing a mutation in the constant (Cmu) region, was separated from the integrated single copy wild-type donor Cmu region by approximately 1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cmu region occurred with high frequency, correcting the mutant chromosomal mu-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cmu region, as they contained the three Cmu-bearing fragments expected from this recombination, the original donor Cmu region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cmu region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cmu regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover. PMID:10353910

  14. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    PubMed

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528

  15. Recovery of arrested replication forks by homologous recombination is error-prone.

    PubMed

    Iraqui, Ismail; Chekkal, Yasmina; Jmari, Nada; Pietrobon, Violena; Fréon, Karine; Costes, Audrey; Lambert, Sarah A E

    2012-01-01

    Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination. PMID:23093942

  16. Inhibition of Homologous Recombination with Vorinostat Synergistically Enhances Ganciclovir Cytotoxicity

    PubMed Central

    Ladd, Brendon; Ackroyd, Jeffrey J.; Hicks, J. Kevin; Canman, Christine E.; Flanagan, Sheryl A.; Shewach, Donna S.

    2014-01-01

    The nucleoside analog ganciclovir (GCV) elicits cytotoxicity in tumor cells via a novel mechanism in which drug incorporation into DNA produces minimal disruption of replication, but numerous DNA double strand breaks occur during the second S-phase after drug exposure. We propose that homologous recombination (HR), a major repair pathway for DNA double strand breaks, can prevent GCV-induced DNA damage, and that inhibition of HR will enhance cytotoxicity with GCV. Survival after GCV treatment in cells expressing a herpes simplex virus thymidine kinase was strongly dependent on HR (>14-fold decrease in IC50 in HR-deficient vs. HR-proficient CHO cells). In a homologous recombination reporter assay, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), decreased HR repair events up to 85%. SAHA plus GCV produced synergistic cytotoxicity in U251tk human glioblastoma cells. Elucidation of the synergistic mechanism demonstrated that SAHA produced a concentration-dependent decrease in the HR proteins Rad51 and CtIP. GCV alone produced numerous Rad51 foci, demonstrating activation of HR. However, the addition of SAHA blocked GCV-induced Rad51 foci formation completely and increased γH2AX, a marker of DNA double strand breaks. SAHA plus GCV also produced synergistic cytotoxicity in HR-proficient CHO cells, but the combination was antagonistic or additive in HR-deficient CHO cells. Collectively, these data demonstrate that HR promotes survival with GCV and compromise of HR by SAHA results in synergistic cytotoxicity, revealing a new mechanism for enhancing anticancer activity with GCV. PMID:24231389

  17. DNA sequence alignment by microhomology sampling during homologous recombination

    PubMed Central

    Qi, Zhi; Redding, Sy; Lee, Ja Yil; Gibb, Bryan; Kwon, YoungHo; Niu, Hengyao; Gaines, William A.; Sung, Patrick

    2015-01-01

    Summary Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair ssDNA with a homologous dsDNA template. Here we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real-time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a 9th nucleotide coincides with an additional reduction in binding free energy and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination. PMID:25684365

  18. The Landscape of Realized Homologous Recombination in Pathogenic Bacteria

    PubMed Central

    Yahara, Koji; Didelot, Xavier; Jolley, Keith A.; Kobayashi, Ichizo; Maiden, Martin C.J.; Sheppard, Samuel K.; Falush, Daniel

    2016-01-01

    Recombination enhances the adaptive potential of organisms by allowing genetic variants to be tested on multiple genomic backgrounds. Its distribution in the genome can provide insight into the evolutionary forces that underlie traits, such as the emergence of pathogenicity. Here, we examined landscapes of realized homologous recombination of 500 genomes from ten bacterial species and found all species have “hot” regions with elevated rates relative to the genome average. We examined the size, gene content, and chromosomal features associated with these regions and the correlations between closely related species. The recombination landscape is variable and evolves rapidly. For example in Salmonella, only short regions of around 1 kb in length are hot whereas in the closely related species Escherichia coli, some hot regions exceed 100 kb, spanning many genes. Only Streptococcus pyogenes shows evidence for the positive correlation between GC content and recombination that has been reported for several eukaryotes. Genes with function related to the cell surface/membrane are often found in recombination hot regions but E. coli is the only species where genes annotated as “virulence associated” are consistently hotter. There is also evidence that some genes with “housekeeping” functions tend to be overrepresented in cold regions. For example, ribosomal proteins showed low recombination in all of the species. Among specific genes, transferrin-binding proteins are recombination hot in all three of the species in which they were found, and are subject to interspecies recombination. PMID:26516092

  19. Recombination walking: Genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination

    SciTech Connect

    Miller, A.M.; Savinelli, E.A.; Couture, S.M.; Hannigan, G.M.; Han, Z.; Selden, R.F.; Treco, D.A. )

    1993-09-01

    Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unorderd) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. 29 refs., 4 figs., 1 tab.

  20. A mechanism for the suppression of homologous recombination in G1 cells.

    PubMed

    Orthwein, Alexandre; Noordermeer, Sylvie M; Wilson, Marcus D; Landry, Sébastien; Enchev, Radoslav I; Sherker, Alana; Munro, Meagan; Pinder, Jordan; Salsman, Jayme; Dellaire, Graham; Xia, Bing; Peter, Matthias; Durocher, Daniel

    2015-12-17

    DNA repair by homologous recombination is highly suppressed in G1 cells to ensure that mitotic recombination occurs solely between sister chromatids. Although many homologous recombination factors are cell-cycle regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 to constrain BRCA2 function to the S/G2 phases in human cells. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein, in complex with cullin-3 (CUL3)-RBX1 (ref. 6). PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA-end resection is sufficient to induce homologous recombination in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR-Cas9-based gene-targeting assay. We conclude that the mechanism prohibiting homologous recombination in G1 minimally consists of the suppression of DNA-end resection coupled with a multi-step block of the recruitment of BRCA2 to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce homologous recombination in G1 cells with defined factors could spur the development of gene-targeting applications in non-dividing cells. PMID:26649820

  1. Homologous recombination within the capsid gene of porcine circovirus type 2 subgroup viruses via natural co-infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several studies had reported homologous recombination between porcine circovirus type 2 (PCV2)-group 1 (Gp1) and -group 2 (Gp2) viruses. Interestingly, the recombination events described thus far mapped either within the Rep gene sequences or the sequences flanking the Rep gene region. Previously, ...

  2. Accurate modification of a chromosomal plasmid by homologous recombination in human cells

    SciTech Connect

    Song, K.Y.; Schwartz, F.; Maeda, N.; Smithies, O.; Kucherlapati, R.

    1987-10-01

    The authors have examined the consequences of modifying mammalian cellular DAN sequences by homologous recombination. A plasmid carrying a 248-base-pair deletion in the neomycin phosphotransferase (neo) gene was introduced into hamster and human cells. The integrated, defective neo gene was used as a target for modification by a second round of transfection with a plasmid carrying a different (283-base-pair) deletion in the neo gene. Recombinants resulting in an intact neo gene were selected by their G418 resistance phenotype. The best ratio of homologous to nonhomologous recombination events was about 1:80. Analysis of the functional neo genes in various independent cell lines establish that simple crossovers (single and double) generated the wild-type neo genes.

  3. Induction of Homologous Recombination Following in utero Exposure to DNA-Damaging Agents

    PubMed Central

    Karia, Bijal; Martinez, Jo Ann; Bishop, Alexander J. R.

    2013-01-01

    Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures. PMID:24029142

  4. Induction of homologous recombination following in utero exposure to DNA-damaging agents.

    PubMed

    Karia, Bijal; Martinez, Jo Ann; Bishop, Alexander J R

    2013-11-01

    Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures. PMID:24029142

  5. Homologous recombination maintenance of genome integrity during DNA damage tolerance

    PubMed Central

    Prado, Félix

    2014-01-01

    The DNA strand exchange protein Rad51 provides a safe mechanism for the repair of DNA breaks using the information of a homologous DNA template. Homologous recombination (HR) also plays a key role in the response to DNA damage that impairs the advance of the replication forks by providing mechanisms to circumvent the lesion and fill in the tracks of single-stranded DNA that are generated during the process of lesion bypass. These activities postpone repair of the blocking lesion to ensure that DNA replication is completed in a timely manner. Experimental evidence generated over the last few years indicates that HR participates in this DNA damage tolerance response together with additional error-free (template switch) and error-prone (translesion synthesis) mechanisms through intricate connections, which are presented here. The choice between repair and tolerance, and the mechanism of tolerance, is critical to avoid increased mutagenesis and/or genome rearrangements, which are both hallmarks of cancer. PMID:27308329

  6. Homologous Recombination Assay for Interstrand Cross-Link Repair

    PubMed Central

    Nakanishi, Koji; Cavallo, Francesca; Brunet, Erika; Jasin, Maria

    2012-01-01

    DNA interstrand cross-links (ICLs) covalently link both strands of the DNA duplex, impeding cellular processes like DNA replication. Homologous recombination (HR) is considered to be a major pathway for the repair of ICLs in mammalian cells as mutants for HR components are highly sensitive to DNA-damaging agents that cause ICLs. This chapter describes GFP assays to measure HR following site-specific ICL formation with psoralen through DNA triplex technology. This approach can be used to determine the genetic requirements for ICL-induced HR in relation to those involved in HR repair of other DNA lesions such as double-strand breaks. PMID:21660700

  7. Homologous recombination deficiency: Exploiting the fundamental vulnerability of ovarian cancer

    PubMed Central

    Konstantinopoulos, Panagiotis A.; Ceccaldi, Raphael; Shapiro, Geoffrey I.; D’Andrea, Alan D.

    2015-01-01

    Approximately 50% of epithelial ovarian cancers (EOCs) exhibit defective DNA repair via homologous recombination (HR) due to genetic and epigenetic alterations of HR pathway genes. Defective HR is an important therapeutic target in EOC as exemplified by the efficacy of platinum analogues in this disease, as well as the advent of poly-ADP ribose polymerase inhibitors which exhibit synthetic lethality when applied to HR deficient cells. Here, we describe the genotypic and phenotypic characteristics of HR deficient EOCs, discuss current and emerging approaches for targeting these tumors, and present challenges associated with these approaches focusing on development and overcoming resistance. PMID:26463832

  8. Inhibition of homologous recombination by the PCNA-interacting protein PARI.

    PubMed

    Moldovan, George-Lucian; Dejsuphong, Donniphat; Petalcorin, Mark I R; Hofmann, Kay; Takeda, Shunichi; Boulton, Simon J; D'Andrea, Alan D

    2012-01-13

    Inappropriate homologous recombination (HR) causes genomic instability and cancer. In yeast, the UvrD family helicase Srs2 is recruited to sites of DNA replication by SUMO-modified PCNA, where it acts to restrict HR by disassembling toxic RAD51 nucleofilaments. How human cells control recombination at replication forks is unknown. Here, we report that the protein PARI, containing a UvrD-like helicase domain, is a PCNA-interacting partner required for preservation of genome stability in human and DT40 chicken cells. Using cell-based and biochemical assays, we show that PARI restricts unscheduled recombination by interfering with the formation of RAD51-DNA HR structures. Finally, we show that PARI knockdown suppresses the genomic instability of Fanconi Anemia/BRCA pathway-deficient cells. Thus, we propose that PARI is a long sought-after factor that suppresses inappropriate recombination events at mammalian replication forks. PMID:22153967

  9. Homologous recombination is required for AAV-mediated gene targeting

    PubMed Central

    Vasileva, Ana; Linden, R. Michael; Jessberger, Rolf

    2006-01-01

    High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting ∼5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR. PMID:16822856

  10. Multilocus Sequence Typing Reveals Evidence of Homologous Recombination Linked to Antibiotic Resistance in the Genus Salinispora

    PubMed Central

    Freel, Kelle C.; Millán-Aguiñaga, Natalie

    2013-01-01

    The three closely related species that currently comprise the genus Salinispora were analyzed using a multilocus sequence typing approach targeting 48 strains derived from four geographic locations. Phylogenetic congruence and a well-supported concatenated tree provide strong support for the delineation of the three species as currently described and the basal relationship of Salinispora arenicola to the more recently diverged sister taxa S. tropica and S. pacifica. The phylogeny of the initial region of the rpoB gene sequenced was atypical, placing the related genera Micromonospora and Verrucosispora within the Salinispora clade. This phylogenetic incongruence was subsequently ascribed to a homologous-recombination event in a portion of the gene associated with resistance to compounds in the rifamycin class, which target RpoB. All S. arenicola strains produced compounds in this class and possessed resistance-conferring amino acid changes in RpoB. The phylogeny of a region of the rpoB gene that is not associated with rifamycin resistance was congruent with the other housekeeping genes. The link between antibiotic resistance and homologous recombination suggests that incongruent phylogenies provide opportunities to identify the molecular targets of secondary metabolites, an observation with potential relevance for drug discovery efforts. Low ratios of interspecies recombination to mutation, even among cooccurring strains, coupled with high levels of within-species recombination suggest that the three species have been described in accordance with natural barriers to recombination. PMID:23892741

  11. Microbial antigenic variation mediated by homologous DNA recombination.

    PubMed

    Vink, Cornelis; Rudenko, Gloria; Seifert, H Steven

    2012-09-01

    Pathogenic microorganisms employ numerous molecular strategies in order to delay or circumvent recognition by the immune system of their host. One of the most widely used strategies of immune evasion is antigenic variation, in which immunogenic molecules expressed on the surface of a microorganism are continuously modified. As a consequence, the host is forced to constantly adapt its humoral immune response against this pathogen. An antigenic change thus provides the microorganism with an opportunity to persist and/or replicate within the host (population) for an extended period of time or to effectively infect a previously infected host. In most cases, antigenic variation is caused by genetic processes that lead to the modification of the amino acid sequence of a particular antigen or to alterations in the expression of biosynthesis genes that induce changes in the expression of a variant antigen. Here, we will review antigenic variation systems that rely on homologous DNA recombination and that are found in a wide range of cellular, human pathogens, including bacteria (such as Neisseria spp., Borrelia spp., Treponema pallidum, and Mycoplasma spp.), fungi (such as Pneumocystis carinii) and parasites (such as the African trypanosome Trypanosoma brucei). Specifically, the various DNA recombination-based antigenic variation systems will be discussed with a focus on the employed mechanisms of recombination, the DNA substrates, and the enzymatic machinery involved. PMID:22212019

  12. Inference of Homologous Recombination in Bacteria Using Whole-Genome Sequences

    PubMed Central

    Didelot, Xavier; Lawson, Daniel; Darling, Aaron; Falush, Daniel

    2010-01-01

    Bacteria and archaea reproduce clonally, but sporadically import DNA into their chromosomes from other organisms. In many of these events, the imported DNA replaces an homologous segment in the recipient genome. Here we present a new method to reconstruct the history of recombination events that affected a given sample of bacterial genomes. We introduce a mathematical model that represents both the donor and the recipient of each DNA import as an ancestor of the genomes in the sample. The model represents a simplification of the previously described coalescent with gene conversion. We implement a Monte Carlo Markov chain algorithm to perform inference under this model from sequence data alignments and show that inference is feasible for whole-genome alignments through parallelization. Using simulated data, we demonstrate accurate and reliable identification of individual recombination events and global recombination rate parameters. We applied our approach to an alignment of 13 whole genomes from the Bacillus cereus group. We find, as expected from laboratory experiments, that the recombination rate is higher between closely related organisms and also that the genome contains several broad regions of elevated levels of recombination. Application of the method to the genomic data sets that are becoming available should reveal the evolutionary history and private lives of populations of bacteria and archaea. The methods described in this article have been implemented in a computer software package, ClonalOrigin, which is freely available from http://code.google.com/p/clonalorigin/. PMID:20923983

  13. Transcription-coupled homologous recombination after oxidative damage.

    PubMed

    Wei, Leizhen; Levine, Arthur Samuel; Lan, Li

    2016-08-01

    Oxidative DNA damage induces genomic instability and may lead to mutagenesis and carcinogenesis. As severe blockades to RNA polymerase II (RNA POLII) during transcription, oxidative DNA damage and the associated DNA strand breaks have a profoundly deleterious impact on cell survival. To protect the integrity of coding regions, high fidelity DNA repair at a transcriptionally active site in non-dividing somatic cells, (i.e., terminally differentiated and quiescent/G0 cells) is necessary to maintain the sequence integrity of transcribed regions. Recent studies indicate that an RNA-templated, transcription-associated recombination mechanism is important to protect coding regions from DNA damage-induced genomic instability. Here, we describe the discovery that G1/G0 cells exhibit Cockayne syndrome (CS) B (CSB)-dependent assembly of homologous recombination (HR) factors at double strand break (DSB) sites within actively transcribed regions. This discovery is a challenge to the current dogma that HR occurs only in S/G2 cells where undamaged sister chromatids are available as donor templates. PMID:27233112

  14. Nonsense-mediated decay regulates key components of homologous recombination

    PubMed Central

    Janke, Ryan; Kong, Jeremy; Braberg, Hannes; Cantin, Greg; Yates, John R.; Krogan, Nevan J.; Heyer, Wolf-Dietrich

    2016-01-01

    Cells frequently experience DNA damage that requires repair by homologous recombination (HR). Proteins involved in HR are carefully coordinated to ensure proper and efficient repair without interfering with normal cellular processes. In Saccharomyces cerevisiae, Rad55 functions in the early steps of HR and is regulated in response to DNA damage through phosphorylation by the Mec1 and Rad53 kinases of the DNA damage response. To further identify regulatory processes that target HR, we performed a high-throughput genetic interaction screen with RAD55 phosphorylation site mutants. Genes involved in the mRNA quality control process, nonsense-mediated decay (NMD), were found to genetically interact with rad55 phospho-site mutants. Further characterization revealed that RAD55 transcript and protein levels are regulated by NMD. Regulation of HR by NMD extends to multiple targets beyond RAD55, including RAD51, RAD54 and RAD57. Finally, we demonstrate that loss of NMD results in an increase in recombination rates and resistance to the DNA damaging agent methyl methanesulfonate, suggesting this pathway negatively regulates HR under normal growth conditions. PMID:27001511

  15. Attenuating homologous recombination stimulates an AID-induced antileukemic effect

    PubMed Central

    Lamont, Kristin R.; Hasham, Muneer G.; Donghia, Nina M.; Branca, Jane; Chavaree, Margaret; Chase, Betsy; Breggia, Anne; Hedlund, Jacquelyn; Emery, Ivette; Cavallo, Francesca; Jasin, Maria; Rüter, Jens

    2013-01-01

    Activation-induced cytidine deaminase (AID) is critical in normal B cells to initiate somatic hypermutation and immunoglobulin class switch recombination. Accumulating evidence suggests that AID is also prooncogenic, inducing cancer-promoting mutations or chromosome rearrangements. In this context, we find that AID is expressed in >40% of primary human chronic lymphocytic leukemia (CLL) cases, consistent with other reports. Using a combination of human B lymphoid leukemia cells and mouse models, we now show that AID expression can be harnessed for antileukemic effect, after inhibition of the RAD51 homologous recombination (HR) factor with 4,4′-diisothiocyanatostilbene-2-2′-disulfonic acid (DIDS). As a proof of principle, we show that DIDS treatment inhibits repair of AID-initiated DNA breaks, induces apoptosis, and promotes cytotoxicity preferentially in AID-expressing human CLL. This reveals a novel antineoplastic role of AID that can be triggered by inhibition of HR, suggesting a potential new paradigm to treat AID-expressing tumors. Given the growing list of tumor types with aberrant AID expression, this novel therapeutic approach has potential to impact a significant patient population. PMID:23589568

  16. Nonsense-mediated decay regulates key components of homologous recombination.

    PubMed

    Janke, Ryan; Kong, Jeremy; Braberg, Hannes; Cantin, Greg; Yates, John R; Krogan, Nevan J; Heyer, Wolf-Dietrich

    2016-06-20

    Cells frequently experience DNA damage that requires repair by homologous recombination (HR). Proteins involved in HR are carefully coordinated to ensure proper and efficient repair without interfering with normal cellular processes. In Saccharomyces cerevisiae, Rad55 functions in the early steps of HR and is regulated in response to DNA damage through phosphorylation by the Mec1 and Rad53 kinases of the DNA damage response. To further identify regulatory processes that target HR, we performed a high-throughput genetic interaction screen with RAD55 phosphorylation site mutants. Genes involved in the mRNA quality control process, nonsense-mediated decay (NMD), were found to genetically interact with rad55 phospho-site mutants. Further characterization revealed that RAD55 transcript and protein levels are regulated by NMD. Regulation of HR by NMD extends to multiple targets beyond RAD55, including RAD51, RAD54 and RAD57 Finally, we demonstrate that loss of NMD results in an increase in recombination rates and resistance to the DNA damaging agent methyl methanesulfonate, suggesting this pathway negatively regulates HR under normal growth conditions. PMID:27001511

  17. Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.

    PubMed

    Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D; Waldman, Alan S

    2008-10-01

    Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death

  18. Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage λ-Td Intron Model System

    PubMed Central

    Parker, M. M.; Court, D. A.; Preiter, K.; Belfort, M.

    1996-01-01

    Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage λ model system was developed to analyze exon homology effects on intron homing and determine the role of the λ 5'-3' exonuclease complex (Redαβ) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type λ, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology. PMID:8807281

  19. The Saccharomyces cerevisiae recombination enhancer biases recombination during interchromosomal mating-type switching but not in interchromosomal homologous recombination.

    PubMed Central

    Houston, Peter; Simon, Peter J; Broach, James R

    2004-01-01

    Haploid Saccharomyces can change mating type through HO-endonuclease cleavage of an expressor locus, MAT, followed by gene conversion using one of two repository loci, HML or HMR, as donor. The mating type of a cell dictates which repository locus is used as donor, with a cells using HML and alpha cells using HMR. This preference is established in part by RE, a locus on the left arm of chromosome III that activates the surrounding region, including HML, for recombination in a cells, an activity suppressed by alpha 2 protein in alpha cells. We have examined the ability of RE to stimulate different forms of interchromosomal recombination. We found that RE exerted an effect on interchromosomal mating-type switching and on intrachromosomal homologous recombination but not on interchromosomal homologous recombination. Also, even in the absence of RE, MAT alpha still influenced donor preference in interchromosomal mating-type switching, supporting a role of alpha 2 in donor preference independent of RE. These results suggest a model in which RE affects competition between productive and nonproductive recombination outcomes. In interchromosome gene conversion, RE enhances both productive and nonproductive pathways, whereas in intrachromosomal gene conversion and mating-type switching, RE enhances only the productive pathway. PMID:15082540

  20. Manipulation of Homologous and Homoeologous Chromosome Recombination in Wheat.

    PubMed

    Lukaszewski, Adam J

    2016-01-01

    Given the sizes of the three genomes in wheat (A, B, and D) and a limited number of chiasmata formed in meiosis, recombination by crossing-over is a very rare event. It is also restricted to very similar homologues; the pairing homoeologous (Ph) system of wheat prevents differentiated chromosomes from pairing and crossing-over. This chapter presents an overview and describes several systems by which the frequency or density of crossing-over can be increased, both in homologues and homoeologues. It also presents the standard system of E.R. Sears for engineering alien chromosome transfers into wheat. PMID:27511168

  1. Is homologous recombination really an error-free process?

    PubMed Central

    Guirouilh-Barbat, Josée; Lambert, Sarah; Bertrand, Pascale; Lopez, Bernard S.

    2014-01-01

    Homologous recombination (HR) is an evolutionarily conserved process that plays a pivotal role in the equilibrium between genetic stability and diversity. HR is commonly considered to be error-free, but several studies have shown that HR can be error-prone. Here, we discuss the actual accuracy of HR. First, we present the product of genetic exchanges (gene conversion, GC, and crossing over, CO) and the mechanisms of HR during double strand break repair and replication restart. We discuss the intrinsic capacities of HR to generate genome rearrangements by GC or CO, either during DSB repair or replication restart. During this process, abortive HR intermediates generate genetic instability and cell toxicity. In addition to genome rearrangements, HR also primes error-prone DNA synthesis and favors mutagenesis on single stranded DNA, a key DNA intermediate during the HR process. The fact that cells have developed several mechanisms protecting against HR excess emphasize its potential risks. Consistent with this duality, several pro-oncogenic situations have been consistently associated with either decreased or increased HR levels. Nevertheless, this versatility also has advantages that we outline here. We conclude that HR is a double-edged sword, which on one hand controls the equilibrium between genome stability and diversity but, on the other hand, can jeopardize the maintenance of genomic integrity. Therefore, whether non-homologous end joining (which, in contrast with HR, is not intrinsically mutagenic) or HR is the more mutagenic process is a question that should be re-evaluated. Both processes can be “Dr. Jekyll” in maintaining genome stability/variability and “Mr. Hyde” in jeopardizing genome integrity. PMID:24966870

  2. DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

    PubMed Central

    Kuzminov, Andrei

    2001-01-01

    Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair. PMID:11459990

  3. BRCA1-directed, enhanced and aberrant homologous recombination

    PubMed Central

    Dever, Seth M; White, E Railey; Hartman, Matthew CT

    2012-01-01

    Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitin-ligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors. PMID:22306997

  4. Homologous Recombination Is Necessary for Normal Lymphocyte Development▿

    PubMed Central

    Caddle, Lura B.; Hasham, Muneer G.; Schott, William H.; Shirley, Bobbi-Jo; Mills, Kevin D.

    2008-01-01

    Primary immunodeficiencies are rare but serious diseases with diverse genetic causes. Accumulating evidence suggests that defects in DNA double-strand break (DSB) repair can underlie many of these syndromes. In this context, the nonhomologous end joining pathway of DSB repair is absolutely required for lymphoid development, but possible roles for the homologous recombination (HR) pathway have remained more controversial. While recent evidence suggests that HR may indeed be important to suppress lymphoid transformation, the specific relationship of HR to normal lymphocyte development remains unclear. We have investigated roles of the X-ray cross-complementing 2 (Xrcc2) HR gene in lymphocyte development. We show that HR is critical for normal B-cell development, with Xrcc2 nullizygosity leading to p53-dependent early S-phase arrest. In the absence of p53 (encoded by Trp53), Xrcc2-null B cells can fully develop but show high rates of chromosome and chromatid fragmentation. We present a molecular model wherein Xrcc2 is important to preserve or restore replication forks during rapid clonal expansion of developing lymphocytes. Our findings demonstrate a key role for HR in lymphoid development and suggest that Xrcc2 defects could underlie some human primary immunodeficiencies. PMID:18212067

  5. Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair

    PubMed Central

    Peng, Guang; Lin, Curtis Chun-Jen; Mo, Wei; Dai, Hui; Park, Yun-Yong; Kim, Soo-Mi; Peng, Yang; Mo, Qianxing; Siwko, Stefan; Hu, Ruozhen; Lee, Ju-Seog; Hennessy, Bryan; Hanash, Samir; Mills, Gordon B.; Lin, Shiaw-Yih

    2014-01-01

    Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer. PMID:24553445

  6. Accelerated protein engineering for chemical biotechnology via homologous recombination.

    PubMed

    Nordwald, Erik M; Garst, Andrew; Gill, Ryan T; Kaar, Joel L

    2013-12-01

    Protein engineering has traditionally relied on random mutagenesis strategies to generate diverse libraries, which require high-throughput screening or selection methods to identify rare variants. Alternatively, approaches to semi-rational library construction can be used to minimize the screening load and enhance the efficiency by which improved mutants may be identified. Such methods are typically limited to characterization of relatively few variants due to the difficulties in generating large rational libraries. New tools from synthetic biology, namely multiplexed DNA synthesis and homologous recombination, provide a promising avenue to rapidly construct large, rational libraries. These technologies also enable incorporation of synthetically encoded features that permit efficient characterization of the fitness of each mutant. Extension of these tools to protein library design could complement rational protein design cycles in an effort to more systematically search complex fitness landscapes. The highly parallelized nature with which such libraries can be generated also has the potential to expand directed protein evolution from single protein targets to protein networks whose concerted activities are required for the biological function of interest. PMID:23540421

  7. ATR inhibition preferentially targets homologous recombination-deficient tumor cells.

    PubMed

    Krajewska, M; Fehrmann, R S N; Schoonen, P M; Labib, S; de Vries, E G E; Franke, L; van Vugt, M A T M

    2015-06-01

    Homologous recombination (HR) is required for faithful repair of double-strand DNA breaks. Defects in HR repair cause severe genomic instability and challenge cellular viability. Paradoxically, various cancers are HR defective and have apparently acquired characteristics to survive genomic instability. We aimed to identify these characteristics to uncover therapeutic targets for HR-deficient cancers. Cytogenetic analysis of 1143 ovarian cancers showed that the degree of genomic instability was correlated to amplification of replication checkpoint genes ataxia telangiectasia and Rad3-related kinase (ATR) and CHEK1. To test whether genomic instability leads to increased reliance on replication checkpoint signaling, we inactivated Rad51 to model HR-related genomic instability. Rad51 inactivation caused defective HR repair and induced aberrant replication dynamics. Notably, inhibition of Rad51 led to increased ATR/checkpoint kinase-1 (Chk1)-mediated replication stress signaling. Importantly, inhibition of ATR or Chk1 preferentially killed HR-deficient cancer cells. Combined, our data show that defective HR caused by Rad51 inhibition results in differential sensitivity for ATR and Chk1 inhibitors, implicating replication checkpoint kinases as potential drug targets for HR-defective cancers. PMID:25174396

  8. Shu1 Promotes Homolog Bias of Meiotic Recombination in Saccharomyces cerevisiae

    PubMed Central

    Hong, Soogil; Kim, Keun Pil

    2013-01-01

    Homologous recombination occurs closely between homologous chromatids with highly ordered recombinosomes through RecA homologs and mediators. The present study demonstrates this relationship during the period of “partner choice” in yeast meiotic recombination. We have examined the formation of recombination intermediates in the absence or presence of Shu1, a member of the PCSS complex, which also includes Psy3, Csm2, and Shu2. DNA physical analysis indicates that Shu1 is essential for promoting the establishment of homolog bias during meiotic homologous recombination, and the partner choice is switched by Mek1 kinase activity. Furthermore, Shu1 promotes both crossover (CO) and non-crossover (NCO) pathways of meiotic recombination. The inactivation of Mek1 kinase allows for meiotic recombination to progress efficiently, but is lost in homolog bias where most double-strand breaks (DSBs) are repaired via stable intersister joint molecules. Moreover, the Srs2 helicase deletion cells in the budding yeast show slightly reduced COs and NCOs, and Shu1 promotes homolog bias independent of Srs2. Our findings reveal that Shu1 and Mek1 kinase activity have biochemically distinct roles in partner choice, which in turn enhances the understanding of the mechanism associated with the precondition for homolog bias. PMID:24213600

  9. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

    PubMed

    Wilhelm, Therese; Ragu, Sandrine; Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S

    2016-05-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  10. Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

    PubMed Central

    Magdalou, Indiana; Machon, Christelle; Dardillac, Elodie; Técher, Hervé; Guitton, Jérôme; Debatisse, Michelle; Lopez, Bernard S.

    2016-01-01

    Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation

  11. Genetic Manipulation of Homologous Recombination In Vivo Attenuates Intestinal Tumorigenesis.

    PubMed

    McIlhatton, Michael A; Murnan, Kevin; Carson, Daniel; Boivin, Gregory P; Croce, Carlo M; Groden, Joanna

    2015-07-01

    Although disruption of DNA repair capacity is unquestionably associated with cancer susceptibility in humans and model organisms, it remains unclear if the inherent tumor phenotypes of DNA repair deficiency syndromes can be regulated by manipulating DNA repair pathways. Loss-of-function mutations in BLM, a member of the RecQ helicase family, cause Bloom's syndrome (BS), a rare, recessive genetic disorder that predisposes to many types of cancer. BLM functions in many aspects of DNA homeostasis, including the suppression of homologous recombination (HR) in somatic cells. We investigated whether BLM overexpression, in contrast with loss-of-function mutations, attenuated the intestinal tumor phenotypes of Apc(Min/+) and Apc(Min/+);Msh2(-/-) mice, animal models of familial adenomatous polyposis coli (FAP). We constructed a transgenic mouse line expressing human BLM (BLM-Tg) and crossed it onto both backgrounds. BLM-Tg decreased adenoma incidence in a dose-dependent manner in our Apc(Min/) (+) model of FAP, although levels of GIN were unaffected and concomitantly increased animal survival over 50%. It did not reduce intestinal tumorigenesis in Apc(Min/) (+);Msh2(-/-) mice. We used the pink-eyed unstable (p(un)) mouse model to demonstrate that increasing BLM dosage in vivo lowered endogenous levels of HR by 2-fold. Our data suggest that attenuation of the Min phenotype is achieved through a direct effect of BLM-Tg on the HR repair pathway. These findings demonstrate that HR can be manipulated in vivo to modulate tumor formation at the organismal level. Our data suggest that lowering HR frequencies may have positive therapeutic outcomes in the context of specific hereditary cancer predisposition syndromes, exemplified by FAP. PMID:25908507

  12. A role for homologous recombination proteins in cell cycle regulation

    PubMed Central

    Kostyrko, Kaja; Bosshard, Sandra; Urban, Zuzanna; Mermod, Nicolas

    2015-01-01

    Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling. PMID:26125600

  13. Ectopic recombination within homologous immunoglobulin mu gene constant regions in a mouse hybridoma cell line.

    PubMed Central

    Baker, M D; Read, L R

    1992-01-01

    We have transferred a pSV2neo vector containing the wild-type constant region of the immunoglobulin mu gene (C mu) into the mutant hybridoma igm482, which bears a 2-bp deletion in the third constant-region exon of its haploid chromosomal mu gene (C mu 3). Independent igm482 transformants contain the wild-type immunoglobulin C mu region stably integrated in ectopic chromosomal positions. We report here that the wild-type immunoglobulin C mu region can function as the donor sequence in a gene conversion event which corrects the 2-bp deletion in the mutant igm482 chromosomal C mu 3 exon. The homologous recombination event restores normal immunoglobulin M production in the mutant cell. Images PMID:1406631

  14. Widespread interspecies homologous recombination reveals reticulate evolution within the genus Streptomyces.

    PubMed

    Cheng, Kun; Rong, Xiaoying; Huang, Ying

    2016-09-01

    Homologous recombination is increasingly being recognized as a driving force in microbial evolution. However, recombination in streptomycetes, a rich source of diverse secondary metabolites, particularly among different species, remains minimally investigated. In this study, the largest sample of Streptomyces species to date, consisting of 142 type strains spanning the genus, with available sequences of 16S rRNA, atpD, gyrB, recA, rpoB and trpB genes, were collected and subjected to a comprehensive population genetic analysis to generate an overall estimate of the level of Streptomyces interspecies genetic exchange and its effect on the evolution of this genus. The results indicate frequent homologous recombination among Streptomyces species, which occurred three times more frequently and was nearly 14 times more important than point mutation in nucleotide sequence divergence (ρ/θw=3.10, r/m=13.74). As a result, a facilitating effect on the evolutionary process and confusion in phylogenetic relationships were observed, as well as a number of specific transfer events of the six gene fragments. A resultant phylogenetic network depicted extensive horizontal genetic exchange which decays clonality in streptomycetes. Moreover, seven evolutionary lineage groups were identified in the present sample in the Structure analysis, generally consistent with morphological and physiological data, and the contribution of recombination was detected to be varied among them. Our analyses demonstrated a reticulate evolution within Streptomyces due to the high level of interspecies gene exchange, which greatly challenges the traditional tree-shaped phylogeny in this genus and may advance our evolutionary understanding of a genuine Streptomyces species. PMID:27329941

  15. Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.

    PubMed

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Horii, Masae; Hamana, Hiroshi; Nagai, Terumi; Muraguchi, Atsushi

    2014-02-14

    Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis. PMID:24462869

  16. p21 controls patterning but not homologous recombination in RPE development.

    PubMed

    Bishop, A J R; Kosaras, B; Hollander, M C; Fornace, A; Sidman, R L; Schiestl, R H

    2006-01-01

    p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53. PMID:16202662

  17. Remodeling and Control of Homologous Recombination by DNA Helicases and Translocases that Target Recombinases and Synapsis.

    PubMed

    Northall, Sarah J; Ivančić-Baće, Ivana; Soultanas, Panos; Bolt, Edward L

    2016-01-01

    Recombinase enzymes catalyse invasion of single-stranded DNA (ssDNA) into homologous duplex DNA forming "Displacement loops" (D-loops), a process called synapsis. This triggers homologous recombination (HR), which can follow several possible paths to underpin DNA repair and restart of blocked and collapsed DNA replication forks. Therefore, synapsis can be a checkpoint for controlling whether or not, how far, and by which pathway, HR proceeds to overcome an obstacle or break in a replication fork. Synapsis can be antagonized by limiting access of a recombinase to ssDNA and by dissociation of D-loops or heteroduplex formed by synapsis. Antagonists include DNA helicases and translocases that are identifiable in eukaryotes, bacteria and archaea, and which target synaptic and pre-synaptic DNA structures thereby controlling HR at early stages. Here we survey these events with emphasis on enabling DNA replication to be resumed from sites of blockage or collapse. We also note how knowledge of anti-recombination activities could be useful to improve efficiency of CRISPR-based genome editing. PMID:27548227

  18. Remodeling and Control of Homologous Recombination by DNA Helicases and Translocases that Target Recombinases and Synapsis

    PubMed Central

    Northall, Sarah J.; Ivančić-Baće, Ivana; Soultanas, Panos; Bolt, Edward L.

    2016-01-01

    Recombinase enzymes catalyse invasion of single-stranded DNA (ssDNA) into homologous duplex DNA forming “Displacement loops” (D-loops), a process called synapsis. This triggers homologous recombination (HR), which can follow several possible paths to underpin DNA repair and restart of blocked and collapsed DNA replication forks. Therefore, synapsis can be a checkpoint for controlling whether or not, how far, and by which pathway, HR proceeds to overcome an obstacle or break in a replication fork. Synapsis can be antagonized by limiting access of a recombinase to ssDNA and by dissociation of D-loops or heteroduplex formed by synapsis. Antagonists include DNA helicases and translocases that are identifiable in eukaryotes, bacteria and archaea, and which target synaptic and pre-synaptic DNA structures thereby controlling HR at early stages. Here we survey these events with emphasis on enabling DNA replication to be resumed from sites of blockage or collapse. We also note how knowledge of anti-recombination activities could be useful to improve efficiency of CRISPR-based genome editing. PMID:27548227

  19. Homologous recombination can restore normal immunoglobulin production in a mutant hybridoma cell line.

    PubMed Central

    Baker, M D; Pennell, N; Bosnoyan, L; Shulman, M J

    1988-01-01

    We report here the occurrence of homologous recombination between transferred and chromosomal immunoglobulin genes. Specifically, we have corrected a chromosomal immunoglobulin gene mutation by transferring pSV2neo vectors encoding the constant region of the immunoglobulin mu heavy chain to mutant hybridoma cells that bear a 2-base-pair deletion in the third constant region exon of their chromosomal mu gene. After DNA transfer, we detected G418-resistant transformants that produce normal IgM. Analysis of the DNA structure of the mu gene in these transformants indicates that in four of five cases the mu gene has been restored as a result of the integration of a single copy of the transfer vector by a reciprocal homologous recombination event; the fifth case seems to have resulted from gene conversion or double crossover. These results suggest that this technology might be adapted for mapping immunoglobulin gene mutations by marker rescue and for more convenient engineering of specifically altered immunoglobulin. Images PMID:2842771

  20. Intermolecular Transposition of Is10 Causes Coupled Homologous Recombination at the Transposition Site

    PubMed Central

    Eichenbaum, Z.; Livneh, Z.

    1995-01-01

    Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid. This was detected by the activity of the tet gene expressed from the phage 434 P(R) promoter. Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site. Cointegrate formation was abolished in δrecA strains, although simple insertions of IS10 were observed. This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor. Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event. Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome. This was accompanied by the formation of an additional copy of IS10 in the chromosome. Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10. PMID:7672587

  1. Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity

    PubMed Central

    Kirkman, Laura A.; Lawrence, Elizabeth A.; Deitsch, Kirk W.

    2014-01-01

    Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-homologous end joining (C-NHEJ) pathway were identified. To better understand how malaria parasites repair DSBs and maintain genome integrity, we modified the yeast I-SceI endonuclease system to generate inducible, site-specific DSBs within the parasite’s genome. Analysis of repaired genomic DNA showed that parasites possess both a typical HR pathway resulting in gene conversion events as well as an end joining (EJ) pathway for repair of DSBs when no homologous sequence is available. The products of EJ were limited in number and identical products were observed in multiple independent experiments. The repair junctions frequently contained short insertions also found in the surrounding sequences, suggesting the possibility of a templated repair process. We propose that an alternative end-joining pathway rather than C-NHEJ, serves as a primary method for repairing DSBs in malaria parasites. PMID:24089143

  2. Investigation of the effects of aging on homologous recombination in long-term bone marrow cultures.

    PubMed

    Epperly, Michael W; Rugo, Rebecca; Cao, Shaonan; Wang, Hong; Franicola, Darcy; Goff, Julie P; Shen, Hongmei; Zhang, Xichen; Wiktor-Brown, Dominika; Engelward, Bevin P; Greenberger, Joel S

    2009-01-01

    Fluorescent yellow direct repeat (FYDR) mice carry a transgenic reporter for homologous recombination (HR) and have been used to reveal an age-dependent increase in HR in the pancreas. An established in vitro model system for accelerated aging of the marrow is the mouse long-term bone marrow culture (LTBMC) system. To determine whether the FYDR system, in which an HR event can lead to a fluorescent cell, can be used to study the effects of aging in LTBMCs, clonally expanded hematopoietic and marrow stromal cells in FYDR, positive control FYDR-Recombined (FYDR-Rec), and negative control wild-type C57BL/6NHsd (WT) LTBMCs were analysed. All groups of cultures demonstrated equivalent parameters of continuous hematopoiesis including generation of multilineage colony forming CFU-GM progenitor cells for over 22 weeks and age associated senescence of hematopoiesis. Results indicate that low expression of the FYDR transgene in bone marrow cells in vivo and in vitro prevents the use of the FYDR mice to study rare combination events in bone marrow. Using an alternative approach for detecting HR, namely the sister chromatid exchange (SCE) assay, a statistically significant increase in the number of SCEs per chromosome was observed in adherent cells subcultured from 20-week-compared to 4-week-old LTBMCs. These data suggest that adherent marrow stromal cells from LTBMCs become increasingly susceptible to HR events during aging. PMID:19779099

  3. Emergence of Pathogenic Coronaviruses in Cats by Homologous Recombination between Feline and Canine Coronaviruses

    PubMed Central

    Terada, Yutaka; Matsui, Nobutaka; Noguchi, Keita; Kuwata, Ryusei; Shimoda, Hiroshi; Soma, Takehisa; Mochizuki, Masami; Maeda, Ken

    2014-01-01

    Type II feline coronavirus (FCoV) emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV). In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3′-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5′-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently. PMID:25180686

  4. BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks.

    PubMed

    Willis, Nicholas A; Chandramouly, Gurushankar; Huang, Bin; Kwok, Amy; Follonier, Cindy; Deng, Chuxia; Scully, Ralph

    2014-06-26

    Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks. Although BRCA1 and BRCA2 affect replication fork processing, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11-two Brca1 elements implicated in tumour suppression-control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of 'long-tract' gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells. PMID:24776801

  5. The role of AtMSH2 in homologous recombination in Arabidopsis thaliana

    PubMed Central

    Emmanuel, Eyal; Yehuda, Elizabeth; Melamed-Bessudo, Cathy; Avivi-Ragolsky, Naomi; Levy, Avraham A

    2006-01-01

    During homologous recombination (HR), a heteroduplex DNA is formed as a consequence of strand invasion. When the two homologous strands differ in sequence, a mismatch is generated. Earlier studies showed that mismatched heteroduplex often triggers abortion of recombination and that a pivotal component of this pathway is the mismatch repair Msh2 protein. In this study, we analysed the roles of AtMSH2 in suppression of recombination in Arabidopsis. We report that AtMSH2 has a broad range of anti-recombination effects: it suppresses recombination between divergent direct repeats in somatic cells or between homologues from different ecotypes during meiosis. This is the first example of a plant gene that affects HR as a function of sequence divergence and that has an anti-recombination meiotic effect. We discuss the implications of these results for plant improvement by gene transfer across species. PMID:16311517

  6. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    SciTech Connect

    Larionov, V.; Kouprina, N. |; Edlarov, M. |; Perkins, E.; Porter, G.; Resnick, M.A.

    1993-12-31

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RADS2, RADI and the RNCI genes,

  7. Positive genetic selection for gene disruption in mammalian cells by homologous recombination.

    PubMed Central

    Sedivy, J M; Sharp, P A

    1989-01-01

    Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA. Images PMID:2536156

  8. Meiotic recombination at the Lmp2 hotspot tolerates minor sequence divergence between homologous chromosomes

    SciTech Connect

    Yoshino, Masayasu; Sagai, Tomoko; Shiroishi, Toshihiko

    1996-06-01

    Recombination is widely considered to linearly depend on the length of the homologous sequences. An 11% mismatch decreases the rate of phage-plasmid recombination 240-fold. Two single nucleotide mismatches, which reduce the longest uninterrupted stretch of similarity from 232 base pairs (bp) to 134 bp, reduce gene conversion in mouse L cells 20-fold. The efficiency of gene targeting through homologous recombination in mouse embryonic stem cells can be increased by using an isogenic, rather than a non-isogenic, DNA construct. In this study we asked whether a high degree of sequence identity between homologous mouse chromosomes enhances meiotic recombination at a hotspot. Sites of meiotic recombination in the mouse major histocompatibility complex (MHC) class II region are not randomly distributed but are almost all clustered within short segments known as recombinational hotspots. The wm7 MHC haplotype, derived from Japanese wild mice Mus musculus molossinus, enhances meiotic recombination at a hotspot near the Lmp2 gene. Heterozygotes between the wm7 haplotype and the b or k haplotypes have yielded a high frequency of recombination (2.1%) in 1.3 kilobase kb segment of this hotspot. 20 refs., 2 figs.

  9. A mitochondrial gene is lost via homologous recombination during reversion of CMS T maize to fertility

    PubMed Central

    Rottmann, W. H.; Brears, T.; Hodge, T. P.; Lonsdale, D. M.

    1987-01-01

    The Texas (T) male sterile cytoplasm of maize is distinguished by a mitochondrially synthesized 13-kd polypeptide and a high susceptibility to the toxin produced by the fungal pathogen Helminthosporium maydis. Fertile, toxin-resistant revertants show an altered restriction profile for mitochondrial DNA and do not produce the 13-kd polypeptide. Characterization of cosmid clones from CMS T maize and a revertant shows that a heavily transcribed open reading frame named T-URF13, potentially coding a 13-kd product, is deleted in the revertant mitochondria. Six transcripts present in CMS T mitochondria, 4000, 3000, 2000, 1800, 1500 and 1200 nucleotides in length, are lacking in revertant mitochondria. T-URF25, an open reading frame coding for a 25-kd product, lies to the 3' end of T-URF13 but is retained in the revertants. T-URF13 and T-URF25 are co-transcribed in CMS T mitochondria; in the revertant T-URF25 is present on a 3100-nucleotide species. The recombination that caused these changes involved a 127-bp repeated sequence. Homologous recombination took place within the central 55 bp of this imperfect repeat. Hybridization analysis of DNA and RNA from other revertants demonstrates that a similar or identical event has taken place independently in these revertants. ImagesFig. 2.Fig. 4.Fig. 5. PMID:16453770

  10. DNA Polymerase δ Is Preferentially Recruited during Homologous Recombination To Promote Heteroduplex DNA Extension▿

    PubMed Central

    Maloisel, Laurent; Fabre, Francis; Gangloff, Serge

    2008-01-01

    DNA polymerases play a central role during homologous recombination (HR), but the identity of the enzyme(s) implicated remains elusive. The pol3-ct allele of the gene encoding the catalytic subunit of DNA polymerase δ (Polδ) has highlighted a role for this polymerase in meiotic HR. We now address the ubiquitous role of Polδ during HR in somatic cells. We find that pol3-ct affects gene conversion tract length during mitotic recombination whether the event is initiated by single-strand gaps following UV irradiation or by site-specific double-strand breaks. We show that the pol3-ct effects on gene conversion are completely independent of mismatch repair, indicating that shorter gene conversion tracts in pol3-ct correspond to shorter extensions of primed DNA synthesis. Interestingly, we find that shorter repair tracts do not favor synthesis-dependent strand annealing at the expense of double-strand-break repair. Finally, we show that the DNA polymerases that have been previously suspected to mediate HR repair synthesis (Polɛ and Polη) do not affect gene conversion during induced HR, including in the pol3-ct background. Our results argue strongly for the preferential recruitment of Polδ during HR. PMID:18086882

  11. p53 modulates homologous recombination by transcriptional regulation of the RAD51 gene

    PubMed Central

    Arias-Lopez, Carmen; Lazaro-Trueba, Iciar; Kerr, Peter; Lord, Christopher J; Dexter, Tim; Iravani, Marjan; Ashworth, Alan; Silva, Augusto

    2006-01-01

    DNA repair by homologous recombination is involved in maintaining genome stability. Previous data report that wild-type p53 suppresses homologous recombination and physically interacts with Rad51. Here, we show the in vivo binding of wild-type p53 to a p53 response element in the promoter of Rad51 and the downregulation of Rad51 messenger RNA and protein by wild-type p53, favoured by DNA damage. Moreover, wild-type p53 inhibits Rad51 foci formation in response to double-strand breaks, whereas p53 contact mutant R280K fails to repress Rad51 mRNA and protein expression and Rad51 foci formation. We propose that transcriptional repression of Rad51 by p53 participates in regulating homologous recombination, and impaired Rad51 repression by p53 mutants may contribute to malignant transformation. PMID:16322760

  12. On the influence of protein-DNA register during homologous recombination.

    PubMed

    Greene, Eric C

    2016-01-17

    Homologous recombination enables the exchange of genetic information between related DNA molecules and is a driving force in evolution. Using single-molecule optical microscopy we have recently shown that members of the Rad51/RecA family of recombinases stabilize paired homologous strand of DNA in precise 3-nt increments. Here we discuss an interesting conceptual implication of these results, which is that the recombinases may actively sense and reorganize their alignment register relative to the bound DNA sequences to ensure optimal base triplet pairing interactions during the early stages of recombination. PMID:26652653

  13. Homologous Recombination in E3 Genes of Human Adenovirus Species D

    PubMed Central

    Singh, Gurdeep; Robinson, Christopher M.; Dehghan, Shoaleh; Jones, Morris S.; Dyer, David W.; Seto, Donald

    2013-01-01

    Genes within the E3 transcription unit of human adenoviruses modulate host immune responses to infection. A comprehensive genomics and bioinformatics analysis of the E3 transcription unit for 38 viruses within human adenovirus species D (HAdV-D) revealed distinct and surprising patterns of homologous recombination. Homologous recombination was identified in open reading frames for E3 CR1α, CR1β, and CR1γ, similar to that previously observed with genes encoding the three major structural capsid proteins, the penton base, hexon, and fiber. PMID:24027303

  14. Effect of limited homology on gene conversion in a Saccharomyces cerevisiae plasmid recombination system

    SciTech Connect

    Ahn, B.Y.; Dornfeld, K.J.; Fagrelius, T.J.; Livingston, D.M.

    1988-06-01

    Plasmids containing heteroallelic copies of the Saccharomyces cerevisiae HIS3 gene undergo intramolecular gene conservation in mitotically dividing S. cerevisiae cels. The authors used this plasmid system to determine the minimum amount of homology required for gene conversion, to examine how conversion tract lengths are affected by limited homology, and to analyze the role of flanking DNA sequences on the pattern of exchange. Plasmids with homologous sequences greater than 2 kilobases have mitotic exchange rates as high as 2 x 10/sup -3/ event soper cell per generation. As the homology is reduced, the exchange rate decreases dramatically. A plasmid with 26 base pairs (bp) of homology undergones gene conversion at a rate of approximately 1 x 10/sup -10/ events per cell per generation. These studies have also shown that an 8-bp insertion mutation 13 bp from a border between homologous and nonhomologous sequences undergoes conversion, but that a similar 8-bp insertion 5 bp from a border does not. Examination of independent conversion events which occurred in plasmids with heteroallelic copies of the HIS3 gene shows that markers within 280 bp of a border between homologous and nonhomologous sequences undergo conversion less frequently than the same markers within a more extensive homologous sequence. Thus, proximity to a border between homologous and nonhomologous sequences shortens the conversion tract length.

  15. Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

    PubMed Central

    Symington, Lorraine S.

    2002-01-01

    The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination. PMID:12456786

  16. Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination.

    PubMed

    Onyango, David O; Howard, Sean M; Neherin, Kashfia; Yanez, Diana A; Stark, Jeremy M

    2016-07-01

    We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3' ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt)-induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF), which requires end resection. Furthermore, XAB2 mediates specific aspects of the DNA damage response associated with end resection proficiency: CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF. XAB2 also promotes histone acetylation events linked to HR proficiency. From truncation mutation analysis, the capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. This XAB2 complex localizes to punctate structures consistent with interchromatin granules that show a striking adjacent-localization to the DSB marker γH2AX. In summary, we suggest that the XAB2 complex mediates DNA damage response events important for the end resection step of HR, and speculate that its adjacent-localization relative to DSBs marked by γH2AX is important for this function. PMID:27084940

  17. Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination

    PubMed Central

    Onyango, David O.; Howard, Sean M.; Neherin, Kashfia; Yanez, Diana A.; Stark, Jeremy M.

    2016-01-01

    We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3′ ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt)-induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF), which requires end resection. Furthermore, XAB2 mediates specific aspects of the DNA damage response associated with end resection proficiency: CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF. XAB2 also promotes histone acetylation events linked to HR proficiency. From truncation mutation analysis, the capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. This XAB2 complex localizes to punctate structures consistent with interchromatin granules that show a striking adjacent-localization to the DSB marker γH2AX. In summary, we suggest that the XAB2 complex mediates DNA damage response events important for the end resection step of HR, and speculate that its adjacent-localization relative to DSBs marked by γH2AX is important for this function. PMID:27084940

  18. Heteroduplex Formation, Mismatch Resolution, and Genetic Sectoring During Homologous Recombination in the Hyperthermophilic Archaeon Sulfolobus Acidocaldarius

    PubMed Central

    Mao, Dominic; Grogan, Dennis W.

    2012-01-01

    Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR) remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldarius pyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr+ clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells, or to conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which regions of heteroduplex DNA formed and then segregated after partial resolution of inter-strand differences. Surprisingly, sectoring was also frequent in cells transformed with single-stranded DNAs. Oligonucleotides produced more sectored transformants when electroporated as single strands than as a duplex, although all forms of donor DNA (positive-strand, negative-strand, and duplex) produced a diversity of genotypes, despite the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells. Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs. PMID:22679441

  19. Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System

    PubMed Central

    An, Mahru C.; O'Brien, Robert N.; Zhang, Ningzhe; Patra, Biranchi N.; De La Cruz, Michael; Ray, Animesh; Ellerby, Lisa M.

    2014-01-01

    We have previously reported the genetic correction of Huntington’s disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. PMID:24761311

  20. Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the present study was to gain new insights into the evolution, homologous recombination and selection pressures imposed on the porcine torovirus (PToV), by examining changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerge...

  1. Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status

    SciTech Connect

    Sprong, Debbie; Janssen, Hilde L.; Vens, Conchita; Begg, Adrian C. . E-mail: a.begg@nki.nl

    2006-02-01

    Purpose: To determine the role of DNA repair in hypoxic radioresistance. Methods and Materials: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. Results: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. Conclusion: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity.

  2. The BARD1/HP1 interaction: Another clue to heterochromatin involvement in homologous recombination

    PubMed Central

    Fukuda, Takayo; Tsuruga, Tomoko; Kuroda, Takako; Takeuchi, Jun; Wu, Wenwen; Ohta, Tomohiko

    2016-01-01

    Chromatin compaction represents a barrier for the repair of DNA double-strand breaks (DSBs). However, heterochromatin components are also required for DSB repair by homologous recombination. The BARD1/HP1 interaction, required for the retention of BRCA1, CTIP, and RAD51 at DSB sites, may play a critical role in the crosstalk between chromatin compaction and DSB repair. PMID:27308582

  3. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.

    PubMed

    Dickinson, Daniel J; Ward, Jordan D; Reiner, David J; Goldstein, Bob

    2013-10-01

    Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here we report a method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism. PMID:23995389

  4. Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium

    SciTech Connect

    Sollewijn Gelpke, M.D.; Mayfield-Gambill, M.; Lin Cereghino, G.P.; Gold, M.H.

    1999-04-01

    The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter was used to drive expression of lip2, the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of Phanerochaete chrysosporium. The expression vector, pUGL, also contained the Schizophyllum commune ura1 gene as a selectable marker. pUGL was used to transform a P. chrysosporium Ura11 auxotroph to prototrophy. Ura{sup +} transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPH8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.

  5. Biased Gene Conversion in Rhizobium etli Is Caused by Preferential Double-Strand Breaks on One of the Recombining Homologs

    PubMed Central

    Yáñez-Cuna, Fares Osam; Castellanos, Mildred

    2015-01-01

    ABSTRACT Gene conversion, the nonreciprocal transfer of information during homologous recombination, is the main process that maintains identity between members of multigene families. Gene conversion in the nitrogenase (nifH) multigene family of Rhizobium etli was analyzed by using a two-plasmid system, where each plasmid carried a copy of nifH. One of the nifH copies was modified, creating restriction fragment length polymorphisms (RFLPs) spaced along the gene. Once the modified plasmid was introduced into R. etli, selection was done for cointegration with a resident plasmid lacking the RFLPs. Most of the cointegrate molecules harbor gene conversion events, biased toward a gain of RFLPs. This bias may be explained under the double-strand break repair model by proposing that the nifH gene lacking the RFLPs suffers a DNA double-strand break, so the incoming plasmid functions as a template for repairing the homolog on the resident plasmid. To support this proposal, we cloned an SceI site into the nifH homolog that had the RFLPs used for scoring gene conversion. In vivo expression of the meganuclease I-SceI allowed the generation of a double-strand break on this homolog. Upon introduction of this modified plasmid into an R. etli strain lacking I-SceI, biased gene conversion still favored the retention of markers on the incoming plasmid. In contrast, when the recipient strain ectopically expressed I-SceI, a dramatic reversal in gene conversion bias was seen, favoring the preservation of resident sequences. These results show that biased gene conversion is caused by preferential double-strand breaks on one of the recombining homologs. IMPORTANCE In this work, we analyzed gene conversion by using a system that entails horizontal gene transfer followed by homologous recombination in the recipient cell. Most gene conversion events are biased toward the acquisition of the incoming sequences, ranging in size from 120 bp to 800 bp. This bias is due to preferential cutting of

  6. A yeast-based genetic screening to identify human proteins that increase homologous recombination.

    PubMed

    Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

    2008-05-01

    To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR. PMID:18248415

  7. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    SciTech Connect

    Larionov, V.; Kouprina, N. |; Eldarov, M. |; Perkins, E.; Porter, G.; Resnick, M.A.

    1994-10-01

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic-growth. The frequency of recombination is partly dependent on the method of transformation In that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RAD52, RAD1 and the RNC1 genes.

  8. PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways

    PubMed Central

    Guirouilh Barbat, Josée; Bonnet, Marie-Elise; Illuzzi, Giuditta; Ronde, Philippe; Gauthier, Laurent R.; Magroun, Najat; Rajendran, Anbazhagan; Lopez, Bernard S.; Scully, Ralph; Boussin, François D.; Schreiber, Valérie; Dantzer, Françoise

    2014-01-01

    The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites and induces an imbalance between BRCA1 and 53BP1. Both events result in compromised accurate C-NHEJ and a concomitant increase in DNA end resection. Nevertheless, HR is significantly reduced upon PARP3 silencing while the enhanced end resection causes mutagenic deletions during A-EJ. As a result, the absence of PARP3 confers hypersensitivity to anti-tumoral drugs generating DSB. PMID:24598253

  9. Cohesin Is Limiting for the Suppression of DNA Damage–Induced Recombination between Homologous Chromosomes

    PubMed Central

    Covo, Shay; Westmoreland, James W.

    2010-01-01

    Double-strand break (DSB) repair through homologous recombination (HR) is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin) is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage–induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G2/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G2/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage–induced recombinants in G2/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer. PMID:20617204

  10. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

    PubMed

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia; Altmannova, Veronika; Sebesta, Marek; Pacesa, Martin; Fugger, Kasper; Sorensen, Claus Storgaard; Lee, Marietta Y W T; Haracska, Lajos; Krejci, Lumir

    2016-04-20

    Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HRin vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy. PMID:26792895

  11. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

    PubMed Central

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia; Altmannova, Veronika; Sebesta, Marek; Pacesa, Martin; Fugger, Kasper; Sorensen, Claus Storgaard; Lee, Marietta Y.W.T.; Haracska, Lajos; Krejci, Lumir

    2016-01-01

    Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy. PMID:26792895

  12. Sister cohesion and structural axis components mediate homolog bias of meiotic recombination

    PubMed Central

    Kim, Keun P.; Weiner, Beth M.; Zhang, Liangran; Jordan, Amy; Dekker, Job; Kleckner, Nancy

    2010-01-01

    SUMMARY Meiotic recombination occurs between one chromatid of each maternal and paternal homolog (homolog bias) versus between sister chromatids (sister bias). Physical DNA analysis reveals that meiotic cohesin/axis component Rec8 promotes sister bias, likely via its cohesion activity. Two meiosis-specific axis components, Red1/Mek1kinase, counteract this effect. With this precondition satisfied, other molecules directly specify homolog bias per se. Rec8 also acts positively to maintain homolog bias during crossover recombination. These observations point to sequential release of double-strand break ends from association with their sister. Red1 and Rec8 are found to play distinct roles for sister cohesion, DSB formation and recombination progression kinetics. Also, the two components are enriched in spatially distinct domains of axial structure that develop prior to DSB formation. We propose that Red1 and Rec8 domains provide functionally complementary environments whereby inputs evolved from DSB repair and late-stage chromosome morphogenesis are integrated to give the complete meiotic chromosomal program. PMID:21145459

  13. Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica.

    PubMed

    Charcas-Lopez, Ma del Socorro; Garcia-Morales, Lorena; Pezet-Valdez, Marisol; Lopez-Camarillo, Cesar; Zamorano-Carrillo, Absalom; Marchat, Laurence A

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan. PMID:24534563

  14. Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica

    PubMed Central

    del Socorro Charcas-Lopez, Ma.; Garcia-Morales, Lorena; Pezet-Valdez, Marisol; Lopez-Camarillo, Cesar; Zamorano-Carrillo, Absalom; Marchat, Laurence A.

    2014-01-01

    Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan. PMID:24534563

  15. Multiple Evolutionary Events Involved in Maintaining Homologs of Resistance to Powdery Mildew 8 in Brassica napus.

    PubMed

    Li, Qin; Li, Jing; Sun, Jin-Long; Ma, Xian-Feng; Wang, Ting-Ting; Berkey, Robert; Yang, Hui; Niu, Ying-Ze; Fan, Jing; Li, Yan; Xiao, Shunyuan; Wang, Wen-Ming

    2016-01-01

    The Resistance to Powdery Mildew 8 (RPW8) locus confers broad-spectrum resistance to powdery mildew in Arabidopsis thaliana. There are four Homologous to RPW8s (BrHRs) in Brassica rapa and three in Brassica oleracea (BoHRs). Brassica napus (Bn) is derived from diploidization of a hybrid between B. rapa and B. oleracea, thus should have seven homologs of RPW8 (BnHRs). It is unclear whether these genes are still maintained or lost in B. napus after diploidization and how they might have been evolved. Here, we reported the identification and sequence polymorphisms of BnHRs from a set of B. napus accessions. Our data indicated that while the BoHR copy from B. oleracea is highly conserved, the BrHR copy from B. rapa is relatively variable in the B. napus genome owing to multiple evolutionary events, such as gene loss, point mutation, insertion, deletion, and intragenic recombination. Given the overall high sequence homology of BnHR genes, it is not surprising that both intragenic recombination between two orthologs and two paralogs were detected in B. napus, which may explain the loss of BoHR genes in some B. napus accessions. When ectopically expressed in Arabidopsis, a C-terminally truncated version of BnHRa and BnHRb, as well as the full length BnHRd fused with YFP at their C-termini could trigger cell death in the absence of pathogens and enhanced resistance to powdery mildew disease. Moreover, subcellular localization analysis showed that both BnHRa-YFP and BnHRb-YFP were mainly localized to the extra-haustorial membrane encasing the haustorium of powdery mildew. Taken together, our data suggest that the duplicated BnHR genes might have been subjected to differential selection and at least some may play a role in defense and could serve as resistance resource in engineering disease-resistant plants. PMID:27493652

  16. Multiple Evolutionary Events Involved in Maintaining Homologs of Resistance to Powdery Mildew 8 in Brassica napus

    PubMed Central

    Li, Qin; Li, Jing; Sun, Jin-Long; Ma, Xian-Feng; Wang, Ting-Ting; Berkey, Robert; Yang, Hui; Niu, Ying-Ze; Fan, Jing; Li, Yan; Xiao, Shunyuan; Wang, Wen-Ming

    2016-01-01

    The Resistance to Powdery Mildew 8 (RPW8) locus confers broad-spectrum resistance to powdery mildew in Arabidopsis thaliana. There are four Homologous to RPW8s (BrHRs) in Brassica rapa and three in Brassica oleracea (BoHRs). Brassica napus (Bn) is derived from diploidization of a hybrid between B. rapa and B. oleracea, thus should have seven homologs of RPW8 (BnHRs). It is unclear whether these genes are still maintained or lost in B. napus after diploidization and how they might have been evolved. Here, we reported the identification and sequence polymorphisms of BnHRs from a set of B. napus accessions. Our data indicated that while the BoHR copy from B. oleracea is highly conserved, the BrHR copy from B. rapa is relatively variable in the B. napus genome owing to multiple evolutionary events, such as gene loss, point mutation, insertion, deletion, and intragenic recombination. Given the overall high sequence homology of BnHR genes, it is not surprising that both intragenic recombination between two orthologs and two paralogs were detected in B. napus, which may explain the loss of BoHR genes in some B. napus accessions. When ectopically expressed in Arabidopsis, a C-terminally truncated version of BnHRa and BnHRb, as well as the full length BnHRd fused with YFP at their C-termini could trigger cell death in the absence of pathogens and enhanced resistance to powdery mildew disease. Moreover, subcellular localization analysis showed that both BnHRa-YFP and BnHRb-YFP were mainly localized to the extra-haustorial membrane encasing the haustorium of powdery mildew. Taken together, our data suggest that the duplicated BnHR genes might have been subjected to differential selection and at least some may play a role in defense and could serve as resistance resource in engineering disease-resistant plants. PMID:27493652

  17. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    SciTech Connect

    Henrique Barreta, Marcos; Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de; Ferreira, Rogerio; Oliveira, Joao Francisco de; Goncalves, Paulo Bayard Dias; Bordignon, Vilceu

    2012-10-01

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  18. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

    PubMed

    Kilian, Oliver; Benemann, Christina S E; Niyogi, Krishna K; Vick, Bertrand

    2011-12-27

    Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology. PMID:22123974

  19. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

    PubMed Central

    Kilian, Oliver; Benemann, Christina S. E.; Niyogi, Krishna K.; Vick, Bertrand

    2011-01-01

    Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology. PMID:22123974

  20. A protocol for construction of gene targeting vectors and generation of homologous recombinant ES cells

    PubMed Central

    Bouabe, Hicham; Okkenhaug, Klaus

    2015-01-01

    Summary The completion of human and mouse genome sequencing has confronted us with huge amount of data sequences that certainly need decades and many generations of scientists to be reasonably interpreted and assigned to physiological functions, and subsequently fruitfully translated into medical application. A means to assess the function of genes provides gene targeting in mouse embryonic stem (ES) cells that enables to introduce site-specific modifications in the mouse genome, and analyze their physiological consequences. Gene targeting enables almost any type of genetic modifications of interest, ranging from gene insertion (e.g. insertion of human-specific genes or reporter genes), gene disruption, point mutations, short and long range deletions, inversions. Site-specific modification into the genome of ES cells can be reached by homologous recombination using targeting vectors. Here, we describe a protocol to generate targeting constructs and homologous recombinant ES cells. PMID:23996269

  1. Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

    PubMed Central

    Bindra, Ranjit S.; Schaffer, Paul J.; Meng, Alice; Woo, Jennifer; Måseide, Kårstein; Roth, Matt E.; Lizardi, Paul; Hedley, David W.; Bristow, Robert G.; Glazer, Peter M.

    2004-01-01

    There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxia-mediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies. PMID:15367671

  2. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination.

    PubMed

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-03-18

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  3. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    PubMed Central

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  4. Multiple regulation of Rad51-mediated homologous recombination by fission yeast Fbh1.

    PubMed

    Tsutsui, Yasuhiro; Kurokawa, Yumiko; Ito, Kentaro; Siddique, Md Shahjahan P; Kawano, Yumiko; Yamao, Fumiaki; Iwasaki, Hiroshi

    2014-08-01

    Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated. PMID:25165823

  5. Mutagenic Organized Recombination Process by Homologous In Vivo Grouping (MORPHING) for Directed Enzyme Evolution

    PubMed Central

    Gonzalez-Perez, David; Molina-Espeja, Patricia; Garcia-Ruiz, Eva; Alcalde, Miguel

    2014-01-01

    Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence. PMID:24614282

  6. Genotypic effects on the frequency of homoeologous and homologous recombination in Brassica napus × B. carinata hybrids.

    PubMed

    Mason, Annaliese S; Nelson, Matthew N; Castello, Marie-Claire; Yan, Guijun; Cowling, Wallace A

    2011-02-01

    We investigated the influence of genotype on homoeologous and homologous recombination frequency in eight different Brassica napus (AAC(n)C(n)) × B. carinata (BBC(c)C(c)) interspecific hybrids (genome composition C(n)C(c)AB). Meiotic recombination events were assessed through microsatellite marker analysis of 67 unreduced microspore-derived progeny. Thirty-four microsatellite markers amplified 83 A-, B-, C(n)- and C(c)-genome alleles at 64 loci, of which a subset of seven markers amplifying 26 alleles could be used to determine allele copy number. Hybrid genotypes varied significantly in loss of A- and B-genome alleles (P < 0.0001), which ranged from 6 to 22% between hybrid progeny sets. Allele copy number analysis revealed 19 A-C, 3 A-B and 10 B-C duplication/deletion events attributed to homoeologous recombination. Additionally, 55 deletions and 19 duplications without an accompanying dosage change in homoeologous alleles were detected. Hybrid progeny sets varied in observed frequencies of loss, gain and exchange of alleles across the A and B genomes as well as in the diploid C genome. Self-fertility in hybrid progeny decreased as the loss of B-genome loci (but not A-genome loci) increased. Hybrid genotypes with high levels of homologous and homoeologous exchange may be exploited for genetic introgressions between B. carinata and B. napus (canola), and those with low levels may be used to develop stable synthetic Brassica allopolyploids. PMID:21046065

  7. Involvement of Caveolin-1 in Repair of DNA Damage through Both Homologous Recombination and Non-Homologous End Joining

    PubMed Central

    Zhu, Hua; Yue, Jingyin; Pan, Zui; Wu, Hao; Cheng, Yan; Lu, Huimei; Ren, Xingcong; Yao, Ming; Shen, Zhiyuan; Yang, Jin-Ming

    2010-01-01

    Background Caveolin-1 (Cav-1), the major component of caveolae, is a 21–24 kDa integral membrane protein that interacts with a number of signaling molecules. By acting as a scaffolding protein, Cav-1 plays crucial roles in the regulation of various physiologic and patho-physiologic processes including oncogenic transformation and tumorigenesis, and tumor invasion and metastasis. Methodology/Principal Findings In the present study we sought to explore the role of Cav-1 in response to DNA damage and the mechanism involved. We found that the level of Cav-1 was up-regulated rapidly in cells treated with ionizing radiation. The up-regulation of Cav-1 following DNA damage occurred only in cells expressing endogenous Cav-1, and was associated with the activation of DNA damage response pathways. Furthermore, we demonstrated that the expression of Cav-1 protected cells against DNA damage through modulating the activities of both the homologous recombination (HR) and non-homologous end joining (NHEJ) repair systems, as evidenced by the inhibitory effects of the Cav-1-targeted siRNA on cell survival, HR frequency, phosphorylation of DNA-dependent protein kinase (DNA-PK), and nuclear translocation of epidermal growth factor receptor (EGFR) following DNA damage, and by the stimulatory effect of the forced expression of Cav-1 on NHEJ frequency. Conclusion/Significance Our results indicate that Cav-1 may play a critical role in sensing genotoxic stress and in orchestrating the response of cells to DNA damage through regulating the important molecules involved in maintaining genomic integrity. PMID:20700465

  8. Optimal cloning of PCR fragments by homologous recombination in Escherichia coli.

    PubMed

    Jacobus, Ana Paula; Gross, Jeferson

    2015-01-01

    PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5' termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories. PMID:25774528

  9. Topoisomerase II-Mediated DNA Damage Is Differently Repaired during the Cell Cycle by Non-Homologous End Joining and Homologous Recombination

    PubMed Central

    de Campos-Nebel, Marcelo; Larripa, Irene; González-Cid, Marcela

    2010-01-01

    Topoisomerase II (Top2) is a nuclear enzyme involved in several metabolic processes of DNA. Chemotherapy agents that poison Top2 are known to induce persistent protein-mediated DNA double strand breaks (DSB). In this report, by using knock down experiments, we demonstrated that Top2α was largely responsible for the induction of γH2AX and cytotoxicity by the Top2 poisons idarubicin and etoposide in normal human cells. As DSB resulting from Top2 poisons-mediated damage may be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), we aimed to analyze both DNA repair pathways. We found that DNA-PKcs was rapidly activated in human cells, as evidenced by autophosphorylation at serine 2056, following Top2-mediated DNA damage. The chemical inhibition of DNA-PKcs by wortmannin and vanillin resulted in an increased accumulation of DNA DSB, as evaluated by the comet assay. This was supported by a hypersensitive phenotype to Top2 poisons of Ku80- and DNA-PKcs- defective Chinese hamster cell lines. We also showed that Rad51 protein levels, Rad51 foci formation and sister chromatid exchanges were increased in human cells following Top2-mediated DNA damage. In support, BRCA2- and Rad51C- defective Chinese hamster cells displayed hypersensitivity to Top2 poisons. The analysis by immunofluorescence of the DNA DSB repair response in synchronized human cell cultures revealed activation of DNA-PKcs throughout the cell cycle and Rad51 foci formation in S and late S/G2 cells. Additionally, we found an increase of DNA-PKcs-mediated residual repair events, but not Rad51 residual foci, into micronucleated and apoptotic cells. Therefore, we conclude that in human cells both NHEJ and HR are required, with cell cycle stage specificity, for the repair of Top2-mediated reversible DNA damage. Moreover, NHEJ-mediated residual repair events are more frequently associated to irreversibly damaged cells. PMID:20824055

  10. Production of a heterozygous mutant cell line by homologous recombination (single knockout).

    PubMed

    Mortensen, R

    2001-05-01

    Formerly UNIT 9.16, this unit takes a more appropriate place in Chapter 23, and has been updated and revised for this publication. Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. In this unit, the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker (e.g., neo). Expression of neo is obtained by including the phosphoglycerate kinase (PGK) promoter in the construct. To enrich for clones in which the target gene has undergone homologous recombination over those in which random integration of the construct has occurred, a negative selectable marker, herpes simplex virus thymidine kinase (HSV-TK), is included in the construct outside the region of homology to the target gene. Depending upon the target gene, it may be easier to assemble the construct by adding the neo and TK genes to the cloned target gene or by adding two fragments of the target gene to a plasmid containing the neo and TK genes. PMID:18265206

  11. Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs.

    PubMed

    Shedge, Vikas; Arrieta-Montiel, Maria; Christensen, Alan C; Mackenzie, Sally A

    2007-04-01

    For >20 years, the enigmatic behavior of plant mitochondrial genomes has been well described but not well understood. Chimeric genes appear, and occasionally are differentially replicated or expressed, with significant effects on plant phenotype, most notably on male fertility, yet the mechanisms of DNA replication, chimera formation, and recombination have remained elusive. Using mutations in two important genes of mitochondrial DNA metabolism, we have observed reproducible asymmetric recombination events occurring at specific locations in the mitochondrial genome. Based on these experiments and existing models of double-strand break repair, we propose a model for plant mitochondrial DNA replication, chimeric gene formation, and the illegitimate recombination events that lead to stoichiometric changes. We also address the physiological and developmental effects of aberrant events in mitochondrial genome maintenance, showing that mitochondrial genome rearrangements, when controlled, influence plant reproduction, but when uncontrolled, lead to aberrant growth phenotypes and dramatic reduction of the cell cycle. PMID:17468263

  12. Enhanced homologous recombination is induced by alpha-particle radiation in somatic cells of Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Bian, Po; Liu, Ping; Wu, Yuejin

    Almost 9 percent of cosmic rays which strike the earth's atmosphere are alpha particles. As one of the ionizing radiations (IR), its biological effects have been widely studied. However, the plant genomic instability induced by alpha-particle radiation was not largely known. In this research, the Arabidopsis thaliana transgenic for GUS recombination substrate was used to evaluate the genomic instability induced by alpha-particle radiation (3.3MeV). The pronounced effects of systemic exposure to alpha-particle radiation on the somatic homologous recombination frequency (HRF) were found at different doses. The 10Gy dose of radiation induced the maximal HRF which was 1.9-fold higher than the control. The local radiation of alpha-particle (10Gy) on root also resulted in a 2.5-fold increase of somatic HRF in non-radiated aerial plant, indicating that the signal(s) of genomic instability was transferred to non-radiated parts and initiated their genomic instability. Concurrent treatment of seedlings of Arabidopsis thaliana with alpha-particle and DMSO(ROS scavenger) both in systemic and local radiation signifi- cantly suppressed the somatic HR, indicating that the free radicals produced by alpha-particle radiation took part in the production of signal of genomic instability rather than the signal transfer. Key words: alpha-particle radiation, somatic homologous recombination, genomic instability

  13. Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination

    SciTech Connect

    Hoang, Margaret L.; Tan, Frederick J.; Lai, David C.; Celniker, Sue E.; Hoskins, Roger A.; Dunham, Maitreya J.; Zheng, Yixian; Koshland, Douglas

    2010-08-27

    Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR-dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR-dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer.

  14. Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells.

    PubMed Central

    Wu, H; Liu, X; Jaenisch, R

    1994-01-01

    A subtle mutation that rendered type I collagen resistant to mammalian collagenase has been introduced into the murine Col1a-1 (recently redesignated Cola-1) gene by homologous recombination in embryonic stem (ES) cells. Initially, a "hit and run" procedure was used. Since two steps were required for introducing each mutation and more than one mutation was to be introduced in the same genomic region independently, we have developed a streamlined procedure that involves two sequential replacement-type homologous recombination events. In the first step, an internal deletion was introduced into the Col1a-1 locus along with the positive and negative selectable markers, neo and tk, to mark the region of interest. G418-resistant homologous recombinants were isolated and used in the second step in which the deleted Col1a-1 allele was replaced with a construct containing the desired mutation. Homologous recombinants containing the mutation were identified among the Tk- ES clones after selection with FIAU [1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (called fialuridine)]. Approximately 10% of such clones contained the desired mutation. The double replacement procedure greatly reduces the time and amount of work required to introduce mutations independently into the same or closely linked regions. Once the homologous recombinants derived from the first step are established, the introduction of other mutations into the deleted region becomes a one-step procedure. For X number of introduced mutations, 2X selections are required with the "hit and run" approach, but only X + 1 are required with the double-replacement method. This innovative procedure could be very useful in studies of gene structure and function as well as gene expression and regulation. Images PMID:8146196

  15. Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures

    SciTech Connect

    Chen, Zhucheng; Yang, Haijuan; Pavletich, Nikola P

    2008-07-08

    The RecA family of ATPases mediates homologous recombination, a reaction essential for maintaining genomic integrity and for generating genetic diversity. RecA, ATP and single-stranded DNA (ssDNA) form a helical filament that binds to double-stranded DNA (dsDNA), searches for homology, and then catalyses the exchange of the complementary strand, producing a new heteroduplex. Here we have solved the crystal structures of the Escherichia coli RecA-ssDNA and RecA-heteroduplex filaments. They show that ssDNA and ATP bind to RecA-RecA interfaces cooperatively, explaining the ATP dependency of DNA binding. The ATP {gamma}-phosphate is sensed across the RecA-RecA interface by two lysine residues that also stimulate ATP hydrolysis, providing a mechanism for DNA release. The DNA is underwound and stretched globally, but locally it adopts a B-DNA-like conformation that restricts the homology search to Watson-Crick-type base pairing. The complementary strand interacts primarily through base pairing, making heteroduplex formation strictly dependent on complementarity. The underwound, stretched filament conformation probably evolved to destabilize the donor duplex, freeing the complementary strand for homology sampling.

  16. Complex in vivo Ligation Using Homologous Recombination and High-efficiency Plasmid Rescue from Saccharomyces cerevisiae

    PubMed Central

    Finnigan, Gregory C.; Thorner, Jeremy

    2015-01-01

    The protocols presented here allow for the facile generation of a wide variety of complex multipart DNA constructs (tagged gene products, gene fusions, chimeric proteins, and other variants) using homologous recombination and in vivo ligation in budding yeast (Saccharomyces cerevisiae). This method is straightforward, efficient and cost-effective, and can be used both for vector creation and for subsequent one-step, high frequency integration into a chromosomal locus in yeast. The procedure utilizes PCR with extended oligonucleotide “tails” of homology between multiple fragments to allow for reassembly in yeast in a single transformation followed by a method for highly efficient plasmid extraction from yeast (for transformation into bacteria). The latter is an improvement on existing methods of yeast plasmid extraction, which, historically, has been a limiting step in recovery of desired constructs. We describe the utility and convenience of our techniques, and provide several examples. PMID:26523287

  17. Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

    PubMed Central

    Ishizaki, Kimitsune; Johzuka-Hisatomi, Yasuyo; Ishida, Sakiko; Iida, Shigeru; Kohchi, Takayuki

    2013-01-01

    The liverwort Marchantia polymorpha is an emerging model organism on account of its ideal characteristics for molecular genetics in addition to occupying a crucial position in the evolution of land plants. Here we describe a method for gene targeting by applying a positive/negative selection system for reduction of non-homologous random integration to an efficient Agrobacterium-mediated transformation system using M. polymorpha sporelings. The targeting efficiency was evaluated by knocking out the NOP1 gene, which impaired air-chamber formation. Homologous recombination was observed in about 2% of the thalli that passed the positive/negative selection. With the advantage of utilizing the haploid gametophytic generation, this strategy should facilitate further molecular genetic analysis of M. polymorpha, in which many of the mechanisms found in land plants are conserved, yet in a less complex form. PMID:23524944

  18. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs.

    PubMed

    Nagy, Zita; Kalousi, Alkmini; Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-02-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  19. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

    PubMed Central

    Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-01-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  20. CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase

    PubMed Central

    Luo, Kuntian; Deng, Min; Li, Yunhui; Wu, Chenming; Xu, Ziwen; Yuan, Jian; Lou, Zhenkun

    2015-01-01

    There are the two major pathways responsible for the repair of DNA double-strand breaks (DSBs): non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ operates throughout the cell-cycle, while HR is primarily active in the S/G2 phases suggesting that there are cell cycle-specific mechanisms that regulate the balance between NHEJ and HR. Here we reported that CDK2 could phosphorylate RNF4 on T26 and T112 and enhance RNF4 E3 ligase activity, which is important for MDC1 degradation and proper HR repair during S phase. Mutation of the RNF4 phosphorylation sites results in MDC1 stabilization, which in turn compromised HR during S-phase. These results suggest that in addition to drive cell cycle progression, CDK also targets RNF4, which is involved in the regulatory network of DSBs repair. PMID:25948581

  1. CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase.

    PubMed

    Luo, Kuntian; Deng, Min; Li, Yunhui; Wu, Chenming; Xu, Ziwen; Yuan, Jian; Lou, Zhenkun

    2015-06-23

    There are the two major pathways responsible for the repair of DNA double-strand breaks (DSBs): non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ operates throughout the cell-cycle, while HR is primarily active in the S/G2 phases suggesting that there are cell cycle-specific mechanisms that regulate the balance between NHEJ and HR. Here we reported that CDK2 could phosphorylate RNF4 on T26 and T112 and enhance RNF4 E3 ligase activity, which is important for MDC1 degradation and proper HR repair during S phase. Mutation of the RNF4 phosphorylation sites results in MDC1 stabilization, which in turn compromised HR during S-phase. These results suggest that in addition to drive cell cycle progression, CDK also targets RNF4, which is involved in the regulatory network of DSBs repair. PMID:25948581

  2. Srs2 and Mus81-Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates.

    PubMed

    Keyamura, Kenji; Arai, Kota; Hishida, Takashi

    2016-07-01

    Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ), and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81-Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability. PMID:27390022

  3. Mutations in homologous recombination genes rescue top3 slow growth in Saccharomyces cerevisiae.

    PubMed Central

    Shor, Erika; Gangloff, Serge; Wagner, Marisa; Weinstein, Justin; Price, Gavrielle; Rothstein, Rodney

    2002-01-01

    In budding yeast, loss of topoisomerase III, encoded by the TOP3 gene, leads to a genomic instability phenotype that includes slow growth, hyper-sensitivity to genotoxic agents, mitotic hyper-recombination, increased chromosome missegregation, and meiotic failure. Slow growth and other defects of top3 mutants are suppressed by mutation of SGS1, which encodes the only RecQ helicase in S. cerevisiae. sgs1 is epistatic to top3, suggesting that the two proteins act in the same pathway. To identify other factors that function in the Sgs1-Top3 pathway, we undertook a genetic screen for non-sgs1 suppressors of top3 defects. We found that slow growth and DNA damage sensitivity of top3 mutants are suppressed by mutations in RAD51, RAD54, RAD55, and RAD57. In contrast, top3 mutants show extreme synergistic growth defects with mutations in RAD50, MRE11, XRS2, RDH54, and RAD1. We also analyzed recombination at the SUP4-o region, showing that in a rad51, rad54, rad55, or rad57 background top3Delta does not increase recombination to the same degree as in a wild-type strain. These results suggest that the presence of the Rad51 homologous recombination complex in a top3 background facilitates creation of detrimental intermediates by Sgs1. We present a model wherein Rad51 helps recruit Sgs1-Top3 to sites of replicative damage. PMID:12399378

  4. A novel non-homologous recombination-mediated mechanism for Escherichia coli unilateral flagellar phase variation.

    PubMed

    Liu, Bin; Hu, Bo; Zhou, Zhemin; Guo, Dan; Guo, Xi; Ding, Peng; Feng, Lu; Wang, Lei

    2012-05-01

    Flagella contribute to the virulence of bacteria through chemotaxis, adhesion to and invasion of host surfaces. Flagellar phase variation is believed to facilitate bacterial evasion of the host immune response. In this study, the flnA gene that encodes Escherichia coli H17 flagellin was examined by whole genome sequencing and genetic deletion analysis. Unilateral flagellar phase variation has been reported in E. coli H3, H47 and H17 strains, although the mechanism for phase variation in the H17 strain has not been previously understood. Analysis of phase variants indicated that the flagellar phase variation in the H17 strain was caused by the deletion of an ∼35 kb DNA region containing the flnA gene from diverse excision sites. The presence of covalently closed extrachromosomal circular forms of this excised 35 kb region was confirmed by the two-step polymerase chain reaction. The deletion and complementation test revealed that the Int1157 integrase, a tyrosine recombinase, mediates the excision of this region. Unlike most tyrosine recombinases, Int1157 is suggested to recognize diverse sites and mediate recombination between non-homologous DNA sequences. This is the first report of non-homologous recombination mediating flagellar phase variation. PMID:22287625

  5. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    PubMed Central

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior. PMID:27148349

  6. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

    PubMed

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-08-19

    Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions. PMID:27220470

  7. Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells.

    PubMed

    Schusser, Benjamin; Collarini, Ellen J; Yi, Henry; Izquierdo, Shelley Mettler; Fesler, Jeffrey; Pedersen, Darlene; Klasing, Kirk C; Kaspers, Bernd; Harriman, William D; van de Lavoir, Marie-Cecile; Etches, Robert J; Leighton, Philip A

    2013-12-10

    Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery. PMID:24282302

  8. Deficiency in Homologous Recombination Renders Mammalian Cells More Sensitive to Proton Versus Photon Irradiation

    SciTech Connect

    Grosse, Nicole; Fontana, Andrea O.; Hug, Eugen B.; Lomax, Antony; Coray, Adolf; Augsburger, Marc; Paganetti, Harald; Sartori, Alessandro A.; Pruschy, Martin

    2014-01-01

    Purpose: To investigate the impact of the 2 major DNA repair machineries on cellular survival in response to irradiation with the 2 types of ionizing radiation. Methods and Materials: The DNA repair and cell survival endpoints in wild-type, homologous recombination (HR)-deficient, and nonhomologous end-joining-deficient cells were analyzed after irradiation with clinically relevant, low-linear energy transfer (LET) protons and 200-keV photons. Results: All cell lines were more sensitive to proton irradiation compared with photon irradiation, despite no differences in the induction of DNA breaks. Interestingly, HR-deficient cells and wild-type cells with small interfering RNA-down-regulated Rad51 were markedly hypersensitive to proton irradiation, resulting in an increased relative biological effectiveness in comparison with the relative biological effectiveness determined in wild-type cells. In contrast, lack of nonhomologous end-joining did not result in hypersensitivity toward proton irradiation. Repair kinetics of DNA damage in wild-type cells were equal after both types of irradiation, although proton irradiation resulted in more lethal chromosomal aberrations. Finally, repair kinetics in HR-deficient cells were significantly delayed after proton irradiation, with elevated amounts of residual γH2AX foci after irradiation. Conclusion: Our data indicate a differential quality of DNA damage by proton versus photon irradiation, with a specific requirement for homologous recombination for DNA repair and enhanced cell survival. This has potential relevance for clinical stratification of patients carrying mutations in the DNA damage response pathways.

  9. Homologous recombination causes the spontaneous deletion of AVR-Pia in Magnaporthe oryzae.

    PubMed

    Sone, Teruo; Takeuchi, Saori; Miki, Shinsuke; Satoh, Yuki; Ohtsuka, Keisuke; Abe, Ayumi; Asano, Kozo

    2013-02-01

    AVR-Pia, an avirulence gene in the genome of the rice blast fungus Magnaporthe oryzae, triggers a hypersensitive reaction in rice cultivars harbouring the resistance gene Pia. The copy number of AVR-Pia was revealed to vary from one to three among M. oryzae isolates avirulent to Pia rice, and three copies of the gene were located on a single chromosome in strain Ina168, from which the gene was originally cloned. The spontaneous avr-Pia mutant originated from Ina168, named Ina168m95-1, which lacks the AVR-Pia gene, and was therefore used to elucidate the molecular mechanism of the deletion of all three copies of AVR-Pia. Screening and analysis of cosmid clones indicated that two copies of the DNA-type transposon Occan (Occan(9E12) and Occan(3A3) ) were located on the same chromosome, and three copies of AVR-Pia were located in between the two Occan elements. Ina168m95-1 contains a conserved Occan element, named Occan(m95-1) , between sequences homologous to the 5'-flanking region of Occan(3A3) and the 3'-flanking region of Occan(9E12) . In addition, sequence polymorphisms indicated a homologous recombination between Occan(3A3) and Occan(9E12) , which resulted in Occan(m95-1) . Based on these observations, we propose the hypothesis that homologous recombination in the two Occan elements leads to the deletion of AVR-Pia in Ina168m95-1. PMID:23198972

  10. Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

    PubMed Central

    Parvin, Jeffrey; Chiba, Natsuko; Ransburgh, Derek

    2011-01-01

    The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was

  11. A dominant mutation in human RAD51 reveals its function in DNA interstrand crosslink repair independent of homologous recombination

    PubMed Central

    Wang, Anderson T.; Kim, Taeho; Wagner, John E.; Conti, Brooke A.; Lach, Francis P.; Huang, Athena L.; Molina, Henrik; Sanborn, Erica M.; Zierhut, Heather; Cornes, Belinda K.; Abhyankar, Avinash; Sougnez, Carrie; Gabriel, Stacey B.; Auerbach, Arleen D.; Kowalczykowski, Stephen C.; Smogorzewska, Agata

    2015-01-01

    Summary Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. In vitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity and a co-dominant negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio of mutant to wildtype RAD51 in cells. Instead, patient cells are sensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity. PMID:26253028

  12. A Dominant Mutation in Human RAD51 Reveals Its Function in DNA Interstrand Crosslink Repair Independent of Homologous Recombination.

    PubMed

    Wang, Anderson T; Kim, Taeho; Wagner, John E; Conti, Brooke A; Lach, Francis P; Huang, Athena L; Molina, Henrik; Sanborn, Erica M; Zierhut, Heather; Cornes, Belinda K; Abhyankar, Avinash; Sougnez, Carrie; Gabriel, Stacey B; Auerbach, Arleen D; Kowalczykowski, Stephen C; Smogorzewska, Agata

    2015-08-01

    Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. In vitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity, and a co-dominant-negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio of mutant to wild-type RAD51 in cells. Instead, patient cells are sensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity. PMID:26253028

  13. [The Engineering of a Yarrowia lipolytica Yeast Strain Capable of Homologous Recombination of the Mitochondrial Genome].

    PubMed

    Isakova, E P; Epova, E Yu; Sekova, V Yu; Trubnikova, E V; Kudykina, Yu K; Zylkova, M V; Guseva, M A; Deryabina, Yu I

    2015-01-01

    None of the studied eukaryotic species has a natural system for homologous recombination of the mitochondrial genome. We propose an integrated genetic construct pQ-SRUS, which allows introduction of the recA gene from Bacillus subtilis into the nuclear genome of an extremophilic yeast, Yarrowia lipolytica. The targeting of recombinant RecA to the yeast mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibits affinity to the outer mitochondrial membrane and thus provides cotranslational transport of RecA to the inner space of the mitochondria. The Y. lipolytica strain bearing the pQ-SRUS construct has the unique ability to integrate DNA constructs into the mitochondrial genome. This fact was confirmed using a tester construct, pQ-NIHN, intended for the introduction of the EYFP gene into the translation initiation region of the Y. lipolytica ND1 mitochondrial gene. The Y. lipolytica strain bearing pQ-SRUS makes it possible to engineer recombinant producers based on Y. lipolytica bearing transgenes in the mitochondrial genome. They are promising for the construction of a genetic system for in vivo replication and modification of the human mitochondrial genome. These strains may be used as a tool for the treatment of human mitochondrial diseases (including genetically inherited ones). PMID:26204776

  14. Molecular and population analyses of a recombination event in the catabolic plasmid pJP4.

    PubMed

    Larraín-Linton, Juanita; De la Iglesia, Rodrigo; Melo, Francisco; González, Bernardo

    2006-10-01

    Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level. PMID:16980481

  15. Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9.

    PubMed

    Chakrapani, Vemulawada; Patra, Swagat Kumar; Panda, Rudra Prasanna; Rasal, Kiran Dashrath; Jayasankar, Pallipuram; Barman, Hirak Kumar

    2016-08-01

    Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes. PMID:27079451

  16. Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination.

    PubMed

    Chen, Changchun; Fenk, Lorenz A; de Bono, Mario

    2013-11-01

    Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR-Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR-CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism. PMID:24013562

  17. Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

    PubMed Central

    Chen, Changchun; Fenk, Lorenz A.; de Bono, Mario

    2013-01-01

    Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism. PMID:24013562

  18. Generation of TALEN-Mediated GRdim Knock-In Rats by Homologous Recombination

    PubMed Central

    Ponce de León, Verónica; Mérillat, Anne-Marie; Tesson, Laurent; Anegón, Ignacio; Hummler, Edith

    2014-01-01

    Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GRdim, that prevents receptor homodimerization in the mouse. TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos. Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes. PMID:24523878

  19. PCAT-1, a long noncoding RNA, regulates BRCA2 and controls homologous recombination in cancer.

    PubMed

    Prensner, John R; Chen, Wei; Iyer, Matthew K; Cao, Qi; Ma, Teng; Han, Sumin; Sahu, Anirban; Malik, Rohit; Wilder-Romans, Kari; Navone, Nora; Logothetis, Christopher J; Araujo, John C; Pisters, Louis L; Tewari, Ashutosh K; Canman, Christine E; Knudsen, Karen E; Kitabayashi, Naoki; Rubin, Mark A; Demichelis, Francesca; Lawrence, Theodore S; Chinnaiyan, Arul M; Feng, Felix Y

    2014-03-15

    Impairment of double-stranded DNA break (DSB) repair is essential to many cancers. However, although mutations in DSB repair proteins are common in hereditary cancers, mechanisms of impaired DSB repair in sporadic cancers remain incompletely understood. Here, we describe the first role for a long noncoding RNA (lncRNA) in DSB repair in prostate cancer. We identify PCAT-1, a prostate cancer outlier lncRNA, which regulates cell response to genotoxic stress. PCAT-1 expression produces a functional deficiency in homologous recombination through its repression of the BRCA2 tumor suppressor, which, in turn, imparts a high sensitivity to small-molecule inhibitors of PARP1. These effects reflected a posttranscriptional repression of the BRCA2 3'UTR by PCAT-1. Our observations thus offer a novel mechanism of "BRCAness" in sporadic cancers. PMID:24473064

  20. Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination

    PubMed Central

    Bonnefoy, Nathalie; Fox, Thomas D.

    2009-01-01

    Saccharomyces cerevisiae is currently the only species in which genetic transformation of mitochondria can be used to generate a wide variety of defined alterations in mtDNA. DNA sequences can be delivered into yeast mitochondria by microprojectile bombardment (biolistic transformation) and subsequently incorporated into mtDNA by the highly active homologous recombination machinery present in the organelle. While transformation frequencies are relatively low, the availability of strong mitochondrial selectable markers for the yeast system, both natural and synthetic, makes the isolation of transformants routine. The strategies and procedures reviewed here allow the researcher to insert defined mutations into endogenous mitochondrial genes, and to insert new genes into mtDNA. These methods provide powerful in vivo tools for the study of mitochondrial biology. PMID:18314724

  1. Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination.

    PubMed

    Frank-Vaillant, Marie; Marcand, Stéphane

    2002-11-01

    The stability of DNA ends generated by the HO endonuclease in yeast is surprisingly high with a half-life of more than an hour. This transient stability is unaffected by mutations that abolish nonhomologous end joining (NHEJ). The unprocessed ends interact with Yku70p and Yku80p, two proteins required for NHEJ, but not significantly with Rad52p, a protein involved in homologous recombination (HR). Repair of a double-strand break by NHEJ is unaffected by the possibility of HR, although the use of HR is increased in NHEJ-defective cells. Partial in vitro 5' strand processing suppresses NHEJ but not HR. These results show that NHEJ precedes HR temporally, and that the availability of substrate dictates the particular pathway used. We propose that transient stability of DNA ends is a foundation for the permanent stability of telomeres. PMID:12453425

  2. Interplay between Fanconi anemia and homologous recombination pathways in genome integrity.

    PubMed

    Michl, Johanna; Zimmer, Jutta; Tarsounas, Madalena

    2016-05-01

    The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error-free pathway for double-strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication-associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability. PMID:27037238

  3. Changes to DNA methylation and homologous recombination frequency in the progeny of stressed plants.

    PubMed

    Migicovsky, Zoë; Kovalchuk, Igor

    2013-02-01

    Plants undergo changes in response to biotic and abiotic stresses that help them adjust and survive. Some of these changes may even be passed on to progeny and eventually lead to adaptive evolution. Transgenerational changes in response to stress include alterations in DNA methylation and changes in homologous recombination frequency (HRF). The progeny of plants that were stressed often show elevated HRF as well as genomic hypermethylation, although specific loci that are beneficial in times of stress may be hypomethylated. One of the possible mechanisms responsible for passing the memory to the progeny involves small interfering RNAs; Dicer-like proteins, DCL2 and DCL3, are in part required for this process. However, while epigenetic modifications are often present in the untreated progeny of stressed plants, they are not usually sustained for multiple unexposed generations. Still, transgenerational inheritance of such changes has already begun to provide evidence for an important role of epigenetics in enhancing stress resistance. PMID:23442135

  4. Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.

    PubMed

    Xue, Haipeng; Wu, Jianbo; Li, Shenglan; Rao, Mahendra S; Liu, Ying

    2016-01-01

    Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability. PMID:24615461

  5. A role for human homologous recombination factors in suppressing microhomology-mediated end joining.

    PubMed

    Ahrabi, Sara; Sarkar, Sovan; Pfister, Sophia X; Pirovano, Giacomo; Higgins, Geoff S; Porter, Andrew C G; Humphrey, Timothy C

    2016-07-01

    DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection. PMID:27131361

  6. Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum

    PubMed Central

    Genois, Marie-Michelle; Plourde, Marie; Éthier, Chantal; Roy, Gaétan; Poirier, Guy G.; Ouellette, Marc; Masson, Jean-Yves

    2015-01-01

    To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival. PMID:25712090

  7. BRCA1 functions independently of homologous recombination in DNA interstrand cross-link repair

    PubMed Central

    Bunting, Samuel F; Callen, Elsa; Kozak, Marina L; Kim, Jung-Min; Wong, Nancy; Lopez-Contreras, Andres J; Ludwig, Thomas; Baer, Richard; Faryabi, Robert B; Malhowski, Amy; Chen, Hua-Tang; Fernandez-Capetillo, Oscar; D’Andrea, Alan; Nussenzweig, Andre

    2012-01-01

    Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice, and rescues HR deficiency, as measured by hypersensitivity to PARP (polyADP-ribose polymerase) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand cross-links (ICLs), indicating that BRCA1 has an additional role in DNA cross-link repair that is distinct from HR. Disruption of the non-homologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi Anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair, whereas FANCD2 provides a key activity that can not be bypassed by ablation of 53BP1 or Ku. PMID:22445484

  8. Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells

    SciTech Connect

    Sekine-Suzuki, Emiko; Yu, Dong; Kubota, Nobuo; Okayasu, Ryuichi; Anzai, Kazunori

    2008-12-12

    Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and {gamma}-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.

  9. Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae

    PubMed Central

    Lee, Min-Soo; Yu, Mi; Kim, Kyoung-Yeon; Park, Geun-Hee; Kwack, KyuBum; Kim, Keun P.

    2015-01-01

    Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer. PMID:25938495

  10. Genome-Wide Screen Reveals Replication Pathway for Quasi-Palindrome Fragility Dependent on Homologous Recombination

    PubMed Central

    Zhang, Yu; Saini, Natalie; Sheng, Ziwei; Lobachev, Kirill S.

    2013-01-01

    Inverted repeats capable of forming hairpin and cruciform structures present a threat to chromosomal integrity. They induce double strand breaks, which lead to gross chromosomal rearrangements, the hallmarks of cancers and hereditary diseases. Secondary structure formation at this motif has been proposed to be the driving force for the instability, albeit the mechanisms leading to the fragility are not well-understood. We carried out a genome-wide screen to uncover the genetic players that govern fragility of homologous and homeologous Alu quasi-palindromes in the yeast Saccharomyces cerevisiae. We found that depletion or lack of components of the DNA replication machinery, proteins involved in Fe-S cluster biogenesis, the replication-pausing checkpoint pathway, the telomere maintenance complex or the Sgs1-Top3-Rmi1 dissolvasome augment fragility at Alu-IRs. Rad51, a component of the homologous recombination pathway, was found to be required for replication arrest and breakage at the repeats specifically in replication-deficient strains. These data demonstrate that Rad51 is required for the formation of breakage-prone secondary structures in situations when replication is compromised while another mechanism operates in DSB formation in replication-proficient strains. PMID:24339793

  11. A role for human homologous recombination factors in suppressing microhomology-mediated end joining

    PubMed Central

    Ahrabi, Sara; Sarkar, Sovan; Pfister, Sophia X.; Pirovano, Giacomo; Higgins, Geoff S.; Porter, Andrew C.G.; Humphrey, Timothy C.

    2016-01-01

    DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2–6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection. PMID:27131361

  12. Genome-wide screen reveals replication pathway for quasi-palindrome fragility dependent on homologous recombination.

    PubMed

    Zhang, Yu; Saini, Natalie; Sheng, Ziwei; Lobachev, Kirill S

    2013-01-01

    Inverted repeats capable of forming hairpin and cruciform structures present a threat to chromosomal integrity. They induce double strand breaks, which lead to gross chromosomal rearrangements, the hallmarks of cancers and hereditary diseases. Secondary structure formation at this motif has been proposed to be the driving force for the instability, albeit the mechanisms leading to the fragility are not well-understood. We carried out a genome-wide screen to uncover the genetic players that govern fragility of homologous and homeologous Alu quasi-palindromes in the yeast Saccharomyces cerevisiae. We found that depletion or lack of components of the DNA replication machinery, proteins involved in Fe-S cluster biogenesis, the replication-pausing checkpoint pathway, the telomere maintenance complex or the Sgs1-Top3-Rmi1 dissolvasome augment fragility at Alu-IRs. Rad51, a component of the homologous recombination pathway, was found to be required for replication arrest and breakage at the repeats specifically in replication-deficient strains. These data demonstrate that Rad51 is required for the formation of breakage-prone secondary structures in situations when replication is compromised while another mechanism operates in DSB formation in replication-proficient strains. PMID:24339793

  13. GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    PubMed Central

    Takizawa, Yoshimasa; Qing, Yong; Takaku, Motoki; Ishida, Takako; Morozumi, Yuichi; Tsujita, Takashi; Kogame, Toshiaki; Hirota, Kouji; Takahashi, Masayuki; Shibata, Takehiko; Kurumizaka, Hitoshi; Takeda, Shunichi

    2010-01-01

    RAD51 is a key factor in homologous recombination (HR) and plays an essential role in cellular proliferation by repairing DNA damage during replication. The assembly of RAD51 at DNA damage is strictly controlled by RAD51 mediators, including BRCA1 and BRCA2. We found that human RAD51 directly binds GEMIN2/SIP1, a protein involved in spliceosome biogenesis. Biochemical analyses indicated that GEMIN2 enhances the RAD51–DNA complex formation by inhibiting RAD51 dissociation from DNA, and thereby stimulates RAD51-mediated homologous pairing. GEMIN2 also enhanced the RAD51-mediated strand exchange, when RPA was pre-bound to ssDNA before the addition of RAD51. To analyze the function of GEMIN2, we depleted GEMIN2 in the chicken DT40 line and in human cells. The loss of GEMIN2 reduced HR efficiency and resulted in a significant decrease in the number of RAD51 subnuclear foci, as observed in cells deficient in BRCA1 and BRCA2. These observations and our biochemical analyses reveal that GEMIN2 regulates HR as a novel RAD51 mediator. PMID:20403813

  14. Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum.

    PubMed

    Genois, Marie-Michelle; Plourde, Marie; Éthier, Chantal; Roy, Gaétan; Poirier, Guy G; Ouellette, Marc; Masson, Jean-Yves

    2015-03-11

    To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival. PMID:25712090

  15. Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

    PubMed

    Su, Guan-Chin; Yeh, Hsin-Yi; Lin, Sheng-Wei; Chung, Chan-I; Huang, Yu-Shan; Liu, Yi-Chung; Lyu, Ping-Chiang; Chi, Peter

    2016-07-27

    During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells. PMID:27131790

  16. ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination

    PubMed Central

    Löbrich, Markus

    2013-01-01

    Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes. PMID:23935532

  17. Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans.

    PubMed

    McClendon, T Brooke; Sullivan, Meghan R; Bernstein, Kara A; Yanowitz, Judith L

    2016-05-01

    Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1 Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1 We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability. PMID:26936927

  18. The cohesion protein SOLO associates with SMC1 and is required for synapsis, recombination, homolog bias and cohesion and pairing of centromeres in Drosophila Meiosis.

    PubMed

    Yan, Rihui; McKee, Bruce D

    2013-01-01

    Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome

  19. The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis, Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

    PubMed Central

    Yan, Rihui; McKee, Bruce D.

    2013-01-01

    Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome

  20. Population Genetic Analysis of Streptomyces albidoflavus Reveals Habitat Barriers to Homologous Recombination in the Diversification of Streptomycetes

    PubMed Central

    Cheng, Kun; Rong, Xiaoying; Pinto-Tomás, Adrián A.; Fernández-Villalobos, Marcela; Murillo-Cruz, Catalina

    2014-01-01

    Examining the population structure and the influence of recombination and ecology on microbial populations makes great sense for understanding microbial evolution and speciation. Streptomycetes are a diverse group of bacteria that are widely distributed in nature and a rich source of useful bioactive compounds; however, they are rarely subjected to population genetic investigations. In this study, we applied a five-gene-based multilocus sequence analysis (MLSA) scheme to 41 strains of Streptomyces albidoflavus derived from diverse sources, mainly insects, sea, and soil. Frequent recombination was detected in S. albidoflavus, supported by multiple lines of evidence from the pairwise homoplasy index (Φw) test, phylogenetic discordance, the Shimodaira-Hasegawa (SH) test, and network analysis, underpinning the predominance of homologous recombination within Streptomyces species. A strong habitat signal was also observed in both phylogenetic and Structure 2.3.3 analyses, indicating the importance of ecological difference in shaping the population structure. Moreover, all three habitat-associated groups, particularly the entomic group, demonstrated significantly reduced levels of gene flow with one another, generally revealing habitat barriers to recombination. Therefore, a combined effect of homologous recombination and ecology is inferred for S. albidoflavus, where dynamic evolution is at least partly balanced by the extent that differential distributions of strains among habitats limit genetic exchange. Our study stresses the significance of ecology in microbial speciation and reveals the coexistence of homologous recombination and ecological divergence in the evolution of streptomycetes. PMID:25416769

  1. Population genetic analysis of Streptomyces albidoflavus reveals habitat barriers to homologous recombination in the diversification of streptomycetes.

    PubMed

    Cheng, Kun; Rong, Xiaoying; Pinto-Tomás, Adrián A; Fernández-Villalobos, Marcela; Murillo-Cruz, Catalina; Huang, Ying

    2015-02-01

    Examining the population structure and the influence of recombination and ecology on microbial populations makes great sense for understanding microbial evolution and speciation. Streptomycetes are a diverse group of bacteria that are widely distributed in nature and a rich source of useful bioactive compounds; however, they are rarely subjected to population genetic investigations. In this study, we applied a five-gene-based multilocus sequence analysis (MLSA) scheme to 41 strains of Streptomyces albidoflavus derived from diverse sources, mainly insects, sea, and soil. Frequent recombination was detected in S. albidoflavus, supported by multiple lines of evidence from the pairwise homoplasy index (Φw) test, phylogenetic discordance, the Shimodaira-Hasegawa (SH) test, and network analysis, underpinning the predominance of homologous recombination within Streptomyces species. A strong habitat signal was also observed in both phylogenetic and Structure 2.3.3 analyses, indicating the importance of ecological difference in shaping the population structure. Moreover, all three habitat-associated groups, particularly the entomic group, demonstrated significantly reduced levels of gene flow with one another, generally revealing habitat barriers to recombination. Therefore, a combined effect of homologous recombination and ecology is inferred for S. albidoflavus, where dynamic evolution is at least partly balanced by the extent that differential distributions of strains among habitats limit genetic exchange. Our study stresses the significance of ecology in microbial speciation and reveals the coexistence of homologous recombination and ecological divergence in the evolution of streptomycetes. PMID:25416769

  2. Formation of large palindromic DNA by homologous recombination of short inverted repeat sequences in Saccharomyces cerevisiae.

    PubMed Central

    Butler, David K; Gillespie, David; Steele, Brandi

    2002-01-01

    Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisiae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1. PMID:12136011

  3. Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.

    PubMed

    Ceccaldi, Raphael; Liu, Jessica C; Amunugama, Ravindra; Hajdu, Ildiko; Primack, Benjamin; Petalcorin, Mark I R; O'Connor, Kevin W; Konstantinopoulos, Panagiotis A; Elledge, Stephen J; Boulton, Simon J; Yusufzai, Timur; D'Andrea, Alan D

    2015-02-12

    Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy. PMID:25642963

  4. Homologous Recombination—Experimental Systems, Analysis and Significance

    PubMed Central

    Kuzminov, Andrei

    2014-01-01

    Homologous recombination is the most complex of all recombination events that shape genomes and produce material for evolution. Homologous recombination events are exchanges between DNA molecules in the lengthy regions of shared identity, catalyzed by a group of dedicated enzymes. There is a variety of experimental systems in E. coli and Salmonella to detect homologous recombination events of several different kinds. Genetic analysis of homologous recombination reveals three separate phases of this process: pre-synapsis (the early phase), synapsis (homologous strand exchange) and post-synapsis (the late phase). In E. coli, there are at least two independent pathway of the early phase and at least two independent pathways of the late phase. All this complexity is incongruent with the originally ascribed role of homologous recombination as accelerator of genome evolution: there is simply not enough duplication and repetition in enterobacterial genomes for homologous recombination to have a detectable evolutionary role, and therefore not enough selection to maintain such a complexity. At the same time, the mechanisms of homologous recombination are uniquely suited for repair of complex DNA lesions called chromosomal lesions. In fact, the two major classes of chromosomal lesions are recognized and processed by the two individual pathways at the early phase of homologous recombination. It follows, therefore, that homologous recombination events are occasional reflections of the continual recombinational repair, made possible in cases of natural or artificial genome redundancy. PMID:26442506

  5. Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

    PubMed Central

    Kavanagh, T A; Thanh, N D; Lao, N T; McGrath, N; Peter, S O; Horváth, E M; Dix, P J; Medgyesy, P

    1999-01-01

    Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids. PMID:10388829

  6. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    PubMed

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells. PMID:26280081

  7. TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.

    PubMed

    Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

    2016-05-20

    Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms. PMID:27180265

  8. Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells.

    PubMed

    Browning, Cynthia L; Qin, Qin; Kelly, Deborah F; Prakash, Rohit; Vanoli, Fabio; Jasin, Maria; Wise, John Pierce

    2016-09-01

    Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis. PMID:27449664

  9. Effect of homologous and heterologous prime-boost on the immune response to recombinant plague antigens.

    PubMed

    Glynn, Audrey; Freytag, Lucy C; Clements, John D

    2005-03-14

    Among the pathogens that have been identified as potential agents of biological warfare or bioterrorism, Yersinia pestis is one of the main concerns due to the severity and potential transmissibility of the pneumonic form of the disease in humans. There are no approved vaccines for protection against pneumonic plague, but a Y. pestis-derived fusion protein (F1-V) has shown great promise as a protective antigen in murine studies. In the current study, we examine different prime-boost regimens, including parenteral, mucosal, and transcutaneous delivery, in order to explore the effect of changing the route of prime and boost on the ability of recombinant F1-V to promote the development of long-lasting, high-titer antibodies. The most significant findings of the study reported here are that (1) intranasal and subcutaneous immunizations are both effective and essentially equivalent for induction of serum and bronchioalveolar anti-F1-V IgG1 responses when a single booster dose is administered by the same (homologous) route, (2) heterologous boosting can be as or more effective than homologous boosting for induction of either serum or bronchioalveolar anti-F1-V IgG1 responses, and (3) anti-F1 and anti-V total IgG responses were highest in animals primed intranasally and boosted by any route when compared to animals primed transcutaneously or subcutaneously. As with previously published studies, there were still significant levels of circulating anti-F1-V antibodies 1 year post-primary immunization. These studies provide important insights into the development of new-generation biodefense vaccines. PMID:15734068

  10. ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

    PubMed

    Brookman, K W; Lamerdin, J E; Thelen, M P; Hwang, M; Reardon, J T; Sancar, A; Zhou, Z Q; Walter, C A; Parris, C N; Thompson, L H

    1996-11-01

    ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues. PMID:8887684

  11. Suppression of homologous recombination sensitizes human tumor cells to IGF-1R inhibition.

    PubMed

    Lodhia, Kunal A; Gao, Shan; Aleksic, Tamara; Esashi, Fumiko; Macaulay, Valentine M

    2015-06-15

    Inhibition of type 1 IGF receptor (IGF-1R) sensitizes to DNA-damaging cancer treatments, and delays repair of DNA double strand breaks (DSBs) by non-homologous end-joining and homologous recombination (HR). In a recent screen for mediators of resistance to IGF-1R inhibitor AZ12253801, we identified RAD51, required for the strand invasion step of HR. These findings prompted us to test the hypothesis that IGF-1R-inhibited cells accumulate DSBs formed at endogenous DNA lesions, and depend on residual HR for their repair. Indeed, initial experiments showed time-dependent accumulation of γH2AX foci in IGF-1R -inhibited or -depleted prostate cancer cells. We then tested effects of suppressing HR, and found that RAD51 depletion enhanced AZ12253801 sensitivity in PTEN wild-type prostate cancer cells but not in cells lacking functional PTEN. Similar sensitization was induced in prostate cancer cells by depletion of BRCA2, required for RAD51 loading onto DNA, and in BRCA2(-/-) colorectal cancer cells, compared with isogenic BRCA2(+/-) cells. We also assessed chemical HR inhibitors, finding that RAD51 inhibitor BO2 blocked RAD51 focus formation and sensitized to AZ12253801. Finally, we tested CDK1 inhibitor RO-3306, which impairs HR by inhibiting CDK1-mediated BRCA1 phosphorylation. R0-3306 suppressed RAD51 focus formation consistent with HR attenuation, and sensitized prostate cancer cells to IGF-1R inhibition, with 2.4-fold reduction in AZ12253801 GI50 and 13-fold reduction in GI80. These data suggest that responses to IGF-1R inhibition are enhanced by genetic and chemical approaches to suppress HR, defining a population of cancers (PTEN wild-type, BRCA mutant) that may be intrinsically sensitive to IGF-1R inhibitory drugs. PMID:25388513

  12. Increased sensitivity to ionizing radiation by targeting the homologous recombination pathway in glioma initiating cells.

    PubMed

    Lim, Yi Chieh; Roberts, Tara L; Day, Bryan W; Stringer, Brett W; Kozlov, Sergei; Fazry, Shazrul; Bruce, Zara C; Ensbey, Kathleen S; Walker, David G; Boyd, Andrew W; Lavin, Martin F

    2014-12-01

    Glioblastoma is deemed the most malignant form of brain tumour, particularly due to its resistance to conventional treatments. A small surviving group of aberrant stem cells termed glioma initiation cells (GICs) that escape surgical debulking are suggested to be the cause of this resistance. Relatively quiescent in nature, GICs are capable of driving tumour recurrence and undergo lineage differentiation. Most importantly, these GICs are resistant to radiotherapy, suggesting that radioresistance contribute to their survival. In a previous study, we demonstrated that GICs had a restricted double strand break (DSB) repair pathway involving predominantly homologous recombination (HR) associated with a lack of functional G1/S checkpoint arrest. This unusual behaviour led to less efficient non-homologous end joining (NHEJ) repair and overall slower DNA DSB repair kinetics. To determine whether specific targeting of the HR pathway with small molecule inhibitors could increase GIC radiosensitivity, we used the Ataxia-telangiectasia mutated inhibitor (ATMi) to ablate HR and the DNA-dependent protein kinase inhibitor (DNA-PKi) to inhibit NHEJ. Pre-treatment with ATMi prior to ionizing radiation (IR) exposure prevented HR-mediated DNA DSB repair as measured by Rad51 foci accumulation. Increased cell death in vitro and improved in vivo animal survival could be observed with combined ATMi and IR treatment. Conversely, DNA-PKi treatment had minimal impact on GICs ability to resolve DNA DSB after IR with only partial reduction in cell survival, confirming the major role of HR. These results provide a mechanistic insight into the predominant form of DNA DSB repair in GICs, which when targeted may be a potential translational approach to increase patient survival. PMID:25017126

  13. A Recombinant β-1,3-Glucanosyltransferase Homolog of Coccidioides posadasii Protects Mice against Coccidioidomycosis

    PubMed Central

    Delgado, Nelson; Xue, Jianmin; Yu, Jieh-Juen; Hung, Chiung-Yu; Cole, Garry T.

    2003-01-01

    Coccidioides posadasii is a fungal respiratory pathogen which is responsible for recurrent epidemics of San Joaquin Valley fever (coccidioidomycosis) in desert regions of the southwestern United States. Numerous studies have revealed that the cell wall of the parasitic phase of the fungus is a reservoir of immunoreactive macromolecules and a potential source of a vaccine against this mycosis. A 495-bp fragment of a C. posadasii gene which encodes a putative wall-associated, glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase was identified by computational analysis of the partially sequenced genome of this pathogen. The translated, full-length gene (GEL1) showed high sequence homology to a reported β-1,3-glucanosyltransferase of Aspergillus fumigatus (70% identity, 90% similarity) and was selected for further study. The GEL1 mRNA of C. posadasii was detected at the highest level during the endosporulation stage of the parasitic cycle, and the mature protein was immunolocalized to the surface of endospores. BALB/c or C57BL/6 mice were immunized subcutaneously with the bacterium-expressed recombinant protein (rGel1p) to evaluate its protective efficacy against a lethal challenge of C. posadasii by either the intraperitoneal or intranasal route. In both cases, rGel1p-immune mice infected with the pathogen showed a significant reduction in fungal burden and increased survival compared to nonimmune mice. The recombinant β-1,3-glucanosyltransferase is a valuable addition to an arsenal of immunoreactive proteins which could be incorporated into a human vaccine against coccidioidomycosis. PMID:12761077

  14. Mitochondrial Genome Rearrangements in Glomus Species Triggered by Homologous Recombination between Distinct mtDNA Haplotypes

    PubMed Central

    Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

    2013-01-01

    Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms. AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence, were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigate dpo diversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity. We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants. PMID:23925788

  15. Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination.

    PubMed

    Yao, Youli; Bilichak, Andriy; Golubov, Andrey; Kovalchuk, Igor

    2016-07-01

    Small interfering RNAs (siRNAs) are involved in the regulation of plant development and response to stress. We have previously shown that mutants impaired in Dicer-like 2 (DCL2), DCL3 and DCL4, RDR2, RDR6 and NPRD1 are partially impaired in their response to stress and dcl2 and dcl3 plants are also impaired in transgenerational response to stress, including changes in homologous recombination frequency (HRF). Here, we have analyzed genome stability of dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl3 dcl4 and rdr6 mutants by measuring the non-induced and the stress-induced recombination frequency. We found that all mutants had the lower spontaneous HRF. The analysis of strand breaks showed that all tested Arabidopsis mutants had a higher level of spontaneous strand breaks, suggesting that the lower HRF is not due to the unusually low level of breaks. Exposure to methyl methane sulfonate (MMS) resulted in an increase in the level of strand breaks in wild-type plants and a decrease in mutants. All mutants had the higher methylation of cytosines at CpG sites under non-induced conditions. Exposure to MMS resulted in a decrease in methylation level in wild-type plants and an increase in methylation in all dcl mutants. The expression of several DNA repair genes was altered in dcl4 plants under non-induced and induced conditions. Our data suggest that siRNA biogenesis may be essential for the maintenance of the genome stability and stress response in Arabidopsis. PMID:26901311

  16. p53 modulates homologous recombination at I-SceI-induced double-strand breaks through cell-cycle regulation.

    PubMed

    Rieckmann, T; Kriegs, M; Nitsch, L; Hoffer, K; Rohaly, G; Kocher, S; Petersen, C; Dikomey, E; Dornreiter, I; Dahm-Daphi, J

    2013-02-21

    Inhibition of homologous recombination (HR) is believed to be a transactivation-independent function of p53 that protects from genetic instability. Misrepair by HR can lead to genetic alterations such as translocations, duplications, insertions and loss of heterozygosity, which all bear the risk of driving oncogenic transformation. Regulation of HR by wild-type p53 (wtp53) should prevent these genomic rearrangements. Mutation of p53 is a frequent event during carcinogenesis. In particular, dominant-negative mutants inhibiting wtp53 expressed from the unperturbed allel can drive oncogenic transformation by disrupting the p53-dependent anticancer barrier. Here, we asked whether the hot spot mutants R175H and R273H relax HR control in p53-proficient cells. Utilizing an I-SceI-based reporter assay, we observed a moderate (1.5 × ) stimulation of HR upon expression of the mutant proteins in p53-proficient CV-1, but not in p53-deficient H1299 cells. Importantly, the stimulatory effect was exactly paralleled by an increase in the number of HR competent S- and G2-phase cells, which can well explain the enhanced recombination frequencies. Furthermore, the impact on HR exerted by the transactivation domain double-mutant L22Q/W23S and mutant R273P, both of which were reported to regulate HR independently of G1-arrest execution, is also exactly mirrored by cell-cycle behavior. These results are in contrast to previous concepts stating that the transactivation-independent impact of p53 on HR is a general phenomenon valid for replication-associated and also for directly induced double-strand break. Our data strongly suggest that the latter is largely mediated by cell-cycle regulation, a classical transactivation-dependent function of p53. PMID:22484423

  17. Homologous recombination and retention of a single form of most genes shape the highly chimeric mitochondrial genome of a cybrid plant

    PubMed Central

    Sanchez-Puerta, M. Virginia; Zubko, Mikhajlo K.; Palmer, Jeffrey D.

    2014-01-01

    Summary • The structure and evolution of angiosperm mitochondrial genomes are driven by extremely high rates of recombination and rearrangement. An excellent experimental system for studying these events is offered by cybrid plants, in which parental mitochondria usually fuse and their genomes recombine. Little is known about the extent, nature, and consequences of mitochondrial recombination in these plants. • We conducted the first study in which the organellar genomes of a cybrid – between Nicotiana tabacum and Hyoscyamus niger – were sequenced and compared to those of its parents. • This cybrid mitochondrial genome is highly recombinant, reflecting at least 30 crossovers and five gene conversions between its parental genomes. It is also surprisingly large (41 and 64% larger than the parental genomes), yet contains single alleles for 90% of mitochondrial genes. • Recombination produced a remarkably chimeric cybrid mitochondrial genome and occurred entirely via homologous mechanisms involving the double-strand break repair and/or break-induced replication pathways. Retention of a single form of most genes could be advantageous to minimize intracellular incompatibilities and/or reflect neutral forces that preferentially eliminate duplicated regions. We discuss the relevance of these findings to the surprisingly frequent occurrence of horizontal gene – and genome – transfer in angiosperm mitochondrial DNAs. PMID:25441621

  18. The Knowns Unknowns: Exploring the Homologous Recombination Repair Pathway in Toxoplasma gondii

    PubMed Central

    Fenoy, Ignacio M.; Bogado, Silvina S.; Contreras, Susana M.; Gottifredi, Vanesa; Angel, Sergio O.

    2016-01-01

    Toxoplasma gondii is an apicomplexan parasite of medical and veterinary importance which causes toxoplasmosis in humans. Great effort is currently being devoted toward the identification of novel drugs capable of targeting such illness. In this context, we believe that the thorough understanding of the life cycle of this model parasite will facilitate the identification of new druggable targets in T. gondii. It is important to exploit the available knowledge of pathways which could modulate the sensitivity of the parasite to DNA damaging agents. The homologous recombination repair (HRR) pathway may be of particular interest in this regard as its inactivation sensitizes other cellular models such as human cancer to targeted therapy. Herein we discuss the information available on T. gondii's HRR pathway from the perspective of its conservation with respect to yeast and humans. Special attention was devoted to BRCT domain-containing and end-resection associated proteins in T. gondii as in other experimental models such proteins have crucial roles in early/late steps or HRR and in the pathway choice for double strand break resolution. We conclude that T. gondii HRR pathway is a source of several lines of investigation that allow to to comprehend the extent of diversification of HRR in T. gondii. Such an effort will serve to determine if HRR could represent a potential targer for the treatment of toxoplasmosis. PMID:27199954

  19. Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi.

    PubMed

    Hwang, In Sun; Ahn, Il-Pyung

    2016-06-01

    Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 ), which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi. PMID:27298592

  20. NAP1 family histone chaperones are required for somatic homologous recombination in Arabidopsis.

    PubMed

    Gao, Juan; Zhu, Yan; Zhou, Wangbin; Molinier, Jean; Dong, Aiwu; Shen, Wen-Hui

    2012-04-01

    Homologous recombination (HR) is essential for maintaining genome integrity and variability. To orchestrate HR in the context of chromatin is a challenge, both in terms of DNA accessibility and restoration of chromatin organization after DNA repair. Histone chaperones function in nucleosome assembly/disassembly and could play a role in HR. Here, we show that the NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) family histone chaperones are required for somatic HR in Arabidopsis thaliana. Depletion of either the NAP1 group or NAP1-RELATED PROTEIN (NRP) group proteins caused a reduction in HR in plants under normal growth conditions as well as under a wide range of genotoxic or abiotic stresses. This contrasts with the hyperrecombinogenic phenotype caused by the depletion of the CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) histone chaperone. Furthermore, we show that the hyperrecombinogenic phenotype caused by CAF-1 depletion relies on NRP1 and NRP2, but the telomere shortening phenotype does not. Our analysis of DNA lesions, H3K56 acetylation, and expression of DNA repair genes argues for a role of NAP1 family histone chaperones in nucleosome disassembly/reassembly during HR. Our study highlights distinct functions for different families of histone chaperones in the maintenance of genome stability and establishes a crucial function for NAP1 family histone chaperones in somatic HR. PMID:22534127

  1. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

    PubMed Central

    Crabb, B S; Cowman, A F

    1996-01-01

    Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest. Images Fig. 4 Fig. 5 PMID:8692985

  2. Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation

    PubMed Central

    Bakr, A.; Oing, C.; Köcher, S.; Borgmann, K.; Dornreiter, I.; Petersen, C.; Dikomey, E.; Mansour, W.Y.

    2015-01-01

    Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3′-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR. PMID:25753674

  3. Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation.

    PubMed

    Bakr, A; Oing, C; Köcher, S; Borgmann, K; Dornreiter, I; Petersen, C; Dikomey, E; Mansour, W Y

    2015-03-31

    Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3'-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR. PMID:25753674

  4. Homologous recombination and nonhomologous end-joining repair pathways regulate fragile site stability.

    PubMed

    Schwartz, Michal; Zlotorynski, Eitan; Goldberg, Michal; Ozeri, Efrat; Rahat, Ayelet; le Sage, Carlos; Chen, Benjamin P C; Chen, David J; Agami, Reuven; Kerem, Batsheva

    2005-11-15

    Common fragile sites are specific loci that form gaps and constrictions on metaphase chromosomes exposed to replication stress, which slows DNA replication. These sites have a role in chromosomal rearrangements in tumors; however, the molecular mechanism of their expression is unclear. Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively. Down-regulation of Rad51, DNA-PKcs, or Ligase IV, an additional component of the NHEJ repair pathway, leads to a significant increase in fragile site expression under replication stress. Replication stress also results in focus formation of the DSB markers, MDC1 and gammaH2AX. These foci colocalized with those of Rad51 and phospho-DNA-PKcs. Furthermore, gammaH2AX and phospho-DNA-PKcs foci were localized at expressed fragile sites on metaphase chromosomes. These findings suggest that DSBs are formed at common fragile sites as a result of replication perturbation. The repair of these breaks by both HR and NHEJ pathways is essential for chromosomal stability at these sites. PMID:16291645

  5. Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM.

    PubMed

    Finney-Manchester, Shawn P; Maheshri, Narendra

    2013-05-01

    A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis. PMID:23470991

  6. Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples

    PubMed Central

    Patterson, M J; Sutton, R E; Forrest, I; Sharrock, R; Lane, M; Kaufmann, A; O'Donnell, R; Edmondson, R J; Wilson, B T; Curtin, N J

    2014-01-01

    Background: Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells. Methods: A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of γH2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells. Results: From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples – three NSCLC and one mesothelioma – were HRR defective and eight samples – one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers – were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1. Conclusions: HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi. PMID:24867690

  7. Regulators of homologous recombination repair as novel targets for cancer treatment

    PubMed Central

    Krajewska, Małgorzata; Fehrmann, Rudolf S. N.; de Vries, Elisabeth G. E.; van Vugt, Marcel A. T. M.

    2015-01-01

    To cope with DNA damage, cells possess a complex signaling network called the ‘DNA damage response’, which coordinates cell cycle control with DNA repair. The importance of this network is underscored by the cancer predisposition that frequently goes along with hereditary mutations in DNA repair genes. One especially important DNA repair pathway in this respect is homologous recombination (HR) repair. Defects in HR repair are observed in various cancers, including hereditary breast, and ovarian cancer. Intriguingly, tumor cells with defective HR repair show increased sensitivity to chemotherapeutic reagents, including platinum-containing agents. These observations suggest that HR-proficient tumor cells might be sensitized to chemotherapeutics if HR repair could be therapeutically inactivated. HR repair is an extensively regulated process, which depends strongly on the activity of various other pathways, including cell cycle pathways, protein-control pathways, and growth factor-activated receptor signaling pathways. In this review, we discuss how the mechanistic wiring of HR is controlled by cell-intrinsic or extracellular pathways. Furthermore, we have performed a meta-analysis on available genome-wide RNA interference studies to identify additional pathways that control HR repair. Finally, we discuss how these HR-regulatory pathways may provide therapeutic targets in the context of radio/chemosensitization. PMID:25852742

  8. Regulation of homologous recombinational repair by lamin B1 in radiation-induced DNA damage.

    PubMed

    Liu, Ning-Ang; Sun, Jiying; Kono, Kazuteru; Horikoshi, Yasunori; Ikura, Tsuyoshi; Tong, Xing; Haraguchi, Tokuko; Tashiro, Satoshi

    2015-06-01

    DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation (IR). RAD51-dependent homologous recombination (HR) is one of the most important pathways in DSB repair and genome integrity maintenance. However, the mechanism of HR regulation by RAD51 remains unclear. To understand the mechanism of RAD51-dependent HR, we searched for interacting partners of RAD51 by a proteomics analysis and identified lamin B1 in human cells. Lamins are nuclear lamina proteins that play important roles in the structural organization of the nucleus and the regulation of chromosome functions. Immunoblotting analyses revealed that siRNA-mediated lamin B1 depletion repressed the DNA damage-dependent increase of RAD51 after IR. The repression was abolished by the proteasome inhibitor MG132, suggesting that lamin B1 stabilizes RAD51 by preventing proteasome-mediated degradation in cells with IR-induced DNA damage. We also showed that lamin B1 depletion repressed RAD51 focus formation and decreased the survival rates after IR. On the basis of these results, we propose that lamin B1 promotes DSB repair and cell survival by maintaining the RAD51 protein levels for HR upon DSB induction after IR. PMID:25733566

  9. ATR suppresses endogenous DNA damage and allows completion of homologous recombination repair.

    PubMed

    Brown, Adam D; Sager, Brian W; Gorthi, Aparna; Tonapi, Sonal S; Brown, Eric J; Bishop, Alexander J R

    2014-01-01

    DNA replication fork stalling or collapse that arises from endogenous damage poses a serious threat to genome stability, but cells invoke an intricate signaling cascade referred to as the DNA damage response (DDR) to prevent such damage. The gene product ataxia telangiectasia and Rad3-related (ATR) responds primarily to replication stress by regulating cell cycle checkpoint control, yet it's role in DNA repair, particularly homologous recombination (HR), remains unclear. This is of particular interest since HR is one way in which replication restart can occur in the presence of a stalled or collapsed fork. Hypomorphic mutations in human ATR cause the rare autosomal-recessive disease Seckel syndrome, and complete loss of Atr in mice leads to embryonic lethality. We recently adapted the in vivo murine pink-eyed unstable (pun) assay for measuring HR frequency to be able to investigate the role of essential genes on HR using a conditional Cre/loxP system. Our system allows for the unique opportunity to test the effect of ATR loss on HR in somatic cells under physiological conditions. Using this system, we provide evidence that retinal pigment epithelium (RPE) cells lacking ATR have decreased density with abnormal morphology, a decreased frequency of HR and an increased level of chromosomal damage. PMID:24675793

  10. Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    PubMed Central

    Hwang, In Sun; Ahn, Il-Pyung

    2016-01-01

    Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 ), which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi. PMID:27298592

  11. Role of Schizosaccharomyces pombe RecQ homolog, recombination, and checkpoint genes in UV damage tolerance.

    PubMed Central

    Murray, J M; Lindsay, H D; Munday, C A; Carr, A M

    1997-01-01

    The cellular responses to DNA damage are complex and include direct DNA repair pathways that remove the damage and indirect damage responses which allow cells to survive DNA damage that has not been, or cannot be, removed. We have identified the gene mutated in the rad12.502 strain as a Schizosaccharomyces pombe recQ homolog. The same gene (designated rqh1) is also mutated in the hus2.22 mutant. We show that Rqhl is involved in a DNA damage survival mechanism which prevents cell death when UV-induced DNA damage cannot be removed. This pathway also requires the correct functioning of the recombination machinery and the six checkpoint rad gene products plus the Cdsl kinase. Our data suggest that Rqh1 operates during S phase as part of a mechanism which prevents DNA damage causing cell lethality. This process may involve the bypass of DNA damage sites by the replication fork. Finally, in contrast with the reported literature, we do not find that rqh1 (rad12) mutant cells are defective in UV dimer endonuclease activity. PMID:9372918

  12. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    PubMed

    Feng, Yuxin; Singleton, David; Guo, Chun; Gardner, Amanda; Pakala, Suresh; Kumar, Rakesh; Jensen, Elwood; Zhang, Jinsong; Khan, Sohaib

    2013-01-01

    Estrogen receptor alpha (ERα), a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy. PMID:23874500

  13. Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability.

    PubMed

    Spies, Julian; Waizenegger, Anja; Barton, Olivia; Sürder, Michael; Wright, William D; Heyer, Wolf-Dietrich; Löbrich, Markus

    2016-06-16

    Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phospho-mimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability. PMID:27264870

  14. The Knowns Unknowns: Exploring the Homologous Recombination Repair Pathway in Toxoplasma gondii.

    PubMed

    Fenoy, Ignacio M; Bogado, Silvina S; Contreras, Susana M; Gottifredi, Vanesa; Angel, Sergio O

    2016-01-01

    Toxoplasma gondii is an apicomplexan parasite of medical and veterinary importance which causes toxoplasmosis in humans. Great effort is currently being devoted toward the identification of novel drugs capable of targeting such illness. In this context, we believe that the thorough understanding of the life cycle of this model parasite will facilitate the identification of new druggable targets in T. gondii. It is important to exploit the available knowledge of pathways which could modulate the sensitivity of the parasite to DNA damaging agents. The homologous recombination repair (HRR) pathway may be of particular interest in this regard as its inactivation sensitizes other cellular models such as human cancer to targeted therapy. Herein we discuss the information available on T. gondii's HRR pathway from the perspective of its conservation with respect to yeast and humans. Special attention was devoted to BRCT domain-containing and end-resection associated proteins in T. gondii as in other experimental models such proteins have crucial roles in early/late steps or HRR and in the pathway choice for double strand break resolution. We conclude that T. gondii HRR pathway is a source of several lines of investigation that allow to to comprehend the extent of diversification of HRR in T. gondii. Such an effort will serve to determine if HRR could represent a potential targer for the treatment of toxoplasmosis. PMID:27199954

  15. Suppression of mutagenesis by Rad51D-mediated homologous recombination

    SciTech Connect

    Hinz, J M; Tebbs, R S; Wilson, P F; Nham, P B; Salazar, E P; Nagasawa, H; Urbin, S S; Thompson, L H

    2005-11-15

    Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR efficiency. We constructed and characterized a Rad51D knockout cell line in widely studied CHO cells. The rad51d mutant (51D1) displays sensitivity to a wide spectrum of induced DNA damage, indicating the broad relevance of HRR to genotoxicity. Untreated 51D1 cells exhibit {approx}5-fold elevated chromosomal breaks, a 12-fold increased rate of hprt mutation, and 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. These results explicitly show the quantitative importance of HHR in preventing these types genetic alterations, which are associated with carcinogenesis. Thus, HRR copes in an error-free manner with spontaneous DNA damage encountered during DNA replication, and Rad51D is essential for this fidelity.

  16. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

    PubMed Central

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H.; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A.

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  17. Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells.

    PubMed

    Dimitrov, Lazar; Pedersen, Darlene; Ching, Kathryn H; Yi, Henry; Collarini, Ellen J; Izquierdo, Shelley; van de Lavoir, Marie-Cecile; Leighton, Philip A

    2016-01-01

    The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds. PMID:27099923

  18. The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

    PubMed Central

    Kooistra, R; Vreeken, K; Zonneveld, J B; de Jong, A; Eeken, J C; Osgood, C J; Buerstedde, J M; Lohman, P H; Pastink, A

    1997-01-01

    The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution. PMID:9315669

  19. [A new method employing homologous recombination and YAC rescue to expedite gap filling long range mapping]. Progress report

    SciTech Connect

    Not Available

    1991-12-31

    We have embarked on three areas of research relevant to the telomere rescue strategy mediated by homologous recombination described in this proposal. First, we have constructed the telomere rescue vector. Second, we have carried out tests in yeast and mammalian cells to ascertain whether the various crucial components function. Finally, we have begun to develop the molecular reagents required to target the telomeric regions of chromosome 16. The specific progress in each area is described briefly below.

  20. (A new method employing homologous recombination and YAC rescue to expedite gap filling, long-range, mapping)

    SciTech Connect

    Not Available

    1991-01-01

    We have embarked on three areas of research relevant to the telomere rescue strategy mediated by homologous recombination described in this proposal. First, we have constructed the telomere rescue vector. Second, we have carried out tests in yeast and mammalian cells to ascertain whether the various crucial components function. Finally, we have begun to develop the molecular reagents required to target the telomeric regions of chromosome 16. The specific progress in each area is described briefly. 7 refs., 3 figs.

  1. A marker of homologous recombination predicts pathological complete response to neoadjuvant chemotherapy in primary breast cancer

    PubMed Central

    Graeser, Monika; McCarthy, Afshan; Lord, Christopher J; Savage, Kay; Hills, Margaret; Salter, Janine; Orr, Nicholas; Parton, Marina; Smith, Ian E; Reis-Filho, Jorge S; Dowsett, Mitch; Ashworth, Alan; Turner, Nicholas

    2010-01-01

    Purpose To assess the prevalence of defective homologous recombination (HR) based DNA repair in sporadic primary breast cancers, examine the clincopathological features that correlate of with defective HR and the relationship with neoadjuvant chemotherapy response. Experimental Design We examined a cohort of 68 patients with sporadic primary breast cancer who received neoadjuvant anthracylcine based chemotherapy, with core biopsies taken 24 hours after the first cycle of chemotherapy. We assessed RAD51 focus formation, a marker of HR competence, by immunofluorescence in post chemotherapy biopsies along with geminin as a marker of proliferative cells. We assessed the RAD51 score as the proportion of proliferative cells with RAD51 foci. Results A low RAD51 score was present in 26% of cases (15/57, 95% CI, 15-40%). Low RAD51 score correlated with high histological grade (p=0.031) and high baseline Ki67 (p=0.005). Low RAD51 score was more frequent in triple negative breast cancers compared to ER and/or HER2 positive breast cancer (67% vs 19% respectively, p=0.0036). Low RAD51 score was strongly predictive of pathological complete response to chemotherapy, with 33% low RAD51 score cancers achieving pathological complete response compared to 3% of other cancers (p=0.011). Conclusions Our results suggest that defective HR, as indicated by low RAD51 score, may be one of the factors that underlie sensitivity to anthracycline based chemotherapy. Defective HR is frequent in triple negative breast cancer, but is also present in a subset of other subtypes, identifying breast cancers that may benefit from therapies that target defective HR, such as PARP inhibitors. PMID:20802015

  2. Involvement of homologous recombination repair after proton-induced DNA damage.

    PubMed

    Rostek, C; Turner, E L; Robbins, M; Rightnar, S; Xiao, W; Obenaus, A; Harkness, T A A

    2008-03-01

    Protection from chronic exposure to cosmic radiation, which is primarily composed of protons, in future manned missions to Mars and beyond is considered to be a key unresolved issue. To model the effects of cosmic radiation on a living cell, we used Saccharomyces cerevisiae cells harboring various deletions of DNA repair genes to investigate the response of cells to DNA strand breaks caused by exposure to 250 MeV proton irradiation (linear energy transfer of 0.41 keV/microm). In our study, DNA strand breaks induced by exposure to protons were predominantly repaired via the homologous recombination and postreplication repair pathways. We simulated chronic exposure to proton irradiation by treating cells from colonies that survived proton treatment, after several rounds of subculturing, to a second proton dose, as well as additional cell stressors. In general, cells cultured from proton surviving colonies were not more sensitive to secondary cell stressors. However, cells from rad52delta colonies that survived proton treatment showed increased resistance to secondary stressors, such as gamma-rays (1.17 and 1.33 MeV; 0.267 keV/microm), ultraviolet (UV) and proton irradiation and elevated temperatures. Resistance to secondary stressors was also observed in rad52delta cells that survived exposure to gamma-rays, rather than protons, but this was not observed to occur in rad52delta cells after UV irradiation. rad52delta cells that survived exposure to protons, followed by gamma-rays (proton surviving colonies were cultured prior to gamma-ray exposure), exhibited an additive effect, whereby these cells had a further increase in stress resistance. A genetic analysis indicated that increased stress resistance is most likely due to a second-site mutation that suppresses the rad52delta phenotype. We will discuss possible origins of these second-site mutations. PMID:18267950

  3. Survivin contributes to DNA repair by homologous recombination in breast cancer cells.

    PubMed

    Véquaud, Eloïse; Desplanques, Grégoire; Jézéquel, Pascal; Juin, Philippe; Barillé-Nion, Sophie

    2016-01-01

    Survivin overexpression, frequently found in breast cancers and others, is associated with poor prognosis. Its dual regulation of cell division and apoptosis makes it an attractive therapeutic target but its exact functions that are required for tumor maintenance are still elusive. Survivin protects cancer cells from genotoxic agents and this ability is generally assigned to a universal anti-apoptotic function. However, a specific role in cancer cell protection from DNA damage has been overlooked so far. We assessed DNA damage occurrence in Survivin-depleted breast cancer cells using γH2AX staining and comete assay. QPCR data and a gene conversion assay indicated that homologous recombination (HR) was impaired upon Survivin depletion. We conducted the analysis of Survivin and HR genes' expression in breast tumors. We revealed BRCAness phenotype of Survivin-depleted cells using cell death assays combined to PARP targeting. Survivin silencing leads to DNA double-strand breaks in breast cancer cells and functionally reduces HR. Survivin depletion decreases the transcription of a set of genes involved in HR, decreases RAD51 protein expression and impairs the endonuclease complex MUS81/EME1 involved in the resolution of Holliday junctions. Clinically, EME1, RAD51, EXO1, BLM expressions correlate with that of BIRC5 (coding for Survivin) and are of prognostic value. Functionally, Survivin depletion triggers p53 activation and sensitizes cancer cells to of PARP inhibition. We defined Survivin as a constitutive actor of HR in breast cancers, and implies that its inhibition would enhance cell vulnerability upon PARP inhibition. PMID:26679694

  4. Genome-wide analyses of LINE–LINE-mediated nonallelic homologous recombination

    PubMed Central

    Startek, Michał; Szafranski, Przemyslaw; Gambin, Tomasz; Campbell, Ian M.; Hixson, Patricia; Shaw, Chad A.; Stankiewicz, Paweł; Gambin, Anna

    2015-01-01

    Nonallelic homologous recombination (NAHR), occurring between low-copy repeats (LCRs) >10 kb in size and sharing >97% DNA sequence identity, is responsible for the majority of recurrent genomic rearrangements in the human genome. Recent studies have shown that transposable elements (TEs) can also mediate recurrent deletions and translocations, indicating the features of substrates that mediate NAHR may be significantly less stringent than previously believed. Using >4 kb length and >95% sequence identity criteria, we analyzed of the genome-wide distribution of long interspersed element (LINE) retrotransposon and their potential to mediate NAHR. We identified 17 005 directly oriented LINE pairs located <10 Mbp from each other as potential NAHR substrates, placing 82.8% of the human genome at risk of LINE–LINE-mediated instability. Cross-referencing these regions with CNVs in the Baylor College of Medicine clinical chromosomal microarray database of 36 285 patients, we identified 516 CNVs potentially mediated by LINEs. Using long-range PCR of five different genomic regions in a total of 44 patients, we confirmed that the CNV breakpoints in each patient map within the LINE elements. To additionally assess the scale of LINE–LINE/NAHR phenomenon in the human genome, we tested DNA samples from six healthy individuals on a custom aCGH microarray targeting LINE elements predicted to mediate CNVs and identified 25 LINE–LINE rearrangements. Our data indicate that LINE–LINE-mediated NAHR is widespread and under-recognized, and is an important mechanism of structural rearrangement contributing to human genomic variability. PMID:25613453

  5. Suberoylanilide Hydroxamic Acid (SAHA) enhances olaparib activity by targeting homologous recombination DNA repair in ovarian cancer

    PubMed Central

    Konstantinopoulos, Panagiotis A.; Wilson, Andrew J.; Saskowski, Jeanette; Wass, Erica; Khabele, Dineo

    2015-01-01

    Objectives Approximately 50% of serous epithelial ovarian cancers (EOC) contain molecular defects in homologous recombination (HR) DNA repair pathways. Poly(ADP-ribose) polymerase inhibitors (PARPi) have efficacy in HR-deficient, but not HR-proficient, EOC tumors as a single agent. Our goal was to determine whether the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize HR-proficient ovarian cancer cells to the PARPi AZD-2281 (olaparib). Methods Ovarian cancer cell lines (SKOV-3, OVCAR-8, NCI/ADR-Res, UWB1.289 BRCA1null and UWB1.289 + BRCA1 wild-type) were treated with saline vehicle, olaparib, SAHA or olaparib/SAHA. Sulforhodamine B (SRB) assessed cytotoxicity and immunofluorescence and Western blot assays assessed markers of apoptosis (cleaved PARP) and DNA damage (pH2AX and RAD51). Drug effects were also tested in SKOV-3 xenografts in Nude mice. Affymetrix microarray experiments were performed in vehicle and SAHA-treated SKOV-3 cells. Results In a microarray analysis, SAHA induced coordinated down-regulation of HR pathway genes, including RAD51 and BRCA1. Nuclear co-expression of RAD51 and pH2AX, a marker of efficient HR repair, was reduced approximately 40% by SAHA treatment alone and combined with olaparib. SAHA combined with olaparib induced apoptosis and pH2AX expression to a greater extent than either drug alone. Olaparib reduced cell viability at increasing concentrations and SAHA enhanced these effects in 4 of 5 cell lines, including BRCA1 null and wild-type cells, in vitro and in SKOV-3 xenografts in vivo. Conclusions These results provide preclinical rationale for targeting DNA damage response pathways by combining small molecule PARPi with HDACi as a mechanism for reducing HR efficiency in ovarian cancer. PMID:24631446

  6. Oxidative stress-related DNA damage and homologous recombination repairing induced by N,N-dimethylformamide.

    PubMed

    Wang, Cui; Yang, Jinhuan; Lu, Dezhao; Fan, Yongsheng; Zhao, Meirong; Li, Zhuoyu

    2016-07-01

    The intensified anthropogenic release of N,N-dimethylformamide (DMF) has been proven to have hepatotoxic effects. However, the potential mechanism for DMF-induced toxicity has rarely been investigated. Our research implicated that DMF induced a significantly dose-dependent increase in reactive oxygen species (ROS) in HL-7702 human liver cells. Moreover, oxidative stress-related DNA damage, marked as 8-hydroxy-2'-deoxyguanosine, was increased 1.5-fold at 100 mmol l(-1) . The most severe DNA lesion (double-strand break, DSB), measured as the formation of γH2AX foci, was increased at/above 6.4 mmol l(-1) , and approximately 50% of cells underwent DSB at the peak induction. Subsequently, the DNA repair system triggered by molecules of RAD50 and MRE11A induced the homologous recombination (HR) pathway by upregulation of both gene and protein levels of RAD50, RAD51, XRCC2 and XRCC3 at 16 mmol l(-1) and was attenuated at 40 mmol l(-1) . Consequently, cellular death observed at 40 mmol l(-1) was exaggerated compared with exposure at 16 mmol l(-1) . Although the exact mechanism relying on the DMF-induced hepatotoxicity needs further clarification, oxidative stress and DNA damage involved in DSBs partially explain the reason for DMF-induced liver injury. Oxidative stress-induced DNA damage should be first considered during risk assessment on liver-targeted chemicals. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26387567

  7. Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation.

    PubMed

    Osipov, Andreyan N; Grekhova, Anna; Pustovalova, Margarita; Ozerov, Ivan V; Eremin, Petr; Vorobyeva, Natalia; Lazareva, Natalia; Pulin, Andrey; Zhavoronkov, Alex; Roumiantsev, Sergey; Klokov, Dmitry; Eremin, Ilya

    2015-09-29

    Molecular and cellular responses to protracted ionizing radiation exposures are poorly understood. Using immunofluorescence microscopy, we studied the kinetics of DNA repair foci formation in normal human fibroblasts exposed to X-rays at a dose rate of 4.5 mGy/min for up to 6 h. We showed that both the number of γH2AX foci and their integral fluorescence intensity grew linearly with time of irradiation up to 2 h. A plateau was observed between 2 and 6 h of exposure, indicating a state of balance between formation and repair of DNA double-strand breaks. In contrast, the number and intensity of foci formed by homologous recombination protein RAD51 demonstrated a continuous increase during 6 h of irradiation. We further showed that the enhancement of the homologous recombination repair was not due to redistribution of cell cycle phases. Our results indicate that continuous irradiation of normal human cells triggers DNA repair responses that are different from those elicited after acute irradiation. The observed activation of the error-free homologous recombination DNA double-strand break repair pathway suggests compensatory adaptive mechanisms that may help alleviate long-term biological consequences and could potentially be utilized both in radiation protection and medical practices. PMID:26337087

  8. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast.

    PubMed

    Silva, J V J; Arenhart, S; Santos, H F; Almeida-Queiroz, S R; Silva, A N M R; Trevisol, I M; Bertani, G R; Gil, L H V G

    2014-01-01

    The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

  9. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

    PubMed Central

    Silva, J.V.J.; Arenhart, S.; Santos, H.F.; Almeida-Queiroz, S.R.; Silva, A.N.M.R.; Trevisol, I.M.; Bertani, G.R.; Gil, L.H.V.G.

    2014-01-01

    The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world’s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

  10. Ubiquitylation of Rad51d Mediated by E3 Ligase Rnf138 Promotes the Homologous Recombination Repair Pathway

    PubMed Central

    Han, Deqiang; Liang, Junbo; Lu, Yalan; Xu, Longchang; Miao, Shiying; Lu, Lin-Yu; Song, Wei; Wang, Linfang

    2016-01-01

    Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway. PMID:27195665

  11. Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.

    PubMed

    Wang, Y; Liu, Y; Chen, J; Tang, M J; Zhang, S L; Wei, L N; Li, C H; Wei, D B

    2015-01-01

    In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, and examined the efficacy of RLF on high-throughput multi-plasmid cloning. This method utilizes the precise DNA repair and recombination systems within Escherichia coli, which allows to bypass the in vitro restriction and ligation enzyme reactions commonly included in routine cloning procedures. A homologous arm is linked to the 5'-end of the forward primer used to amplify both the target gene and vector. A different homologous arm is linked to the 5'-end of the reverse primer. Therefore, genes can be cloned into the vectors by homologous recombination after co-transformation of the amplified target gene and the linearized vector, which bear the same homologous arm on either end. More than twenty-four different plasmids were generated by this method, which uses two simple polymerase chain reaction steps. This method is highly efficient in cloning any gene of interest into any vector at any site without sequence constraints, as no restriction and ligation reactions are required. PMID:26505379

  12. Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

    PubMed Central

    Kay, Jennifer E.; Na, Li; Rowland, Elizabeth A.; Winther, Kelly E.; Chow, Danielle N.; Kimoto, Takafumi; Matsuguchi, Tetsuya; Jonnalagadda, Vidya S.; Maklakova, Vilena I.; Singh, Vijay R.; Wadduwage, Dushan N.; Rajapakse, Jagath; So, Peter T. C.; Collier, Lara S.; Engelward, Bevin P.

    2014-01-01

    Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals. PMID:24901438

  13. Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

    PubMed

    Sukup-Jackson, Michelle R; Kiraly, Orsolya; Kay, Jennifer E; Na, Li; Rowland, Elizabeth A; Winther, Kelly E; Chow, Danielle N; Kimoto, Takafumi; Matsuguchi, Tetsuya; Jonnalagadda, Vidya S; Maklakova, Vilena I; Singh, Vijay R; Wadduwage, Dushan N; Rajapakse, Jagath; So, Peter T C; Collier, Lara S; Engelward, Bevin P

    2014-06-01

    Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals. PMID:24901438

  14. The Rate of Nonallelic Homologous Recombination in Males Is Highly Variable, Correlated between Monozygotic Twins and Independent of Age

    PubMed Central

    MacArthur, Jacqueline A. L.; Spector, Timothy D.; Lindsay, Sarah J.; Mangino, Massimo; Gill, Raj; Small, Kerrin S.; Hurles, Matthew E.

    2014-01-01

    Nonallelic homologous recombination (NAHR) between highly similar duplicated sequences generates chromosomal deletions, duplications and inversions, which can cause diverse genetic disorders. Little is known about interindividual variation in NAHR rates and the factors that influence this. We estimated the rate of deletion at the CMT1A-REP NAHR hotspot in sperm DNA from 34 male donors, including 16 monozygotic (MZ) co-twins (8 twin pairs) aged 24 to 67 years old. The average NAHR rate was 3.5×10−5 with a seven-fold variation across individuals. Despite good statistical power to detect even a subtle correlation, we observed no relationship between age of unrelated individuals and the rate of NAHR in their sperm, likely reflecting the meiotic-specific origin of these events. We then estimated the heritability of deletion rate by calculating the intraclass correlation (ICC) within MZ co-twins, revealing a significant correlation between MZ co-twins (ICC = 0.784, p = 0.0039), with MZ co-twins being significantly more correlated than unrelated pairs. We showed that this heritability cannot be explained by variation in PRDM9, a known regulator of NAHR, or variation within the NAHR hotspot itself. We also did not detect any correlation between Body Mass Index (BMI), smoking status or alcohol intake and rate of NAHR. Our results suggest that other, as yet unidentified, genetic or environmental factors play a significant role in the regulation of NAHR and are responsible for the extensive variation in the population for the probability of fathering a child with a genomic disorder resulting from a pathogenic deletion. PMID:24603440

  15. Germinal Excisions of the Maize Transposon Activator Do Not Stimulate Meiotic Recombination or Homology-Dependent Repair at the Bz Locus

    PubMed Central

    Dooner, H. K.; Martinez-Ferez, I. M.

    1997-01-01

    Double-strand breaks have been implicated both in the initiation of meiotic recombination in yeast and as intermediates in the transposition process of nonreplicative transposons. Some transposons of this class, notably P of Drosophila and Tc1 of Caenorhabditis elegans, promote a form of homology-dependent premeiotic gene conversion upon excision. In this work, we have looked for evidence of an interaction between Ac transposition and meiotic recombination at the bz locus in maize. We find that the frequency of meiotic recombination between homologues is not enhanced by the presence of Ac in one of the bz heteroalleles and, conversely, that the presence of a homologous sequence in either trans (homologous chromosome) or cis (tandem duplication) does not promote conversion of the Ac insertion site. However, a tandem duplication of the bz locus may be destabilized by the insertion of Ac. We discuss possible reasons for the lack of interaction between Ac excision and homologous meiotic recombination in maize. PMID:9409847

  16. Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis

    PubMed Central

    Klink, Magdalena; Brzezinska, Marta; Sulowska, Zofia; Dziadek, Jaroslaw

    2014-01-01

    The intracellular pathogen Mycobacterium tuberculosis (Mtb) is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs). These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR) and non-homologous ends joining (NHEJ), in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA), NHEJ [Δ(ku,ligD)], or both DSB repair systems [Δ(ku,ligD,recA)]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA) mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA) mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages. PMID:24658131

  17. Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining.

    PubMed

    Beckta, Jason M; Dever, Seth M; Gnawali, Nisha; Khalil, Ashraf; Sule, Amrita; Golding, Sarah E; Rosenberg, Elizabeth; Narayanan, Aarthi; Kehn-Hall, Kylene; Xu, Bo; Povirk, Lawrence F; Valerie, Kristoffer

    2015-09-29

    Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations. PMID:26320175

  18. Extrachromosomal homologous DNA recombination in plant cells is fast and is not affected by CpG methylation.

    PubMed Central

    Puchta, H; Kocher, S; Hohn, B

    1992-01-01

    Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the beta-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared. Images PMID:1630452

  19. Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli.

    PubMed

    Khairnar, Nivedita P; Kamble, Vidya A; Mangoli, Suhas H; Apte, Shree K; Misra, Hari S

    2007-07-01

    The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli. PMID:17630970

  20. Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases

    PubMed Central

    De Paepe, Marianne; Hutinet, Geoffrey; Son, Olivier; Amarir-Bouhram, Jihane; Schbath, Sophie; Petit, Marie-Agnès

    2014-01-01

    Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like

  1. Srs2 and Mus81–Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates

    PubMed Central

    Keyamura, Kenji; Arai, Kota

    2016-01-01

    Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ), and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81–Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability. PMID:27390022

  2. New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination.

    PubMed

    Jacques, Marie-Agnès; Denancé, Nicolas; Legendre, Bruno; Morel, Emmanuelle; Briand, Martial; Mississipi, Stelly; Durand, Karine; Olivier, Valérie; Portier, Perrine; Poliakoff, Françoise; Crouzillat, Dominique

    2015-01-01

    Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants. PMID:26712553

  3. Chi hotspots trigger a conformational change in the helicase-like domain of AddAB to activate homologous recombination

    PubMed Central

    Gilhooly, Neville S.; Carrasco, Carolina; Gollnick, Benjamin; Wilkinson, Martin; Wigley, Dale B.; Moreno-Herrero, Fernando; Dillingham, Mark S.

    2016-01-01

    In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition. PMID:26762979

  4. Plastome Mutations and Recombination Events in Barley Chloroplast Mutator Seedlings.

    PubMed

    Landau, Alejandra; Lencina, Franco; Pacheco, María G; Prina, Alberto R

    2016-05-01

    The barley chloroplast mutator (cpm) is an allele of a nuclear gene that when homozygous induces several types of cytoplasmically inherited chlorophyll deficiencies. In this work, a plastome Targeting Induced Local Lesions in Genomes (TILLING) strategy based on mismatch digestion was used on families that carried the cpm genotype through many generations. Extensive scanning of 33 plastome genes and a few intergenic regions was conducted. Numerous polymorphisms were detected on both genic and intergenic regions. The detected polymorphisms can be accounted for by at least 61 independent mutational events. The vast majority of the polymorphisms originated in substitutions and small indels (insertions/deletions) in microsatellites. The rpl23 and the rps16 genes were the most polymorphic. Interestingly, the variation observed in the rpl23 gene consisted of several combinations of 5 different one nucleotide polymorphisms. Besides, 4 large indels that have direct repeats at both ends were also observed, which appear to be originated from recombinational events. The cpm mutation spectrum suggests that the CPM gene product is probably involved in plastome mismatch repair. The numerous subtle molecular changes that were localized in a wide range of plastome sites show the cpm as a valuable source of plastome variability for plant research and/or plant breeding. Moreover, the cpm mutant appears to be an interesting experimental material for investigating the mechanisms responsible for maintaining the stability of plant organelle DNA. PMID:26774059

  5. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    SciTech Connect

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G.; Leung, Stanley G.; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; Østvold, Anne Carine; Schild, David; Sung, Patrick; Wiese, Claudia

    2015-08-31

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Finally, our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

  6. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    DOE PAGESBeta

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G.; Leung, Stanley G.; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; et al

    2015-08-31

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintainingmore » wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Finally, our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.« less

  7. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    PubMed Central

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G.; Leung, Stanley G.; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; Østvold, Anne Carine; Schild, David; Sung, Patrick; Wiese, Claudia

    2015-01-01

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression. PMID:26323318

  8. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

    PubMed

    Parplys, Ann C; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G; Leung, Stanley G; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; Østvold, Anne Carine; Schild, David; Sung, Patrick; Wiese, Claudia

    2015-11-16

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression. PMID:26323318

  9. RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination.

    PubMed

    Marin-Vicente, Consuelo; Domingo-Prim, Judit; Eberle, Andrea B; Visa, Neus

    2015-03-15

    The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs. PMID:25632158

  10. Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells

    PubMed Central

    Böttcher, Romy; Hollmann, Manuel; Merk, Karin; Nitschko, Volker; Obermaier, Christina; Philippou-Massier, Julia; Wieland, Isabella; Gaul, Ulrike; Förstemann, Klaus

    2014-01-01

    The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system from Streptococcus pyogenes has been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage by cas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factor lig4. Using cultured Drosophila melanogaster S2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules for S. pyogenes cas9 sgRNA design and demonstrate that the complementarity region allows length variation and 5′-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated. PMID:24748663

  11. Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis.

    PubMed

    Brooker, J D; McCarthy, J M

    1997-09-01

    Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular alpha-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15-20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium. PMID:9236293

  12. Close, stable homolog juxtaposition during meiosis in budding yeast is dependent on meiotic recombination, occurs independently of synapsis, and is distinct from DSB-independent pairing contacts

    PubMed Central

    Peoples, Tamara L.; Dean, Eric; Gonzalez, Oscar; Lambourne, Lindsey; Burgess, Sean M.

    2002-01-01

    A site-specific recombination system that probes the relative probabilities that pairs of chromosomal loci collide with one another in living cells of budding yeast was used to explore the relative contributions of pairing, recombination, synaptonemal complex formation, and telomere clustering to the close juxtaposition of homologous chromosome pairs during meiosis. The level of Cre-mediated recombination between a pair of loxP sites located at an allelic position on homologous chromosomes was 13-fold greater than that between a pair of loxP sites located at ectopic positions on nonhomologous chromosomes. Mutations affecting meiotic recombination initiation and the processing of DNA double-strand breaks (DSBs) into single-end invasions (SEIs) reduced the levels of allelic Cre-mediated recombination levels by three- to sixfold. The severity of Cre/loxP phenotypes is presented in contrast to relatively weak DSB-independent pairing defects as assayed using fluorescence in situ hybridization for these mutants. Mutations affecting synaptonemal complex (SC) formation or crossover control gave wild-type levels of allelic Cre-mediated recombination. A delay in attaining maximum levels of allelic Cre-mediated recombination was observed for a mutant defective in telomere clustering. None of the mutants affected ectopic levels of recombination. These data suggest that stable, close homolog juxtaposition in yeast is distinct from pre-DSB pairing interactions, requires both DSB and SEI formation, but does not depend on crossovers or SC. PMID:12101126

  13. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

    PubMed Central

    Folger, K R; Wong, E A; Wahl, G; Capecchi, M R

    1982-01-01

    We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by

  14. Rapid Acquisition of Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus: Role of Hypermutation and Homologous Recombination

    PubMed Central

    Iguchi, Shigekazu; Mizutani, Tomonori; Hiramatsu, Keiichi; Kikuchi, Ken

    2016-01-01

    Background We previously reported the case of a 64-year-old man with mediastinitis caused by Staphylococcus aureus in which the infecting bacterium acquired linezolid resistance after only 14 days treatment with linezolid. We therefore investigated relevant clinical isolates for possible mechanisms of this rapid acquisition of linezolid resistance. Methods Using clinical S. aureus isolates, we assessed the in vitro mutation rate and performed stepwise selection for linezolid resistance. To investigate homologous recombination, sequences were determined for each of the 23S ribosomal RNA (23S rRNA) loci; analyzed sequences spanned the entirety of each 23S rRNA gene, including domain V, as well as the 16S-23S intergenic spacer regions. We additionally performed next-generation sequencing on clinical strains to identify single-nucleotide polymorphisms compared to the N315 genome. Results Strains isolated from the patient prior to linezolid exposure (M5-M7) showed higher-level linezolid resistance than N315, and the pre-exposure strain (M2) exhibited more rapid acquisition of linezolid resistance than did N315. However, the mutation rates of these and contemporaneous clinical isolates were similar to those of N315, and the isolates did not exhibit any mutations in hypermutation-related genes. Sequences of the 23S rRNA genes and 16S-23S intergenic spacer regions were identical among the pre- and post-exposure clinical strains. Notably, all of the pre-exposure isolates harbored a recQ missense mutation (Glu69Asp) with respect to N315; such a lesion may have affected short sequence recombination (facilitating, for example, recombination among rrn loci). We hypothesize that this mechanism contributed to rapid acquisition of linezolid resistance. Conclusions Hypermutation and homologous recombination of the ribosomal RNA genes, including 23S rRNA genes, appear not to have been sources of the accelerated acquisition of linezolid resistance observed in our clinical case

  15. EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

    PubMed Central

    Wu, Yuehan; Lee, Suk-Hee; Williamson, Elizabeth A.; Reinert, Brian L.; Cho, Ju Hwan; Xia, Fen; Jaiswal, Aruna Shanker; Srinivasan, Gayathri; Patel, Bhavita; Brantley, Alexis; Zhou, Daohong; Shao, Lijian; Pathak, Rupak; Hauer-Jensen, Martin; Singh, Sudha; Kong, Kimi; Wu, Xaiohua; Kim, Hyun-Suk; Beissbarth, Timothy; Gaedcke, Jochen; Burma, Sandeep; Nickoloff, Jac A.; Hromas, Robert A.

    2015-01-01

    Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ. PMID:26684013

  16. The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis

    PubMed Central

    Higgins, James D.; Armstrong, Susan J.; Franklin, F. Christopher H.; Jones, Gareth H.

    2004-01-01

    MSH4, a meiosis-specific member of the MutS-homolog family of genes, is required for normal levels of recombination and fertility in budding yeast, mouse, and Caenorhabditis elegans. In this paper, we report the identification and characterization of the Arabidopsis homolog of MSH4 (AtMSH4). We demonstrate that AtMSH4 expression can only be detected in floral tissues, consistent with a role in reproduction. Immunofluorescence studies indicate that its expression is limited to early meiotic prophase I, preceding the synapsis of homologous chromosomes. A T-DNA insertional mutant (Atmsh4) exhibited normal vegetative growth but a severe reduction in fertility, consistent with a meiotic defect; this was confirmed by cytological analysis of meiosis. RNAi-induced down-regulation of the MSH4 gene resulted in a similar fertility and meiotic phenotype. We demonstrate that prophase I chromosome synapsis is delayed and may be incomplete in Atmsh4, and metaphase I chiasma frequency is greatly reduced to ∼15% of wild type, leading to univalence and nondisjunction. We show that these residual chiasmata are randomly distributed among cells and chromosomes. These features of chiasma frequency and distribution in Atmsh4 show close parallels to MSH4-independent crossovers in budding yeast that have been proposed to originate by a separate pathway. Furthermore, the characteristics of the MSH4-independent chiasmata in the Atmsh4 mutant closely parallel those of second-pathway crossovers that have been postulated from Arabidopsis crossover analysis and mathematical modeling. Taken together, this evidence strongly indicates that Arabidopsis possesses two crossover pathways. PMID:15489296

  17. A system for assaying homologous recombination at the endogenous human thymidine kinase gene

    SciTech Connect

    Benjamin, M.B.; Little, J.B. ); Potter, H. ); Yandell, D.W. Massachusetts Eye and Ear Infirmary, Boston Harvard Medical School, Boston, MA )

    1991-08-01

    A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK{sup +/+} parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or {minus}1 frameshifts. Resulting TK{sup {minus}/{minus}} mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by {approx}8 kilobases. These lines undergo spontaneous reversion to TK{sup +} at a frequency of < 10{sup {minus}7}, and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK{sup +}. The mode of reversion to TK{sup +} was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. The data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.

  18. Soluble Epoxide Hydrolase Homologs in Strongylocentrotus purpuratus Suggest a Gene Duplication Event and Subsequent Divergence

    PubMed Central

    Harris, Todd R.; Aronov, Pavel A.

    2008-01-01

    The mammalian soluble epoxide hydrolase (sEH) is a multidomain enzyme composed of C- and N-terminal regions that contain active sites for epoxide hydrolase (EH) and phosphatase activities, respectively. We report the cloning of two 60 kDa multidomain enzymes from the purple sea urchin Strongylocentrotus purpuratus displaying significant sequence similarity to both the N- and C-terminal domains of the mammalian sEH. While one urchin enzyme did not exhibit EH activity, the second enzyme hydrolyzed several lipid messenger molecules metabolized by the mammalian sEH, including the epoxyeicosatrienoic acids. Neither of the urchin enzymes displayed phosphatase activity. The urchin EH was inhibited by small molecule inhibitors of the mammalian sEH and is the likely ancestor of the enzyme. Sequence comparisons suggest that the urchin sEH homologs are the result of a gene fusion event between a gene encoding for an EH and a gene for an enzyme of undetermined function. This fusion event was followed by a duplication event to produce the urchin enzymes. PMID:18554159

  19. RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

    PubMed Central

    Chen, Su; Wang, Chen; Sun, Luxi; Wang, Da-Liang; Chen, Lu; Huang, Zhuan; Yang, Qi; Gao, Jie; Yang, Xi-Bin; Chang, Jian-Feng; Chen, Ping; Lan, Li

    2014-01-01

    Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients. PMID:25384975

  20. Rapid generation of long tandem DNA repeat arrays by homologous recombination in yeast to study their function in mammalian genomes

    PubMed Central

    2011-01-01

    We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA) of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete. PMID:21982381

  1. A new method to reconstruct recombination events at a genomic scale.

    PubMed

    Melé, Marta; Javed, Asif; Pybus, Marc; Calafell, Francesc; Parida, Laxmi; Bertranpetit, Jaume

    2010-01-01

    Recombination is one of the main forces shaping genome diversity, but the information it generates is often overlooked. A recombination event creates a junction between two parental sequences that may be transmitted to the subsequent generations. Just like mutations, these junctions carry evidence of the shared past of the sequences. We present the IRiS algorithm, which detects past recombination events from extant sequences and specifies the place of each recombination and which are the recombinants sequences. We have validated and calibrated IRiS for the human genome using coalescent simulations replicating standard human demographic history and a variable recombination rate model, and we have fine-tuned IRiS parameters to simultaneously optimize for false discovery rate, sensitivity, and accuracy in placing the recombination events in the sequence. Newer recombinations overwrite traces of past ones and our results indicate more recent recombinations are detected by IRiS with greater sensitivity. IRiS analysis of the MS32 region, previously studied using sperm typing, showed good concordance with estimated recombination rates. We also applied IRiS to haplotypes for 18 X-chromosome regions in HapMap Phase 3 populations. Recombination events detected for each individual were recoded as binary allelic states and combined into recotypes. Principal component analysis and multidimensional scaling based on recotypes reproduced the relationships between the eleven HapMap Phase III populations that can be expected from known human population history, thus further validating IRiS. We believe that our new method will contribute to the study of the distribution of recombination events across the genomes and, for the first time, it will allow the use of recombination as genetic marker to study human genetic variation. PMID:21124860

  2. Loss of CtIP disturbs homologous recombination repair and sensitizes breast cancer cells to PARP inhibitors.

    PubMed

    Wang, Junhui; Ding, Qianshan; Fujimori, Hiroaki; Motegi, Akira; Miki, Yoshio; Masutani, Mitsuko

    2016-02-16

    Breast cancer is one of the leading causes of death worldwide, and therefore, new and improved approaches for the treatment of breast cancer are desperately needed. CtIP (RBBP8) is a multifunctional protein that is involved in various cellular functions, including transcription, DNA replication, DNA repair and the G1 and G2 cell cycle checkpoints. CtIP plays an important role in homologous recombination repair by interacting with tumor suppressor protein BRCA1. Here, we analyzed the expression profile of CtIP by data mining using published microarray data sets. We found that CtIP expression is frequently decreased in breast cancer patients, and the patient group with low-expressing CtIP mRNA is associated with a significantly lower survival rate. The knockdown of CtIP in breast cancer MCF7 cells reduced Rad51 foci numbers and enhanced f H2AX foci formation after f-irradiation, suggesting that deficiency of CtIP decreases homologous recombination repair and delays DNA double strand break repair. To explore the effect of CtIP on PARP inhibitor therapy for breast cancer, CtIP-depleted MCF7 cells were treated with PARP inhibitor olaparib (AZD2281) or veliparib (ABT-888). As in BRCA mutated cells, PARP inhibitors showed cytotoxicity to CtIP-depleted cells by preventing cells from repairing DNA damage, leading to decreased cell viability. Further, a xenograft tumor model in mice with MCF7 cells demonstrated significantly increased sensitivity towards PARP inhibition under CtIP deficiency. In summary, this study shows that low level of CtIP expression is associated with poor prognosis in breast cancer, and provides a rationale for establishing CtIP expression as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. PMID:26713604

  3. The Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex promotes trinucleotide repeat expansions independently of homologous recombination.

    PubMed

    Ye, Yanfang; Kirkham-McCarthy, Lucy; Lahue, Robert S

    2016-07-01

    Trinucleotide repeats (TNRs) are tandem arrays of three nucleotides that can expand in length to cause at least 17 inherited human diseases. Somatic expansions in patients can occur in differentiated tissues where DNA replication is limited and cannot be a primary source of somatic mutation. Instead, mouse models of TNR diseases have shown that both inherited and somatic expansions can be suppressed by the loss of certain DNA repair factors. It is generally believed that these repair factors cause misprocessing of TNRs, leading to expansions. Here we extend this idea to show that the Mre11-Rad50-Xrs2 (MRX) complex of Saccharomyces cerevisiae is a causative factor in expansions of short TNRs. Mutations that eliminate MRX subunits led to significant suppression of expansions whereas mutations that inactivate Rad51 had only a minor effect. Coupled with previous evidence, this suggests that MRX drives expansions of short TNRs through a process distinct from homologous recombination. The nuclease function of Mre11 was dispensable for expansions, suggesting that expansions do not occur by Mre11-dependent nucleolytic processing of the TNR. Epistasis between MRX and post-replication repair (PRR) was tested. PRR protects against expansions, so a rad5 mutant gave a high expansion rate. In contrast, the mre11 rad5 double mutant gave a suppressed expansion rate, indistinguishable from the mre11 single mutant. This suggests that MRX creates a TNR substrate for PRR. Protein acetylation was also tested as a mechanism regulating MRX activity in expansions. Six acetylation sites were identified in Rad50. Mutation of all six lysine residues to arginine gave partial bypass of a sin3 HDAC mutant, suggesting that Rad50 acetylation is functionally important for Sin3-mediated expansions. Overall we conclude that yeast MRX helps drive expansions of short TNRs by a mechanism distinct from its role in homologous recombination and independent of the nuclease function of Mre11. PMID:27173583

  4. Loss of CtIP disturbs homologous recombination repair and sensitizes breast cancer cells to PARP inhibitors

    PubMed Central

    Fujimori, Hiroaki; Motegi, Akira; Miki, Yoshio; Masutani, Mitsuko

    2016-01-01

    Breast cancer is one of the leading causes of death worldwide, and therefore, new and improved approaches for the treatment of breast cancer are desperately needed. CtIP (RBBP8) is a multifunctional protein that is involved in various cellular functions, including transcription, DNA replication, DNA repair and the G1 and G2 cell cycle checkpoints. CtIP plays an important role in homologous recombination repair by interacting with tumor suppressor protein BRCA1. Here, we analyzed the expression profile of CtIP by data mining using published microarray data sets. We found that CtIP expression is frequently decreased in breast cancer patients, and the patient group with low-expressing CtIP mRNA is associated with a significantly lower survival rate. The knockdown of CtIP in breast cancer MCF7 cells reduced Rad51 foci numbers and enhanced f H2AX foci formation after f-irradiation, suggesting that deficiency of CtIP decreases homologous recombination repair and delays DNA double strand break repair. To explore the effect of CtIP on PARP inhibitor therapy for breast cancer, CtIP-depleted MCF7 cells were treated with PARP inhibitor olaparib (AZD2281) or veliparib (ABT-888). As in BRCA mutated cells, PARP inhibitors showed cytotoxicity to CtIP-depleted cells by preventing cells from repairing DNA damage, leading to decreased cell viability. Further, a xenograft tumor model in mice with MCF7 cells demonstrated significantly increased sensitivity towards PARP inhibition under CtIP deficiency. In summary, this study shows that low level of CtIP expression is associated with poor prognosis in breast cancer, and provides a rationale for establishing CtIP expression as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. PMID:26713604

  5. The homologous recombination component EEPD1 is required for genome stability in response to developmental stress of vertebrate embryogenesis

    PubMed Central

    Chun, Changzoon; Wu, Yuehan; Lee, Suk-Hee; Williamson, Elizabeth A.; Reinert, Brian L.; Jaiswal, Aruna Shanker; Nickoloff, Jac A.; Hromas, Robert A.

    2016-01-01

    ABSTRACT Stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR), initiated by nuclease cleavage of branched structures at stalled forks. We previously reported that the 5′ nuclease EEPD1 is recruited to stressed replication forks, where it plays critical early roles in HR initiation by promoting fork cleavage and end resection. HR repair of stressed replication forks prevents their repair by non-homologous end-joining (NHEJ), which would cause genome instability. Rapid cell division during vertebrate embryonic development generates enormous pressure to maintain replication speed and accuracy. To determine the role of EEPD1 in maintaining replication fork integrity and genome stability during rapid cell division in embryonic development, we assessed the role of EEPD1 during zebrafish embryogenesis. We show here that when EEPD1 is depleted, zebrafish embryos fail to develop normally and have a marked increase in death rate. Zebrafish embryos depleted of EEPD1 are far more sensitive to replication stress caused by nucleotide depletion. We hypothesized that the HR defect with EEPD1 depletion would shift repair of stressed replication forks to unopposed NHEJ, causing chromosome abnormalities. Consistent with this, EEPD1 depletion results in nuclear defects including anaphase bridges and micronuclei in stressed zebrafish embryos, similar to BRCA1 deficiency. These results demonstrate that the newly characterized HR protein EEPD1 maintains genome stability during embryonic replication stress. These data also imply that the rapid cell cycle transit seen during embryonic development produces replication stress that requires HR to resolve. PMID:26900729

  6. The Elephant and the Blind Men: Making Sense of PARP Inhibitors in Homologous Recombination Deficient Tumor Cells.

    PubMed

    De Lorenzo, Silvana B; Patel, Anand G; Hurley, Rachel M; Kaufmann, Scott H

    2013-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is an important component of the base excision repair (BER) pathway as well as a regulator of homologous recombination (HR) and non-homologous end-joining (NHEJ). Previous studies have demonstrated that treatment of HR-deficient cells with PARP inhibitors results in stalled and collapsed replication forks. Consequently, HR-deficient cells are extremely sensitive to PARP inhibitors. Several explanations have been advanced to explain this so-called synthetic lethality between HR deficiency and PARP inhibition: (i) reduction of BER activity leading to enhanced DNA double-strand breaks, which accumulate in the absence of HR; (ii) trapping of inhibited PARP1 at sites of DNA damage, which prevents access of other repair proteins; (iii) failure to initiate HR by poly(ADP-ribose) polymer-dependent BRCA1 recruitment; and (iv) activation of the NHEJ pathway, which selectively induces error-prone repair in HR-deficient cells. Here we review evidence regarding these various explanations for the ability of PARP inhibitors to selectively kill HR-deficient cancer cells and discuss their potential implications. PMID:24062981

  7. Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease.

    PubMed Central

    Puchta, H; Dujon, B; Hohn, B

    1993-01-01

    Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination. Images PMID:8255757

  8. Patterns of integration of DNA microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules

    SciTech Connect

    Folger, K.R.; Wong, E.A.; Wahl, G.; Capecchi, M.R.

    1982-11-01

    The authors examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk/sup -/ and RAT-2tk/sup -/ cells to the TK/sup +/ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, the authors were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA.

  9. Promotion of Homologous Recombination and Genomic Stability byRAD51AP1 via RAD51 Recombinase Enhancement

    SciTech Connect

    Wiese, Claudia; Dray, Eloise; Groesser, Torsten; San Filippo,Joseph; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams,Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-04-11

    Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

  10. Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

    PubMed Central

    Wiese, Claudia; Dray, Eloïse; Groesser, Torsten; Filippo, Joseph San; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams, Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-01-01

    Summary Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds both dsDNA and a D-loop structure, and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement. PMID:17996711

  11. A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.

    PubMed

    Holmberg, Mats A; Gowda, Naveen Kumar Chandappa; Andréasson, Claes

    2014-06-01

    Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs. PMID:24631626

  12. Tetracycline Selective Pressure and Homologous Recombination Shape the Evolution of Chlamydia suis: A Recently Identified Zoonotic Pathogen.

    PubMed

    Joseph, Sandeep J; Marti, Hanna; Didelot, Xavier; Read, Timothy D; Dean, Deborah

    2016-01-01

    Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions. PMID:27576537

  13. Tetracycline Selective Pressure and Homologous Recombination Shape the Evolution of Chlamydia suis: A Recently Identified Zoonotic Pathogen

    PubMed Central

    Joseph, Sandeep J.; Marti, Hanna; Didelot, Xavier; Read, Timothy D.; Dean, Deborah

    2016-01-01

    Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct. Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions. PMID:27576537

  14. A physiological significance of the functional interaction between Mus81 and Rad27 in homologous recombination repair

    PubMed Central

    Thu, Huong Phung Thi; Nguyen, Tuan Anh; Munashingha, Palinda Ruvan; Kwon, Buki; Dao Van, Quy; Seo, Yeon-Soo

    2015-01-01

    Fen1 and Mus81–Mms4 are endonucleases involved in the processing of various DNA structural intermediates, and they were shown to have genetic and functional interactions with each other. Here, we show the in vivo significance of the interactions between Mus81 and Rad27 (yeast Fen1). The N-terminal 120 amino-acid (aa) region of Mus81, although entirely dispensable for its catalytic activity, was essential for the abilities of Mus81 to bind to and be stimulated by Rad27. In the absence of SGS1, the mus81Δ120N mutation lacking the N-terminal 120 aa region exhibited synthetic lethality, and the lethality was rescued by deletion of RAD52, a key homologous recombination mediator. These findings, together with the fact that Sgs1 constitutes a redundant pathway with Mus81–Mms4, indicate that the N-terminus-mediated interaction of Mus81 with Rad27 is physiologically important in resolving toxic recombination intermediates. Mutagenic analyses of the N-terminal region identified two distinct motifs, named N21–26 (aa from 21–26) and N108–114 (aa from 108–114) important for the in vitro and in vivo functions of Mus81. Our findings indicate that the N-terminal region of Mus81 acts as a landing pad to interact with Rad27 and that Mus81 and Rad27 work conjointly for efficient removal of various aberrant DNA structures. PMID:25628354

  15. Northern African Strains of Human T-Lymphotropic Virus Type 1 Arose from a Recombination Event

    PubMed Central

    Desrames, Alexandra; Cassar, Olivier; Gout, Olivier; Hermine, Olivier; Taylor, Graham P.; Afonso, Philippe V.

    2014-01-01

    ABSTRACT Although recombination is a major source of genetic variability in retroviruses, no recombinant strain had been observed for human T-lymphotropic virus type 1 (HTLV-1), the first isolated human-pathogenic retrovirus. Different genotypes exist for HTLV-1: Genotypes b and d to g are restricted to central Africa, while genotype c is only endemic in Australo-Melanesia. In contrast, the cosmopolitan genotype a is widely distributed. We applied a combination of phylogenetics and recombination analysis approaches to a set of new HTLV-1 sequences, which we collected from 19 countries throughout Africa, the continent where the virus has the largest endemic presence. This led us to demonstrate the presence of recombinants in HTLV-1. Indeed, the HTLV-1 strains currently present in North Africa have originated from a recombinant event between strains from Senegal and West Africa. This recombination is estimated to have occurred around 4,000 years ago. This recombination seems to have been generated during reverse transcription. In conclusion, we demonstrate that, albeit rare, recombination can occur in HTLV-1 and may play a role in the evolution of this retrovirus. IMPORTANCE A number of HTLV-1 subtypes have been described in different populations, but none of the genetic differences between these subtypes have been ascribed to recombination events. Here we report an HTLV-1 recombinant virus among infected individuals in North Africa. This demonstrates that, contrary to what was thought, recombination can occur and could play a role in the evolution of HTLV-1. PMID:24942582

  16. Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination.

    PubMed

    Diancourt, Laure; Passet, Virginie; Chervaux, Christian; Garault, Peggy; Smokvina, Tamara; Brisse, Sylvain

    2007-10-01

    Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137(T) (= ATCC 393(T)). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (pi ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei

  17. A murine-ES like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    PubMed Central

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels

    2010-01-01

    Murine embryonic stem cells have been shown to exist in two functionally distinct pluripotent states, embryonic stem cells (ES cell)- and epiblast stem cells (EpiSCs), which are defined by the culture growth factor conditions. Human ES cells appear to exist in an epiblast-like state, which in comparison to their murine counterparts, is relatively difficult to propagate and manipulate. As a result, gene targeting is difficult and to-date only a handful of human knock-in or knock-out cell lines exist. We explored whether an alternative stem cell state exists for human stem cells as well, and demonstrate that manipulation of the growth factor milieu allows the derivation of a novel human stem cell type that displays morphological, molecular and functional properties of murine ES cells and facilitates gene targeting. As such, the murine ES-like state provides a powerful tool for the generation of recombinant human pluripotent stem cell lines. PMID:20569691

  18. Type III Restriction Is Alleviated by Bacteriophage (RecE) Homologous Recombination Function but Enhanced by Bacterial (RecBCD) Function

    PubMed Central

    Handa, Naofumi; Kobayashi, Ichizo

    2005-01-01

    Previous works have demonstrated that DNA breaks generated by restriction enzymes stimulate, and are repaired by, homologous recombination with an intact, homologous DNA region through the function of lambdoid bacteriophages lambda and Rac. In the present work, we examined the effect of bacteriophage functions, expressed in bacterial cells, on restriction of an infecting tester phage in a simple plaque formation assay. The efficiency of plaque formation on an Escherichia coli host carrying EcoRI, a type II restriction system, is not increased by the presence of Rac prophage—presumably because, under the single-infection conditions of the plaque assay, a broken phage DNA cannot find a homologue with which to recombine. To our surprise, however, we found that the efficiency of plaque formation in the presence of a type III restriction system, EcoP1 or EcoP15, is increased by the bacteriophage-mediated homologous recombination functions recE and recT of Rac prophage. This type III restriction alleviation does not depend on lar on Rac, unlike type I restriction alleviation. On the other hand, bacterial RecBCD-homologous recombination function enhances type III restriction. These results led us to hypothesize that the action of type III restriction enzymes takes place on replicated or replicating DNA in vivo and leaves daughter DNAs with breaks at nonallelic sites, that bacteriophage-mediated homologous recombination reconstitutes an intact DNA from them, and that RecBCD exonuclease blocks this repair by degradation from the restriction breaks. PMID:16237019

  19. From gateway to multisite gateway in one recombination event

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We created a construct, pDONR-R4-R3, to easily convert any available Gateway destination vector to a MultiSite Gateway vector in a single recombination reaction. In addition, we designed pDONR-R4-R3 so that DNA fragments already cloned upstream or downstream of the Gateway cassette in the original d...

  20. SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase

    PubMed Central

    Ahn, Jang-Won; Kim, Sunjik; Na, Wooju; Baek, Su-Jin; Kim, Jeong-Hwan; Min, Keehong; Yeom, Jeonghun; Kwak, Hoyun; Jeong, Sunjoo; Lee, Cheolju; Kim, Seon-Young; Choi, Cheol Yong

    2015-01-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase. PMID:26068472

  1. Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells.

    PubMed

    Timiri Shanmugam, Prakash Srinivasan; Nair, Renjith Parameshwaran; De Benedetti, Arrigo; Caldito, Gloria; Abreo, Fleurette; Sunavala-Dossabhoy, Gulshan

    2016-04-01

    Accidental or medical radiation exposure of the salivary glands can gravely impact oral health. Previous studies have shown the importance of Tousled-like kinase 1 (TLK1) and its alternate start variant TLK1B in cell survival against genotoxic stresses. Through a high-throughput library screening of natural compounds, the phenolic phytochemical, gallic acid (GA), was identified as a modulator of TLK1/1B. This small molecule possesses anti-oxidant and free radical scavenging properties, but in this study, we report that in vitro it promotes survival of human salivary acinar cells, NS-SV-AC, through repair of ionizing radiation damage. Irradiated cells treated with GA show improved clonogenic survival compared to untreated controls. And, analyses of DNA repair kinetics by alkaline single-cell gel electrophoresis and γ-H2AX foci immunofluorescence indicate rapid resolution of DNA breaks in drug-treated cells. Study of DR-GFP transgene repair indicates GA facilitates homologous recombinational repair to establish a functional GFP gene. In contrast, inactivation of TLK1 or its shRNA knockdown suppressed resolution of radiation-induced DNA tails in NS-SV-AC, and homology directed repair in DR-GFP cells. Consistent with our results in culture, animals treated with GA after exposure to fractionated radiation showed better preservation of salivary function compared to saline-treated animals. Our results suggest that GA-mediated transient modulation of TLK1 activity promotes DNA repair and suppresses radiation cytoxicity in salivary gland cells. PMID:26855419

  2. Homologous Flare-CME Events and Their Metric Type II Radio Burst Association

    NASA Technical Reports Server (NTRS)

    Yashiro, S.; Gopalswamy, N.; Makela, P.; Akiyama, S.; Uddin, W.; Srivastava, A. K.; Joshi, N. C.; Chandra, R.; Manoharan, P. K.; Mahalakshmi, K.; Dwivedi, V. C.; Jain, R.; Awasthi, A. K.; Nitta, N. V.; Aschwanden, M. J.; Choudhary, D. P.

    2014-01-01

    Active region NOAA 11158 produced many flares during its disk passage. At least two of these flares can be considered as homologous: the C6.6 flare at 06:51 UT and C9.4 flare at 12:41 UT on February 14, 2011. Both flares occurred at the same location (eastern edge of the active region) and have a similar decay of the GOES soft X-ray light curve. The associated coronal mass ejections (CMEs) were slow (334 and 337 km/s) and of similar apparent widths (43deg and 44deg), but they had different radio signatures. The second event was associated with a metric type II burst while the first one was not. The COR1 coronagraphs on board the STEREO spacecraft clearly show that the second CME propagated into the preceding CME that occurred 50 min before. These observations suggest that CME-CME interaction might be a key process in exciting the type II radio emission by slow CMEs.

  3. The study on space-flight induced DNA damage in Arabidopsis thaliana using the related homologous recombination reporter system

    NASA Astrophysics Data System (ADS)

    Sun, Qiao; Nechitailo, Galina S.; Lu, Jinying; Liu, Min; Li, Huasheng

    Usually, phenotype changes of plants were used to analayze the responding genetic damages. However, this method is time-consuming, laborious and needs a long period. Here, we developed an Arabidopsis thaliana homologous recombination reporter system, in which HR frequency and HR-related AtRAD54 gene expression level were used as mutagenic end points. Based on the system, effect of DNA damage by space-flight during the Shenzhou-9 mission was investigated. In this study, an Arabidopsis thaliana-line transgenic for GUS recombination substrates (R3L66, AtRAD54promoter:: GFP + GUS) was used to study the mutagenicity of space-flight, and the results showed that 13 days space-flight exposure of seedlings induced a significant increase in HRF compared with its ground-base three-dimensional clinostat (generally called a random positioning machine or RPM, an effective simulator of microgravity) controls and ground 1g controls. We also observed three-dimensional clinostat induced a significant increase in HRF and HR-related AtRAD54 gene expression level compared with ground 1g controls. Treatment with the ROS scavenger DMSO dramatically reduced the effects of simulated microgravity on the induction of HR and expression of the AtRAD54 gene, suggesting that ROS play a critical role in mediating the simulated microgravity mutagenic effects in plants. In order to understand the combined effects of radiation and microgravity (the main factors in space environment) on DNA damage, we further investigated the effects of modeled microgravity on radiation-induced bystander effects (RIBE) n vivo in A. thaliana plants using the expression level of the HR-related AtRAD54 gene as mutagenic end points. The results showed that the modeled microgravity significantly inhibited the up-regulated expression of the AtRAD54 gene in bystander aerial plants after root irradiation, suggesting a repressive effect of microgravity on RIBE.

  4. Gene targeting by TALEN-induced homologous recombination in goats directs production of β-lactoglobulin-free, high-human lactoferrin milk.

    PubMed

    Cui, Chenchen; Song, Yujie; Liu, Jun; Ge, Hengtao; Li, Qian; Huang, Hui; Hu, Linyong; Zhu, Hongmei; Jin, Yaping; Zhang, Yong

    2015-01-01

    β-Lactoglobulin (BLG) is a major goat's milk allergen that is absent in human milk. Engineered endonucleases, including transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases, enable targeted genetic modification in livestock. In this study, TALEN-mediated gene knockout followed by gene knock-in were used to generate BLG knockout goats as mammary gland bioreactors for large-scale production of human lactoferrin (hLF). We introduced precise genetic modifications in the goat genome at frequencies of approximately 13.6% and 6.09% for the first and second sequential targeting, respectively, by using targeting vectors that underwent TALEN-induced homologous recombination (HR). Analysis of milk from the cloned goats revealed large-scale hLF expression or/and decreased BLG levels in milk from heterozygous goats as well as the absence of BLG in milk from homozygous goats. Furthermore, the TALEN-mediated targeting events in somatic cells can be transmitted through the germline after SCNT. Our result suggests that gene targeting via TALEN-induced HR may expedite the production of genetically engineered livestock for agriculture and biomedicine. PMID:25994151

  5. I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells

    PubMed Central

    Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A.; Panthier, Jean-Jacques

    2012-01-01

    Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells. PMID:22761925

  6. Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination.

    PubMed

    Zhu, Hongmei; Liu, Jun; Cui, Chenchen; Song, Yujie; Ge, Hengtao; Hu, Linyong; Li, Qian; Jin, Yaping; Zhang, Yong

    2016-01-01

    Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as β-lactoglobulin (BLG). Here, we employed transcription activator-like effector nuclease (TALEN)-assisted homologous recombination in goat fibroblasts to introduce human α-lactalbumin (hLA) genes into goat BLG locus. TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection. Specifically, BLG mRNA levels were gradually decreasing in both mo- and bi-allelic goat mammary epithelial cells (GMECs) while hLA demonstrated expression in GMECs in vitro. Gene-targeted fibroblast cells were efficiently used in somatic cell nuclear transfer, resulting in production of hLA knock-in goats directing down-regulated BLG expression and abundant hLA secretion in animal milk. Our findings provide valuable background for animal milk optimization and expedited development for agriculture and biomedicine. PMID:27258157

  7. Combination of FACS and homologous recombination for the generation of stable and high-expression engineered cell lines.

    PubMed

    Shi, Lei; Chen, Xuesi; Tang, Wenying; Li, Zhenyi; Liu, Jin; Gao, Feng; Sang, Jianli

    2014-01-01

    Traditionally, cell line generation requires several months and involves screening of over several hundred cell clones for high productivity before dozens are selected as candidate cell lines. Here, we have designed a new strategy for the generation of stable and high-expression cell lines by combining homologous recombination (HR) and fluorescence-activated cell sorting (FACS). High expression was indicated by the expression of secreted green fluorescent protein (SEGFP). Parental cell lines with the highest expression of SEGFP were then selected by FACS and identified by stability analysis. Consequently, HR vectors were constructed using the cassette for SEGFP as the HR region. After transfecting the HR vector, the cells with negative SEGFP expression were enriched by FACS. The complete exchange between SEGFP and target gene (TNFR-Fc) cassettes was demonstrated by DNA analysis. Compared with the traditional method, by integrating the cassette containing the gene of interest into the pre-selected site, the highest producing cells secreted a more than 8-fold higher titer of target protein. Hence, this new strategy can be applied to isolated stable cell lines with desirable expression of any gene of interest. The stable cell lines can rapidly produce proteins for researching protein structure and function and are even applicable in drug discovery. PMID:24646904

  8. Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

    SciTech Connect

    Noguchi, Miho; Yu, Dong; Hirayama, Ryoichi; Ninomiya, Yasuharu; Sekine, Emiko; Kubota, Nobuo; Ando, Koichi; Okayasu, Ryuichi . E-mail: rokayasu@nirs.go.jp

    2006-12-22

    In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing.

  9. Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability.

    PubMed

    Trego, Kelly S; Groesser, Torsten; Davalos, Albert R; Parplys, Ann C; Zhao, Weixing; Nelson, Michael R; Hlaing, Ayesu; Shih, Brian; Rydberg, Björn; Pluth, Janice M; Tsai, Miaw-Sheue; Hoeijmakers, Jan H J; Sung, Patrick; Wiese, Claudia; Campisi, Judith; Cooper, Priscilla K

    2016-02-18

    XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging. PMID:26833090

  10. Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

    SciTech Connect

    Zafar, Faria; Seidler, Sara B.; Kronenberg, Amy; Schild, David; Wiese, Claudia

    2010-06-29

    To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.

  11. Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis.

    PubMed

    Zahabiun, Farzaneh; Sadjjadi, Seyed Mahmoud; Yunus, Muhammad Hafiznur; Rahumatullah, Anizah; Moghaddam, Mohammad Hosein Falaki; Saidin, Syazwan; Noordin, Rahmah

    2015-08-01

    Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory-secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis. PMID:26033026

  12. PARP1 inhibition radiosensitizes HNSCC cells deficient in homologous recombination by disabling the DNA replication fork elongation response.

    PubMed

    Wurster, Stephanie; Hennes, Fabian; Parplys, Ann C; Seelbach, Jasna I; Mansour, Wael Y; Zielinski, Alexandra; Petersen, Cordula; Clauditz, Till S; Münscher, Adrian; Friedl, Anna A; Borgmann, Kerstin

    2016-03-01

    There is a need to develop new, more efficient therapies for head and neck cancer (HNSCC) patients. It is currently unclear whether defects in DNA repair genes play a role in HNSCCs' resistance to therapy. PARP1 inhibitors (PARPi) were found to be "synthetic lethal" in cancers deficient in BRCA1/2 with impaired homologous recombination. Since tumors rarely have these particular mutations, there is considerable interest in finding alternative determinants of PARPi sensitivity. Effectiveness of combined irradiation and PARPi olaparib was evaluated in ten HNSCC cell lines, subdivided into HR-proficient and HR-deficient cell lines using a GFP-based reporter assay. Both groups were equally sensitive to PARPi alone. Combined treatment revealed stronger synergistic interactions in the HR-deficient group. Because HR is mainly active in S-Phase, replication processes were analyzed. A stronger impact of treatment on replication processes (p = 0.04) and an increased number of radial chromosomes (p = 0.003) were observed in the HR-deficient group. We could show that radiosensitization by inhibition of PARP1 strongly correlates with HR competence in a replication-dependent manner. Our observations indicate that PARP1 inhibitors are promising candidates for enhancing the therapeutic ratio achieved by radiotherapy via disabling DNA replication processes in HR-deficient HNSCCs. PMID:26799421

  13. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks

    PubMed Central

    Vriend, Lianne E.M.; Prakash, Rohit; Chen, Chun-Chin; Vanoli, Fabio; Cavallo, Francesca; Zhang, Yu; Jasin, Maria; Krawczyk, Przemek M.

    2016-01-01

    DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR (nickHR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of nickHR are largely unexplored. Here, we applied Cas9 nickases to study nickHR in mammalian cells. We find that nickHR is unaffected by inhibition of major damage signaling kinases and that it is not suppressed by nonhomologous end-joining (NHEJ) components, arguing that nick processing does not require a DSB intermediate to trigger HR. Relative to a single nick, nicking both strands enhances HR, consistent with a DSB intermediate, even when nicks are induced up to ∼1kb apart. Accordingly, HR and NHEJ compete for repair of these paired nicks, but, surprisingly, only when 5' overhangs or blunt ends can be generated. Our study advances the understanding of molecular mechanisms driving nick and paired-nick repair in mammalian cells and clarify phenomena associated with Cas9-mediated genome editing. PMID:27001513

  14. PARP1 inhibition radiosensitizes HNSCC cells deficient in homologous recombination by disabling the DNA replication fork elongation response

    PubMed Central

    Parplys, Ann C.; Seelbach, Jasna I.; Mansour, Wael Y.; Zielinski, Alexandra; Petersen, Cordula; Clauditz, Till S.; Münscher, Adrian; Friedl, Anna A.; Borgmann, Kerstin

    2016-01-01

    There is a need to develop new, more efficient therapies for head and neck cancer (HNSCC) patients. It is currently unclear whether defects in DNA repair genes play a role in HNSCCs' resistance to therapy. PARP1 inhibitors (PARPi) were found to be “synthetic lethal” in cancers deficient in BRCA1/2 with impaired homologous recombination. Since tumors rarely have these particular mutations, there is considerable interest in finding alternative determinants of PARPi sensitivity. Effectiveness of combined irradiation and PARPi olaparib was evaluated in ten HNSCC cell lines, subdivided into HR-proficient and HR-deficient cell lines using a GFP-based reporter assay. Both groups were equally sensitive to PARPi alone. Combined treatment revealed stronger synergistic interactions in the HR-deficient group. Because HR is mainly active in S-Phase, replication processes were analyzed. A stronger impact of treatment on replication processes (p = 0.04) and an increased number of radial chromosomes (p = 0.003) were observed in the HR-deficient group. We could show that radiosensitization by inhibition of PARP1 strongly correlates with HR competence in a replication-dependent manner. Our observations indicate that PARP1 inhibitors are promising candidates for enhancing the therapeutic ratio achieved by radiotherapy via disabling DNA replication processes in HR-deficient HNSCCs. PMID:26799421

  15. Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination

    PubMed Central

    Cui, Chenchen; Song, Yujie; Ge, Hengtao; Hu, Linyong; Li, Qian; Jin, Yaping; Zhang, Yong

    2016-01-01

    Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as β-lactoglobulin (BLG). Here, we employed transcription activator-like effector nuclease (TALEN)-assisted homologous recombination in goat fibroblasts to introduce human α-lactalbumin (hLA) genes into goat BLG locus. TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection. Specifically, BLG mRNA levels were gradually decreasing in both mo- and bi-allelic goat mammary epithelial cells (GMECs) while hLA demonstrated expression in GMECs in vitro. Gene-targeted fibroblast cells were efficiently used in somatic cell nuclear transfer, resulting in production of hLA knock-in goats directing down-regulated BLG expression and abundant hLA secretion in animal milk. Our findings provide valuable background for animal milk optimization and expedited development for agriculture and biomedicine. PMID:27258157

  16. 5-Fluorouracil sensitizes colorectal tumor cells towards double stranded DNA breaks by interfering with homologous recombination repair

    PubMed Central

    Srinivas, Upadhyayula Sai; Dyczkowski, Jerzy; Beißbarth, Tim; Gaedcke, Jochen; Mansour, Wael Y.; Borgmann, Kerstin; Dobbelstein, Matthias

    2015-01-01

    Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy. PMID:25909291

  17. Ataxia telangiectasia mutated (ATM) is dispensable for endonuclease I-SceI-induced homologous recombination in mouse embryonic stem cells.

    PubMed

    Rass, Emilie; Chandramouly, Gurushankar; Zha, Shan; Alt, Frederick W; Xie, Anyong

    2013-03-01

    Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency. PMID:23355489

  18. Disparate requirements for the Walker A and B ATPase motifs ofhuman RAD51D in homologous recombination

    SciTech Connect

    Wiese, Claudia; Hinz, John M.; Tebbs, Robert S.; Nham, Peter B.; Urbin, Salustra S.; Collins, David W.; Thompson, Larry H.; Schild, David

    2006-04-21

    In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks. Ectopic expression of wild type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.

  19. Genomic complexity profiling reveals that HORMAD1 overexpression contributes to homologous recombination deficiency in triple-negative breast cancers

    PubMed Central

    Watkins, Johnathan; Weekes, Daniel; Shah, Vandna; Gazinska, Patrycja; Joshi, Shalaka; Sidhu, Bhavna; Gillett, Cheryl; Pinder, Sarah; Vanoli, Fabio; Jasin, Maria; Mayrhofer, Markus; Isaksson, Anders; Cheang, Maggie C.U.; Mirza, Hasan; Frankum, Jessica; Lord, Christopher J.; Ashworth, Alan; Vinayak, Shaveta; Ford, James M.; Telli, Melinda L.; Grigoriadis, Anita; Tutt, Andrew N.J.

    2015-01-01

    Triple-negative breast cancers (TNBCs) are characterised by a wide spectrum of genomic alterations, some of which might be caused by defects in DNA repair processes such as homologous recombination (HR). Despite this understanding, associating particular patterns of genomic instability with response to therapy has been challenging. Here, we show that Allelic-imbalanced Copy Number Aberrations (AiCNA) are more prevalent in TNBCs that respond to platinum-based chemotherapy, thus providing a candidate predictive biomarker for this disease. Furthermore, we show that a high level of AiCNA is linked with elevated expression of a meiosis-associated gene HORMAD1. Elevated HORMAD1 expression suppresses RAD51-dependent HR and drives the use of alternative forms of DNA repair, the generation of AiCNAs as well as sensitising cancer cells to HR targeting therapies. Our data therefore provides a mechanistic association between HORMAD1 expression, a specific pattern of genomic instability and an association with response to platinum-based chemotherapy in TNBC. PMID:25770156

  20. A Cyclin-Dependent Kinase Inhibitor, Dinaciclib, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition.

    PubMed

    Alagpulinsa, David A; Ayyadevara, Srinivas; Yaccoby, Shmuel; Shmookler Reis, Robert J

    2016-02-01

    PARP1/2 are required for single-strand break repair, and their inhibition causes DNA replication fork collapse and double-strand break (DSB) formation. These DSBs are primarily repaired via homologous recombination (HR), a high-fidelity repair pathway. Should HR be deficient, DSBs may be repaired via error-prone nonhomologous end-joining mechanisms, or may persist, ultimately resulting in cell death. The combined disruption of PARP and HR activities thus produces synthetic lethality. Multiple myeloma cells are characterized by chromosomal instability and pervasive DNA damage, implicating aberrant DNA repair. Cyclin-dependent kinases (CDK), upstream modulators of HR, are dysregulated in multiple myeloma. Here, we show that a CDK inhibitor, dinaciclib, impairs HR repair and sensitizes multiple myeloma cells to the PARP1/2 inhibitor ABT-888. Dinaciclib abolishes ABT-888-induced BRCA1 and RAD51 foci and potentiates DNA damage, indicated by increased γH2AX foci. Dinaciclib treatment reduces expression of HR repair genes, including Rad51, and blocks BRCA1 phosphorylation, a modification required for HR repair, thus inhibiting HR repair of chromosome DSBs. Cotreatment with dinaciclib and ABT-888 in vitro resulted in synthetic lethality of multiple myeloma cells, but not normal CD19(+) B cells, and slowed growth of multiple myeloma xenografts in SCID mice almost two-fold. These findings support combining dinaciclib with PARP inhibitors for multiple myeloma therapy. Mol Cancer Ther; 15(2); 241-50. ©2015 AACR. PMID:26719576

  1. Meiotic Recombination in Somatic Cell Nuclear Transfer Bulls and Their Offspring

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In mammals, homologous chromosome pairing and recombination are essential events for meiosis. The generation of reciprocal exchanges of genetic material ensure both genetic diversity and the proper segregation of homologous chromosomes. With the advent of reproductive biotechnologies such as somat...

  2. Transcription and double-strand breaks induce similar mitotic recombination events in Saccharomyces cerevisiae.

    PubMed Central

    González-Barrera, Sergio; García-Rubio, María; Aguilera, Andrés

    2002-01-01

    We have made a comparative analysis of double-strand-break (DSB)-induced recombination and spontaneous recombination under low- and high-transcription conditions in yeast. We constructed two different recombination substrates, one for the analysis of intermolecular gene conversions and the other for intramolecular gene conversions and inversions. Such substrates were based on the same leu2-HOr allele fused to the tet promoter and containing a 21-bp HO site. Gene conversions and inversions were differently affected by rad1, rad51, rad52, and rad59 single and double mutations, consistent with the actual view that such events occur by different recombination mechanisms. However, the effect of each mutation on each type of recombination event was the same, whether associated with transcription or induced by the HO-mediated DSB. Both the highly transcribed DNA and the HO-cut sequence acted as recipients of the gene conversion events. These results are consistent with the hypothesis that transcription promotes initiation of recombination along the DNA sequence being transcribed. The similarity between transcription-associated and DSB-induced recombination suggests that transcription promotes DNA breaks. PMID:12399375

  3. RecQ helicase and RecJ nuclease provide complementary functions to resect DNA for homologous recombination

    PubMed Central

    Morimatsu, Katsumi; Kowalczykowski, Stephen C.

    2014-01-01

    Recombinational DNA repair by the RecF pathway of Escherichia coli requires the coordinated activities of RecA, RecFOR, RecQ, RecJ, and single-strand DNA binding (SSB) proteins. These proteins facilitate formation of homologously paired joint molecules between linear double-stranded (dsDNA) and supercoiled DNA. Repair starts with resection of the broken dsDNA by RecQ, a 3′→5′ helicase, RecJ, a 5′→3′ exonuclease, and SSB protein. The ends of a dsDNA break can be blunt-ended, or they may possess either 5′- or 3′-single-stranded DNA (ssDNA) overhangs of undefined length. Here we show that RecJ nuclease alone can initiate nucleolytic resection of DNA with 5′-ssDNA overhangs, and that RecQ helicase can initiate resection of DNA with blunt-ends or 3′-ssDNA overhangs by DNA unwinding. We establish that in addition to its well-known ssDNA exonuclease activity, RecJ can display dsDNA exonuclease activity, degrading 100–200 nucleotides of the strand terminating with a 5′-ssDNA overhang. The dsDNA product, with a 3′-ssDNA overhang, is an optimal substrate for RecQ, which unwinds this intermediate to reveal the complementary DNA strand with a 5′-end that is degraded iteratively by RecJ. On the other hand, RecJ cannot resect duplex DNA that is either blunt-ended or terminated with 3′-ssDNA; however, such DNA is unwound by RecQ to create ssDNA for RecJ exonuclease. RecJ requires interaction with SSB for exonucleolytic degradation of ssDNA but not dsDNA. Thus, complementary action by RecJ and RecQ permits initiation of recombinational repair from all dsDNA ends: 5′-overhangs, blunt, or 3′-overhangs. Such helicase–nuclease coordination is a common mechanism underlying resection in all organisms. PMID:25411316

  4. miR-3940-5p enhances homologous recombination after DSB in Cr(VI) exposed 16HBE cell.

    PubMed

    Li, Yang; Hu, Guiping; Li, Ping; Tang, Shichuan; Zhang, Ji; Jia, Guang

    2016-02-17

    Hexavalent chromium (Cr(VI)) is a well-recognized human carcinogen, yet the molecular mechanisms by which cause human cancer are still not well understood. MicroRNAs (miRNAs), which are small non-coding RNAs, are involved in carcinogenesis and DNA damage repair. Previous occupational population study showed that hexavalent chromium (Cr(VI)) downregulated plasma miR-3940-5p level, and a low miR-3940-5p level was associated with high XRCC2 expression in lymphocytes, indicating that miR-3940-5p maybe play a protective effect in Cr(VI) induced DNA damage. Here we investigated miR-3940-5p expression and its roles in DNA repair in Cr(VI)-treated 16HBE cells. miR-3940-5p change was detected by qRT-PCR. Rad51 foci formation and double strand break (DSB) were investigated to assess homologous recombination repair (HR) capacity by Immunofluorescent assay and Neutral Comet assay. XRCC2 expression was also evaluated after miRNA oligonucleotides transfection using Western blot. Cr(VI) treatment suppressed miR-3940-5p level in 16HBE cells. miR-3904-5p mimic downregulated XRCC2 expression. As a result, the formation of Rad51-foci was inhibited and DSB repair was prolonged. The results indicate that miR-3940-5p plays a protective effect in Cr(VI) induced DNA damage. PMID:26860703

  5. Targeting homologous recombination and telomerase in Barrett’s adenocarcinoma: Impact on telomere maintenance, genomic instability, and tumor growth

    PubMed Central

    Lu, Renquan; Pal, Jagannath; Buon, Leutz; Nanjappa, Puru; Shi, Jialan; Fulciniti, Mariateresa; Tai, Yu-Tzu; Guo, Lin; Yu, Min; Gryaznov, Sergei; Munshi, Nikhil C.; Shammas, Masood A.

    2014-01-01

    Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in BAC. The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contribute to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase, makes telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore

  6. Targeting homologous recombination and telomerase in Barrett's adenocarcinoma: impact on telomere maintenance, genomic instability and tumor growth.

    PubMed

    Lu, R; Pal, J; Buon, L; Nanjappa, P; Shi, J; Fulciniti, M; Tai, Y-T; Guo, L; Yu, M; Gryaznov, S; Munshi, N C; Shammas, M A

    2014-03-20

    Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett's esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their

  7. The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80

    PubMed Central

    Typas, Dimitris; Luijsterburg, Martijn S.; Wiegant, Wouter W.; Diakatou, Michaela; Helfricht, Angela; Thijssen, Peter E.; van de Broek, Bram; Mullenders, Leon H.; van Attikum, Haico

    2015-01-01

    The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR. PMID:26101254

  8. Progress in the treatment of ovarian cancer-lessons from homologous recombination deficiency-the first 10 years.

    PubMed

    Kaye, S B

    2016-04-01

    For several years, a major obstacle in the systemic treatment of ovarian cancer has been the lack of a therapeutic strategy tailored to specific biomarkers present in the individual patient's tumour. However, considerable progress has been made recently through the development of drugs targeting cells deficient in the key mechanism of double-strand DNA repair, known as homologous recombination (HRD). These drugs, inhibitors of the enzyme poly (ADP) ribose polymerase (PARP), selectively kill HRD cells through a process known as tumour-selective synthetic lethality. Olaparib is the first such agent, now approved for the treatment of ovarian cancer associated with mutations in the BRCA 1/2 genes, since these are characterised by cells with HRD. Importantly, another group of patients with tumours bearing a similar repair deficiency but without BRCA mutations may also be susceptible to PARP inhibition and efforts to develop an HRD assay are therefore a priority so that these patients can be identified as PARPi candidates. In addition, combination strategies are an area of intense research; these include combinations with antiangiogenic agents and with inhibitors of the P13K/AKT pathway and others are likely to merit assessment since resistance to PARP inhibitors will certainly emerge as the next challenge. While olaparib is the first PARP inhibitor to receive approval for ovarian cancer treatment, others including rucaparib and niraparib are clearly effective in this disease and, within the next year or two, the results of ongoing randomised trials will clarify their respective roles. PARP inhibitors are generally well tolerated; regulatory approval at present supports their use as a maintenance therapy (in Europe) and as treatment for advanced recurrent disease (in the United States), but it is likely that these indications will extend as the results of ongoing trials become available. Ten years have elapsed between the first pre-clinical publications and the

  9. Genetic polymorphisms in homologous recombination repair genes in healthy Slovenian population and their influence on DNA damage

    PubMed Central

    Goricar, Katja; Erculj, Nina; Zadel, Maja; Dolzan, Vita

    2012-01-01

    Background Homologous recombination (HR) repair is an important mechanism involved in repairing double-strand breaks in DNA and for maintaining genomic stability. Polymorphisms in genes coding for enzymes involved in this pathway may influence the capacity for DNA repair. The aim of this study was to select tag single nucleotide polymorphisms (SNPs) in specific genes involved in HR repair, to determine their allele frequencies in a healthy Slovenian population and their influence on DNA damage detected with comet assay. Materials and methods In total 373 individuals were genotyped for nine tag SNPs in three genes: XRCC3 722C>T, XRCC3 -316A>G, RAD51 -98G>C, RAD51 -61G>T, RAD51 1522T>G, NBS1 553G>C, NBS1 1197A>G, NBS1 37117C>T and NBS1 3474A>C using competitive allele-specific amplification (KASPar assay). Comet assay was performed in a subgroup of 26 individuals to determine the influence of selected SNPs on DNA damage. Results We observed that age significantly affected genotype frequencies distribution of XRCC3 -316A>G (P = 0.039) in healthy male blood donors. XRCC3 722C>T (P = 0.005), RAD51 -61G>T (P = 0.023) and NBS1 553G>C (P = 0.008) had a statistically significant influence on DNA damage. Conclusions XRCC3 722C>T, RAD51 -61G>T and NBS1 553G>C polymorphisms significantly affect the repair of damaged DNA and may be of clinical importance as they are common in Slovenian population. PMID:22933979

  10. Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair12

    PubMed Central

    Kausar, Tasneem; Schreiber, Jason S.; Karnak, David; Parsels, Leslie A.; Parsels, Joshua D.; Davis, Mary A.; Zhao, Lili; Maybaum, Jonathan; Lawrence, Theodore S.; Morgan, Meredith A.

    2015-01-01

    To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy. PMID:26585231

  11. Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier.

    PubMed

    Willis, Nicholas A; Scully, Ralph

    2016-07-17

    The Escherichia coli replication fork arrest complex Tus/Ter mediates site-specific replication fork arrest and homologous recombination (HR) on a mammalian chromosome, inducing both conservative "short tract" gene conversion (STGC) and error-prone "long tract" gene conversion (LTGC) products. We showed previously that bidirectional fork arrest is required for the generation of STGC products at Tus/Ter-stalled replication forks and that the HR mediators BRCA1, BRCA2 and Rad51 mediate STGC but suppress LTGC at Tus/Ter-arrested forks. Here, we report the impact of Ter array length on Tus/Ter-induced HR, comparing HR reporters containing arrays of 6, 9, 15 or 21 Ter sites-each targeted to the ROSA26 locus of mouse embryonic stem (ES) cells. Increasing Ter copy number within the array beyond 6 did not affect the magnitude of Tus/Ter-induced HR but biased HR in favor of LTGC. A "lock"-defective Tus mutant, F140A, known to exhibit higher affinity than wild type (wt)Tus for duplex Ter, reproduced these effects. In contrast, increasing Ter copy number within the array reduced HR induced by the I-SceI homing endonuclease, but produced no consistent bias toward LTGC. Thus, the mechanisms governing HR at Tus/Ter-arrested replication forks are distinct from those governing HR at an enzyme-induced chromosomal double strand break (DSB). We propose that increased spatial separation of the 2 arrested forks encountering an extended Tus/Ter barrier impairs the coordination of DNA ends generated by the processing of the stalled forks, thereby favoring aberrant LTGC over conservative STGC. PMID:27136113

  12. The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80.

    PubMed

    Typas, Dimitris; Luijsterburg, Martijn S; Wiegant, Wouter W; Diakatou, Michaela; Helfricht, Angela; Thijssen, Peter E; van de Broek, Bram; Mullenders, Leon H; van Attikum, Haico

    2015-08-18

    The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR. PMID:26101254

  13. Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

    SciTech Connect

    Nagasawa, H; Wilson, P F; Chen, D J; Thompson, L H; Bedford, J S; Little, J B

    2007-10-26

    We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells (Nagasawa et al., Radiat. Res. 164, 141-147, 2005). In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23-0.33 SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after {alpha}-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

  14. Human DNA Helicase B Functions in Cellular Homologous Recombination and Stimulates Rad51-Mediated 5′-3′ Heteroduplex Extension In Vitro

    PubMed Central

    Liu, Hanjian; Yan, Peijun; Fanning, Ellen

    2015-01-01

    Homologous recombination is involved in the repair of DNA damage and collapsed replication fork, and is critical for the maintenance of genomic stability. Its process involves a network of proteins with different enzymatic activities. Human DNA helicase B (HDHB) is a robust 5′-3′ DNA helicase which accumulates on chromatin in cells exposed to DNA damage. HDHB facilitates cellular recovery from replication stress, but its role in DNA damage response remains unclear. Here we report that HDHB silencing results in reduced sister chromatid exchange, impaired homologous recombination repair, and delayed RPA late-stage foci formation induced by ionizing radiation. Ectopically expressed HDHB colocalizes with Rad51, Rad52, RPA, and ssDNA. In vitro, HDHB stimulates Rad51-mediated heteroduplex extension in 5′-3′ direction. A helicase-defective mutant HDHB failed to promote this reaction. Our studies implicate HDHB promotes homologous recombination in vivo and stimulates 5′-3′ heteroduplex extension during Rad51-mediated strand exchange in vitro. PMID:25617833

  15. Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair.

    PubMed

    Harnicek, Dominique; Kampmann, Eric; Lauber, Kirsten; Hennel, Roman; Cardoso Martins, Ana Sofia; Guo, Yang; Belka, Claus; Mörtl, Simone; Gallmeier, Eike; Kanaar, Roland; Mansmann, Ulrich; Hucl, Tomas; Lindner, Lars H; Hiddemann, Wolfgang; Issels, Rolf D

    2016-07-15

    The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency. PMID:26933761

  16. Phylogenetic and molecular epidemiological studies reveal evidence of multiple past recombination events between infectious laryngotracheitis viruses.

    PubMed

    Lee, Sang-Won; Devlin, Joanne M; Markham, John F; Noormohammadi, Amir H; Browning, Glenn F; Ficorilli, Nino P; Hartley, Carol A; Markham, Philip F

    2013-01-01

    In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field. PMID:23383306

  17. Phylogenetic and Molecular Epidemiological Studies Reveal Evidence of Multiple Past Recombination Events between Infectious Laryngotracheitis Viruses

    PubMed Central

    Lee, Sang-Won; Devlin, Joanne M.; Markham, John F.; Noormohammadi, Amir H.; Browning, Glenn F.; Ficorilli, Nino P.; Hartley, Carol A.; Markham, Philip F.

    2013-01-01

    In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field. PMID:23383306

  18. Temporal evolution and potential recombination events in PRRSV strains of Sonora Mexico.

    PubMed

    Burgara-Estrella, Alexel; Reséndiz-Sandoval, Mónica; Cortey, Martí; Mateu, Enric; Hernández, Jesús

    2014-12-01

    The aim of this work was to examine the evolution and potential existence of intragenic recombinations of PRRSV strains in Sonora, Mexico. In this study, 142 serum samples from farms located in Hermosillo (HMO), Cd. Obregón (OBR) and Navojoa (NAV) were sequenced from 2002 to 2012. Ninety non-redundant sequences of ORF5 gene were analyzed for temporal and spatial relationships among strains and the probability of a recombination event. The phylogenetic analysis showed 30 strains grouped into eight groups; 16 strains were closely related among the farms, while 14 were un-related. The first strain in this study was observed in 2002. A number of farms were infected with one or more strains, and in the majority of the strains, the virus was replaced by a new strain. The recombination analysis suggested the presence of four viruses as products of a recombination event; in one case, a virus close related with MLV vaccine was involved as the parent virus. This work shows the evolution of PRRSV in the field, the viral dissemination between farms and the potential recombination events. Our data suggest that PRRSV in Sonora has a specific genetic nature compared with other PRRSV. PMID:25448445

  19. XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

    SciTech Connect

    Yamada, N; Hinz, J; Kopf, V L; Segalle, K; Thompson, L

    2004-02-25

    Homologous recombinational repair is a major DNA repair pathway that preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 appears to exist in a single complex with Rad51C. To begin to examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a non-conservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of the mutants' dysfunction, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon coexpression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in HRR.

  20. Germline Methylation Patterns Determine the Distribution of Recombination Events in the Dog Genome

    PubMed Central

    Berglund, Jonas; Quilez, Javier; Arndt, Peter F.; Webster, Matthew T.

    2015-01-01

    The positive-regulatory domain containing nine gene, PRDM9, which strongly associates with the location of recombination events in several vertebrates, is inferred to be inactive in the dog genome. Here, we address several questions regarding the control of recombination and its influence on genome evolution in dogs. First, we address whether the association between CpG islands (CGIs) and recombination hotspots is generated by lack of methylation, GC-biased gene conversion (gBGC), or both. Using a genome-wide dog single nucleotide polymorphism data set and comparisons of the dog genome with related species, we show that recombination-associated CGIs have low CpG mutation rates, and that CpG mutation rate is negatively correlated with recombination rate genome wide, indicating that nonmethylation attracts the recombination machinery. We next use a neighbor-dependent model of nucleotide substitution to disentangle the effects of CpG mutability and gBGC and analyze the effects that loss of PRDM9 has on these rates. We infer that methylation patterns have been stable during canid genome evolution, but that dog CGIs have experienced a drastic increase in substitution rate due to gBGC, consistent with increased levels of recombination in these regions. We also show that gBGC is likely to have generated many new CGIs in the dog genome, but these mostly occur away from genes, whereas the number of CGIs in gene promoter regions has not increased greatly in recent evolutionary history. Recombination has a major impact on the distribution of CGIs that are detected in the dog genome due to the interaction between methylation and gBGC. The results indicate that germline methylation patterns are the main determinant of recombination rates in the absence of PRDM9. PMID:25527838

  1. A Recombinationally Repressed Region between Mat2 and Mat3 Loci Shares Homology to Centromeric Repeats and Regulates Directionality of Mating-Type Switching in Fission Yeast

    PubMed Central

    Grewal, SIS.; Klar, AJS.

    1997-01-01

    Cells of the fission yeast Schizosaccharomyces pombe switch mating type by replacing genetic information at the transcriptionally active mat1 locus with sequences copied from one of two closely linked silent loci, mat2-P or mat3-M. By a process referred to as directionality of switching, cells predominantly switch to the opposite mat1 allele; the mat1-P allele preferentially recombines with mat3, while mat1-M selects the mat2. In contrast to efficient recombination at mat1, recombination within the adjoining mat2-mat3 interval is undetectable. We defined the role of sequences between mat2 and mat3, designated the K-region, in directionality as well as recombinational suppression. Cloning and sequencing analysis revealed that a part of the K-region is homologous to repeat sequences present at centromeres, which also display transcriptional and recombinational suppression. Replacement of 7.5 kb of the K-region with the ura4(+) gene affected directionality in a variegated manner. Analysis of the swi6-mod locus, which was previously shown to affect directionality, in KΔ::ura4(+) strains suggested the existence of at least two overlapping directionality mechanisms. Our work furthers the model that directionality is regulated by cell-type-specific organization of the heterochromatin-like structure in the mating-type region and provides evidence that the K-region contributes to silencing of the mat2-mat3 interval. PMID:9258669

  2. Homologous Solar Events on 2011 January 27: Build-up and Propagation in a Complex Coronal Environment

    NASA Astrophysics Data System (ADS)

    Pick, M.; Stenborg, G.; Démoulin, P.; Zucca, P.; Lecacheux, A.

    2016-05-01

    In spite of the wealth of imaging observations at the extreme-ultraviolet (EUV), X-ray, and radio wavelengths, there are still relatively few cases where all of the imagery is available to study the full development of a coronal mass ejection (CME) event and its associated shock. The aim of this study is to contribute to the understanding of the role of the coronal environment in the development of CMEs and the formation of shocks, and their propagation. We have analyzed the interactions of a couple of homologous CME events with ambient coronal structures. Both events were launched in a direction far from the local vertical, and exhibited a radical change in their direction of propagation during their progression from the low corona into higher altitudes. Observations at EUV wavelengths from the Atmospheric Imaging Assembly instrument on board the Solar Dynamic Observatory were used to track the events in the low corona. The development of the events at higher altitudes was followed by the white-light coronagraphs on board the Solar and Heliospheric Observatory. Radio emissions produced during the development of the events were well recorded by the Nançay solar instruments. Thanks to their detection of accelerated electrons, the radio observations are an important complement to the EUV imaging. They allowed us to characterize the development of the associated shocks, and helped to unveil the physical processes behind the complex interactions between the CMEs and ambient medium (e.g., compression, reconnection).

  3. A Role for the Malignant Brain Tumour (MBT) Domain Protein LIN-61 in DNA Double-Strand Break Repair by Homologous Recombination

    PubMed Central

    Johnson, Nicholas M.; Lemmens, Bennie B. L. G.; Tijsterman, Marcel

    2013-01-01

    Malignant brain tumour (MBT) domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR) for the repair of DNA double-strand breaks (DSBs). lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT–deficient tumours may also have defective DSB repair. PMID:23505385

  4. Generation of Fibroblasts Lacking the Sal-like 1 Gene by Using Transcription Activator-like Effector Nuclease-mediated Homologous Recombination

    PubMed Central

    Kim, Se Eun; Kim, Ji Woo; Kim, Yeong Ji; Kwon, Deug-Nam; Kim, Jin-Hoi; Kang, Man-Jong

    2016-01-01

    The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5′ arm, 1.8-kb 3′ arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using 300 μg/mL G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3′ and 5′ arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR–restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene. PMID:26949958

  5. Mechanism of radiosensitization by the Chk1/2 inhibitor AZD7762 involves abrogation of the G2 checkpoint and inhibition of homologous recombinational DNA repair.

    PubMed

    Morgan, Meredith A; Parsels, Leslie A; Zhao, Lili; Parsels, Joshua D; Davis, Mary A; Hassan, Maria C; Arumugarajah, Sankari; Hylander-Gans, Linda; Morosini, Deborah; Simeone, Diane M; Canman, Christine E; Normolle, Daniel P; Zabludoff, Sonya D; Maybaum, Jonathan; Lawrence, Theodore S

    2010-06-15

    The median survival for patients with locally advanced pancreatic cancer treated with gemcitabine and radiation is approximately 1 year. To develop improved treatment, we have combined a Chk1/2-targeted agent, AZD7762, currently in phase I clinical trials, with gemcitabine and ionizing radiation in preclinical pancreatic tumor models. We found that in vitro AZD7762 alone or in combination with gemcitabine significantly sensitized MiaPaCa-2 cells to radiation. AZD7762 inhibited Chk1 autophosphorylation (S296 Chk1), stabilized Cdc25A, and increased ATR/ATM-mediated Chk1 phosphorylation (S345 Chk1). Radiosensitization by AZD7762 was associated with abrogation of the G(2) checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent gamma-H2AX expression. AZD7762 was also a radiation sensitizer in multiple tumor xenograft models. In both MiaPaCa-2- and patient-derived xenografts, AZD7762 significantly prolonged the median time required for tumor volume doubling in response to gemcitabine and radiation. Together, our findings suggest that G(2) checkpoint abrogation and homologous recombination repair inhibition both contribute to sensitization by Chk1 inhibition. Furthermore, they support the clinical use of AZD7762 in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer. PMID:20501833

  6. A novel role for the mono-ADP-ribosyltransferase PARP14/ARTD8 in promoting homologous recombination and protecting against replication stress.

    PubMed

    Nicolae, Claudia M; Aho, Erin R; Choe, Katherine N; Constantin, Daniel; Hu, He-Juan; Lee, Deokjae; Myung, Kyungjae; Moldovan, George-Lucian

    2015-03-31

    Genomic instability, a major hallmark of cancer cells, is caused by incorrect or ineffective DNA repair. Many DNA repair mechanisms cooperate in cells to fight DNA damage, and are generally regulated by post-translational modification of key factors. Poly-ADP-ribosylation, catalyzed by PARP1, is a post-translational modification playing a prominent role in DNA repair, but much less is known about mono-ADP-ribosylation. Here we report that mono-ADP-ribosylation plays an important role in homologous recombination DNA repair, a mechanism essential for replication fork stability and double strand break repair. We show that the mono-ADP-ribosyltransferase PARP14 interacts with the DNA replication machinery component PCNA and promotes replication of DNA lesions and common fragile sites. PARP14 depletion results in reduced homologous recombination, persistent RAD51 foci, hypersensitivity to DNA damaging agents and accumulation of DNA strand breaks. Our work uncovered PARP14 as a novel factor required for mitigating replication stress and promoting genomic stability. PMID:25753673

  7. Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells

    PubMed Central

    2014-01-01

    Background Very few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs. Results Recombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells. Conclusions The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs. PMID:25037233

  8. Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

    PubMed Central

    Poncet, S; Bernard, C; Dervyn, E; Cayley, J; Klier, A; Rapoport, G

    1997-01-01

    Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control. PMID:9361428

  9. Polymorphisms of homologous recombination RAD51, RAD51B, XRCC2, and XRCC3 genes and the risk of prostate cancer.

    PubMed

    Nowacka-Zawisza, Maria; Wiśnik, Ewelina; Wasilewski, Andrzej; Skowrońska, Milena; Forma, Ewa; Bryś, Magdalena; Różański, Waldemar; Krajewska, Wanda M

    2015-01-01

    Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland. PMID:26339569

  10. TRF2-RAP1 is required to protect telomeres from engaging in homologous recombination-mediated deletions and fusions.

    PubMed

    Rai, Rekha; Chen, Yong; Lei, Ming; Chang, Sandy

    2016-01-01

    Repressor/activator protein 1 (RAP1) is a highly conserved telomere-interacting protein. Yeast Rap1 protects telomeres from non-homologous end joining (NHEJ), plays important roles in telomere length control and is involved in transcriptional gene regulation. However, a role for mammalian RAP1 in telomere end protection remains controversial. Here we present evidence that mammalian RAP1 is essential to protect telomere from homology directed repair (HDR) of telomeres. RAP1 cooperates with the basic domain of TRF2 (TRF2(B)) to repress PARP1 and SLX4 localization to telomeres. Without RAP1 and TRF2(B), PARP1 and SLX4 HR factors promote rapid telomere resection, resulting in catastrophic telomere loss and the generation of telomere-free chromosome fusions in both mouse and human cells. The RAP1 Myb domain is required to repress both telomere loss and formation of telomere-free fusions. Our results highlight the importance of the RAP1-TRF2 heterodimer in protecting telomeres from inappropriate processing by the HDR pathway. PMID:26941064

  11. TRF2-RAP1 is required to protect telomeres from engaging in homologous recombination-mediated deletions and fusions

    PubMed Central

    Rai, Rekha; Chen, Yong; Lei, Ming; Chang, Sandy

    2016-01-01

    Repressor/activator protein 1 (RAP1) is a highly conserved telomere-interacting protein. Yeast Rap1 protects telomeres from non-homologous end joining (NHEJ), plays important roles in telomere length control and is involved in transcriptional gene regulation. However, a role for mammalian RAP1 in telomere end protection remains controversial. Here we present evidence that mammalian RAP1 is essential to protect telomere from homology directed repair (HDR) of telomeres. RAP1 cooperates with the basic domain of TRF2 (TRF2B) to repress PARP1 and SLX4 localization to telomeres. Without RAP1 and TRF2B, PARP1 and SLX4 HR factors promote rapid telomere resection, resulting in catastrophic telomere loss and the generation of telomere-free chromosome fusions in both mouse and human cells. The RAP1 Myb domain is required to repress both telomere loss and formation of telomere-free fusions. Our results highlight the importance of the RAP1-TRF2 heterodimer in protecting telomeres from inappropriate processing by the HDR pathway. PMID:26941064

  12. Nonhomologous end-joining repair plays a more important role than homologous recombination repair in defining radiosensitivity after exposure to high-LET radiation.

    PubMed

    Takahashi, Akihisa; Kubo, Makoto; Ma, Hongyu; Nakagawa, Akiko; Yoshida, Yukari; Isono, Mayu; Kanai, Tatsuaki; Ohno, Tatsuya; Furusawa, Yoshiya; Funayama, Tomoo; Kobayashi, Yasuhiko; Nakano, Takashi

    2014-09-01

    DNA double-strand breaks (DSBs) induced by ionizing radiation pose a major threat to cell survival. The cell can respond to the presence of DSBs through two major repair pathways: homologous recombination (HR) and nonhomologous end joining (NHEJ). Higher levels of cell death are induced by high-linear energy transfer (LET) radiation when compared to low-LET radiation, even at the same physical doses, due to less effective and efficient DNA repair. To clarify whether high-LET radiation inhibits all repair pathways or specifically one repair pathway, studies were designed to examine the effects of radiation with different LET values on DNA DSB repair and radiosensitivity. Embryonic fibroblasts bearing repair gene (NHEJ-related Lig4 and/or HR-related Rad54) knockouts (KO) were used and their responses were compared to wild-type cells. The cells were exposed to X rays, spread-out Bragg peak (SOBP) carbon ion beams as well as with carbon, iron, neon and argon ions. Cell survival was measured with colony-forming assays. The sensitization enhancement ratio (SER) values were calculated using the 10% survival dose of wild-type cells and repair-deficient cells. Cellular radiosensitivity was listed in descending order: double-KO cells > Lig4-KO cells > Rad54-KO cells > wild-type cells. Although Rad54-KO cells had an almost constant SER value, Lig4-KO cells showed a high-SER value when compared to Rad54-KO cells, even with increasing LET values. These results suggest that with carbon-ion therapy, targeting NHEJ repair yields higher radiosensitivity than targeting homologous recombination repair. PMID:25117625

  13. Multifactorial Resistance of Bacillus subtilis Spores to High-Energy Proton Radiation: Role of Spore Structural Components and the Homologous Recombination and Non-Homologous End Joining DNA Repair Pathways

    PubMed Central

    Reitz, Günther; Li, Zuofeng; Klein, Stuart; Nicholson, Wayne L.

    2012-01-01

    Abstract The space environment contains high-energy charged particles (e.g., protons, neutrons, electrons, α-particles, heavy ions) emitted by the Sun and galactic sources or trapped in the radiation belts. Protons constitute the majority (87%) of high-energy charged particles. Spores of Bacillus species are one of the model systems used for astro- and radiobiological studies. In this study, spores of different Bacillus subtilis strains were used to study the effects of high energetic proton irradiation on spore survival. Spores of the wild-type B. subtilis strain [mutants deficient in the homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways and mutants deficient in various spore structural components such as dipicolinic acid (DPA), α/β-type small, acid-soluble spore protein (SASP) formation, spore coats, pigmentation, or spore core water content] were irradiated as air-dried multilayers on spacecraft-qualified aluminum coupons with 218 MeV protons [with a linear energy transfer (LET) of 0.4 keV/μm] to various final doses up to 2500 Gy. Spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to proton radiation than wild-type spores, indicating that both HR and NHEJ DNA repair pathways are needed for spore survival. Spores lacking DPA, α/β-type SASP, or with increased core water content were also significantly more sensitive to proton radiation, whereas the resistance of spores lacking pigmentation or spore coats was essentially identical to that of the wild-type spores. Our results indicate that α/β-type SASP, core water content, and DPA play an important role in spore resistance to high-energy proton irradiation, suggesting their essential function as radioprotectants of the spore interior. Key Words: Bacillus—Spores—DNA repair—Protection—High-energy proton radiation. Astrobiology 12, 1069–1077. PMID:23088412

  14. Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

    PubMed Central

    Ma, Jian Jun; Xu, Peng; Zhang, Wei; Li, Yu Mei; Fu, Qiang; Zhu, Guang Feng; Xue, Wei; Lei, Yong Hua; Gao, Jing Yu; Wang, Juan Ying; Shao, Chen; Yi, Cheng Gang; Wang, He

    2013-01-01

    Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment. PMID:23950876

  15. Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells.

    PubMed

    Shahar, O D; Raghu Ram, E V S; Shimshoni, E; Hareli, S; Meshorer, E; Goldberg, M

    2012-07-26

    DNA double-strand breaks (DSBs), the most hazardous DNA lesions, may result in genomic instability, a hallmark of cancer cells. The main DSB repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). In mammalian cells, NHEJ, which can lead to inaccurate repair, predominates. HR repair (HRR) is considered accurate and is restricted to S, G2 and M phases of the cell cycle. Despite its importance, many aspects regarding HRR remain unknown. Here, we developed a novel inducible on/off switch cell system that enables, for the first time, to induce a DSB in a rapid and reversible manner in human cells. By limiting the duration of DSB induction, we found that non-persistent endonuclease-induced DSBs are rarely repaired by HR, whereas persistent DSBs result in the published HRR frequencies (non-significant HR frequency versus frequency of ∼10%, respectively). We demonstrate that these DSBs are repaired by an accurate repair mechanism, which is distinguished from HRR (most likely, error-free NHEJ). Notably, our data reveal that HRR frequencies of endonuclease-induced DSBs in human cells are >10-fold lower than what was previously estimated by prevailing methods, which resulted in recurrent DSB formation. Our findings suggest a role for HRR mainly in repairing challenging DSBs, in contrast to uncomplicated lesions that are frequently repaired by NHEJ. Preventing HR from repairing DSBs in the complex and repetitive human genome probably has an essential role in maintaining genomic stability. PMID:22105360

  16. Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

    NASA Technical Reports Server (NTRS)

    Asaad, N. A.; Zeng, Z. C.; Guan, J.; Thacker, J.; Iliakis, G.

    2000-01-01

    The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

  17. Protection against aerosolized Yersinia pestis challenge following homologous and heterologous prime-boost with recombinant plague antigens.

    PubMed

    Glynn, Audrey; Roy, Chad J; Powell, Bradford S; Adamovicz, Jeffrey J; Freytag, Lucy C; Clements, John D

    2005-08-01

    A Yersinia pestis-derived fusion protein (F1-V) has shown great promise as a protective antigen against aerosol challenge with Y. pestis in murine studies. In the current study, we examined different prime-boost regimens with F1-V and demonstrate that (i) boosting by a route other than the route used for the priming dose (heterologous boosting) protects mice as well as homologous boosting against aerosol challenge with Y. pestis, (ii) parenteral immunization is not required to protect mice against aerosolized plague challenge, (iii) the route of immunization and choice of adjuvant influence the magnitude of the antibody response as well as the immunoglobulin G1 (IgG1)/IgG2a ratio, and (iv) inclusion of an appropriate adjuvant is critical for nonparenteral immunization. PMID:16041052

  18. DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination.

    PubMed

    Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S; Lan, Li

    2015-07-01

    Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome. PMID:26100862

  19. Excitation pressure regulates the activation energy for recombination events in the photosystem II reaction centres of Chlamydomonas reinhardtii.

    PubMed

    Pocock, Tessa; Sane, P V; Falk, S; Hüner, N P A

    2007-12-01

    Using in vivo thermoluminescence, we examined the effects of growth irradiance and growth temperature on charge recombination events in photosystem II reaction centres of the model green alga Chlamydomonas reinhardtii. We report that growth at increasing irradiance at either 29 or 15 degrees C resulted in comparable downward shifts in the temperature peak maxima (T(M)) for S2QB- charge pair recombination events, with minimal changes in S2QA- recombination events. This indicates that such growth conditions decrease the activation energy required for S2QB- charge pair recombination events with no concomitant change in the activation energy for S2QA- recombination events. This resulted in a decrease in the DeltaT(M) between S2QA- and S2QB- recombination events, which was reversible when shifting cells from low to high irradiance and back to low irradiance at 29 degrees C. We interpret these results to indicate that the redox potential of QB was modulated independently of QA, which consequently narrowed the redox potential gap between QA and QB in photosystem II reaction centres. Since a decrease in the DeltaT(M) between S2QA- and S2QB- recombination events correlated with growth at increasing excitation pressure, we conclude that acclimation to growth under high excitation pressure narrows the redox potential gap between QA and QB in photosystem II reaction centres, enhancing the probability for reaction center quenching in C. reinhardtii. We discuss the molecular basis for the modulation of the redox state of QB, and suggest that the potential for reaction center quenching complements antenna quenching via the xanthophyll cycle in the photoprotection of C. reinhardtii from excess light. PMID:18059530

  20. Haplotype diversity generated by ancient recombination-like events in the MHC of Indian rhesus macaques.

    PubMed

    Doxiadis, Gaby G M; de Groot, Nanine; Otting, Nel; de Vos-Rouweler, Annemiek J M; Bolijn, Maria J; Heijmans, Corrine M C; de Groot, Natasja G; van der Wiel, Marit K H; Remarque, Edmond J; Vangenot, Christelle; Nunes, José M; Sanchez-Mazas, Alicia; Bontrop, Ronald E

    2013-08-01

    The Mamu-A, Mamu-B, and Mamu-DRB genes of the rhesus macaque show several levels of complexity such as allelic heterogeneity (polymorphism), copy number variation, differential segregation of genes/alleles present on a haplotype (diversity) and transcription level differences. A combination of techniques was implemented to screen a large panel of pedigreed Indian rhesus macaques (1,384 individuals representing the offspring of 137 founding animals) for haplotype diversity in an efficient and inexpensive manner. This approach allowed the definition of 140 haplotypes that display a relatively low degree of region variation as reflected by the presence of only 17 A, 18 B and 22 DRB types, respectively, exhibiting a global linkage disequilibrium comparable to that in humans. This finding contrasts with the situation observed in rhesus macaques from other geographic origins and in cynomolgus monkeys from Indonesia. In these latter populations, nearly every haplotype appears to be characterised by a unique A, B and DRB region. In the Indian population, however, a reshuffling of existing segments generated "new" haplotypes. Since the recombination frequency within the core MHC of the Indian rhesus macaques is relatively low, the various haplotypes were most probably produced by recombination events that accumulated over a long evolutionary time span. This idea is in accord with the notion that Indian rhesus macaques experienced a severe reduction in population during the Pleistocene due to a bottleneck caused by geographic changes. Thus, recombination-like processes appear to be a way to expand a diminished genetic repertoire in an isolated and relatively small founder population. PMID:23715823

  1. Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells.

    PubMed

    Jayakumar, Sundarraj; Pal, Debojyoti; Sandur, Santosh K

    2015-09-01

    Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2. Together, these results suggest the involvement of Nrf2 in DNA repair, a hitherto unknown function of Nrf2, putatively through its influence on HR pathway. PMID:26133502

  2. λ Recombination and Recombineering.

    PubMed

    Murphy, Kenan C

    2016-05-01

    The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821

  3. DNA replication arrest leads to enhanced homologous recombination and cell death in meristems of rice OsRecQl4 mutants

    PubMed Central

    2013-01-01

    Background Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DNA damage has been confirmed. However, the function of AtRecQ4A in the meristem during replication stress has not yet been well elucidated. Results We isolated the BLM counterpart gene OsRecQl4 from rice and analyzed its function using a reverse genetics approach. Osrecql4 mutant plants showed hypersensitivity to DNA damaging agents and enhanced frequency of HR compared to wild-type (WT) plants. We further analyzed the effect of aphidicolin—an inhibitor of S-phase progression via its inhibitory effect on DNA polymerases—on genome stability in the root meristem in osrecql4 mutant plants and corresponding WT plants. The following effects were observed upon aphidicolin treatment: a) comet assay showed induction of DNA double-strand breaks (DSBs) in mutant plants, b) TUNEL assay showed enhanced DNA breaks at the root meristem in mutant plants, c) a recombination reporter showed enhanced HR frequency in mutant calli, d) propidium iodide (PI) staining of root tips revealed an increased incidence of cell death in the meristem of mutant plants. Conclusions These results demonstrate that the aphidicolin-sensitive phenotype of osrecql4 mutants was in part due to induced DSBs and cell death, and that OsRecQl4 plays an important role as a caretaker, maintaining genome stability during DNA replication stress in the rice meristem. PMID:23586618

  4. Gene CATCHR--gene cloning and tagging for Caenorhabditis elegans using yeast homologous recombination: a novel approach for the analysis of gene expression.

    PubMed

    Sassi, Holly E; Renihan, Stephanie; Spence, Andrew M; Cooperstock, Ramona L

    2005-01-01

    Expression patterns of gene products provide important insights into gene function. Reporter constructs are frequently used to analyze gene expression in Caenorhabditis elegans, but the sequence context of a given gene is inevitably altered in such constructs. As a result, these transgenes may lack regulatory elements required for proper gene expression. We developed Gene Catchr, a novel method of generating reporter constructs that exploits yeast homologous recombination (YHR) to subclone and tag worm genes while preserving their local sequence context. YHR facilitates the cloning of large genomic regions, allowing the isolation of regulatory sequences in promoters, introns, untranslated regions and flanking DNA. The endogenous regulatory context of a given gene is thus preserved, producing expression patterns that are as accurate as possible. Gene Catchr is flexible: any tag can be inserted at any position without introducing extra sequence. Each step is simple and can be adapted to process multiple genes in parallel. We show that expression patterns derived from Gene Catchr transgenes are consistent with previous reports and also describe novel expression data. Mutant rescue assays demonstrate that Gene Catchr-generated transgenes are functional. Our results validate the use of Gene Catchr as a valuable tool to study spatiotemporal gene expression. PMID:16254074

  5. Genomic evolution in Barrett’s adenocarcinoma cells: critical roles of elevated hsRAD51, homologous recombination and Alu sequences in the genome

    PubMed Central

    Pal, J; Bertheau, R; Buon, L; Qazi, A; Batchu, RB; Bandyopadhyay, S; Ali-Fehmi, R; Beer, DG; Weaver, DW; Reis, RJ Shmookler; Goyal, RK; Huang, Q; Munshi, NC; Shammas, MA

    2012-01-01

    A prominent feature of most cancers including Barrett’s adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease. PMID:21423218

  6. Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination

    PubMed Central

    Wu, Jianbo; Hunt, Samuel D.; Xue, Haipeng; Liu, Ying; Darabi, Radbod

    2016-01-01

    Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP+ myogenic cells were sorted and characterized. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications. PMID:26729410

  7. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

    PubMed

    Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

    2015-01-01

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. PMID:26473830

  8. Small-molecule survivin inhibitor YM155 enhances radiosensitization in esophageal squamous cell carcinoma by the abrogation of G2 checkpoint and suppression of homologous recombination repair

    PubMed Central

    2014-01-01

    Background Survivin is overexpressed in cancer cells and plays a crucial role in apoptosis evasion. YM155, a small-molecule inhibitor of survivin, could enhance the cytotoxicity of various DNA-damaging agents. Here, we evaluated the radiosensitizaion potential of YM155 in human esophageal squamous cell carcinoma (ESCC). Methods Cell viability was determined by CCK8 assay. The radiosensitization effect of YM155 was evaluated by clonogenic survival and progression of tumor xenograft. Cell cycle progression was determined by flow cytometric analysis. Radiation-induced DNA double strand break (DSB) and homologous recombination repair (HRR) were detected by the staining of γ-H2AX and RAD51, respectively. Expression of survivin and cell cycle regulators was detected by Western blot analysis. Results YM155 induced radiosensitization in ESCC cell lines Eca109 and TE13, associated with the abrogation of radiation induced G2/M checkpoint, impaired Rad51 focus formation, and the prolongation of γ-H2AX signaling. G2/M transition markers, including the activation of cyclinB1/Cdc2 kinase and the suppression of Cdc2 Thr14/Tyr15 phosphorylation were induced by YM155 in irradiated cells. The combination of YM155 plus irradiation delayed the growth of ESCC tumor xenografts to a greater extent compared with either treatment modality alone. Conclusions Our findings suggest that the abrogation of G2 checkpoint and the inhibition of HRR contribute to radiosensitization by YM155 in ESCC cells. PMID:25139395

  9. DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

    PubMed Central

    Kurosawa, Aya; Saito, Shinta; So, Sairei; Hashimoto, Mitsumasa; Iwabuchi, Kuniyoshi; Watabe, Haruka; Adachi, Noritaka

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR. PMID:23967291

  10. The over-expression of the β2 catalytic subunit of the proteasome decreases homologous recombination and impairs DNA double-strand break repair in human cells.

    PubMed

    Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

    2011-01-01

    By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

  11. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    PubMed Central

    Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

    2011-01-01

    By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

  12. Expression of an Oncogenic BARD1 Splice Variant Impairs Homologous Recombination and Predicts Response to PARP-1 Inhibitor Therapy in Colon Cancer

    PubMed Central

    Ozden, Ozkan; Bishehsari, Faraz; Bauer, Jessica; Park, Seong-Hoon; Jana, Arundhati; Baik, Seung Hyun; Sporn, Judith C.; Staudacher, Jonas J.; Yazici, Cemal; Krett, Nancy; Jung, Barbara

    2016-01-01

    BRCA1-associated RING domain protein 1 (BARD1) stabilizes BRCA1 protein by forming a heterodimeric RING-RING complex, and impacts function of BRCA1, including homologous recombination (HR) repair. Although colon cancer cells usually express wild type BRCA1, presence of an oncogenic BARD1 splice variant (SV) in select cancers may render BRCA1 dysfunctional and allow cells to become sensitive to HR targeting therapies. We previously reported association of loss of full-length (FL) BARD1 with poor prognosis in colon cancer as well as expression of various BARD1 SVs with unknown function. Here we show that loss of BARD1 function through the expression of a BARD1 SV, BARD1β, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1β sensitizes colon cancer cells to poly ADP ribose polymerase 1 (PARP-1) inhibition even in a FL BRCA1 background. These results suggest that expression of BARD1β may serve as a future biomarker to assess suitability of colon cancers for HR targeting with PARP-1 inhibitors in treatment of advanced colon cancer. PMID:27197561

  13. PARP targeting counteracts gliomagenesis through induction of mitotic catastrophe and aggravation of deficiency in homologous recombination in PTEN-mutant glioma

    PubMed Central

    Majuelos-Melguizo, Jara; Rodríguez, María Isabel; López-Jiménez, Laura; Rodríguez-Vargas, Jose M.; Martí Martín-Consuegra, Juan M.; Serrano-Sáenz, Santiago; Gavard, Julie; Mariano Ruiz de Almodóvar, J; Javier Oliver, F

    2015-01-01

    Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults and one of the most aggressive cancers. PARP-1 is a nuclear protein involved in multiple facets of DNA repair and transcriptional regulation. In this study we dissected the action of PARP inhibition in different GBM cell lines with either functional or mutated PTEN that confers resistance to diverse therapies. In PTEN mutant cells, PARP inhibition induced a severe genomic instability, exacerbated homologous recombination repair (HR) deficiency and down-regulated the Spindle Assembly Checkpoint (SAC) factor BUBR1, leading to mitotic catastrophe (MC). EGFR gene amplification also represents a signature of genetic abnormality in GBM. To more effectively target GBM cells, co-treatment with a PARP inhibitor and an EGFR blocker, erlotinib, resulted in a strong suppression of ERK1/2 activation and in vivo the combined effect elicited a robust reduction in tumour development. In conclusion, PARP inhibition targets PTEN-deficient GBM cells through accentuation of SAC repression and aggravation of HR deficiency, leading to the induction of genomic instability and eventually deriving to mitotic catastrophe (MC); the inhibition of PARP and co-treatment with an inhibitor of pro-survival pathways strongly retarded in vivo gliomagenesis. PMID:25576921

  14. NuMA promotes homologous recombination repair by regulating the accumulation of the ISWI ATPase SNF2h at DNA breaks

    PubMed Central

    Vidi, Pierre-Alexandre; Liu, Jing; Salles, Daniela; Jayaraman, Swaathi; Dorfman, George; Gray, Matthew; Abad, Patricia; Moghe, Prabhas V.; Irudayaraj, Joseph M.; Wiesmüller, Lisa; Lelièvre, Sophie A.

    2014-01-01

    Chromatin remodeling factors play an active role in the DNA damage response by shaping chromatin to facilitate the repair process. The spatiotemporal regulation of these factors is key to their function, yet poorly understood. We report that the structural nuclear protein NuMA accumulates at sites of DNA damage in a poly[ADP-ribose]ylation-dependent manner and functionally interacts with the ISWI ATPase SNF2h/SMARCA5, a chromatin remodeler that facilitates DNA repair. NuMA coimmunoprecipitates with SNF2h, regulates its diffusion in the nucleoplasm and controls its accumulation at DNA breaks. Consistent with NuMA enabling SNF2h function, cells with silenced NuMA exhibit reduced chromatin decompaction after DNA cleavage, lesser focal recruitment of homologous recombination repair factors, impaired DNA double-strand break repair in chromosomal (but not in episomal) contexts and increased sensitivity to DNA cross-linking agents. These findings reveal a structural basis for the orchestration of chromatin remodeling whereby a scaffold protein promotes genome maintenance by directing a remodeler to DNA breaks. PMID:24753406

  15. Expression of an Oncogenic BARD1 Splice Variant Impairs Homologous Recombination and Predicts Response to PARP-1 Inhibitor Therapy in Colon Cancer.

    PubMed

    Ozden, Ozkan; Bishehsari, Faraz; Bauer, Jessica; Park, Seong-Hoon; Jana, Arundhati; Baik, Seung Hyun; Sporn, Judith C; Staudacher, Jonas J; Yazici, Cemal; Krett, Nancy; Jung, Barbara

    2016-01-01

    BRCA1-associated RING domain protein 1 (BARD1) stabilizes BRCA1 protein by forming a heterodimeric RING-RING complex, and impacts function of BRCA1, including homologous recombination (HR) repair. Although colon cancer cells usually express wild type BRCA1, presence of an oncogenic BARD1 splice variant (SV) in select cancers may render BRCA1 dysfunctional and allow cells to become sensitive to HR targeting therapies. We previously reported association of loss of full-length (FL) BARD1 with poor prognosis in colon cancer as well as expression of various BARD1 SVs with unknown function. Here we show that loss of BARD1 function through the expression of a BARD1 SV, BARD1β, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1β sensitizes colon cancer cells to poly ADP ribose polymerase 1 (PARP-1) inhibition even in a FL BRCA1 background. These results suggest that expression of BARD1β may serve as a future biomarker to assess suitability of colon cancers for HR targeting with PARP-1 inhibitors in treatment of advanced colon cancer. PMID:27197561

  16. SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells.

    PubMed

    Alagoz, Meryem; Katsuki, Yoko; Ogiwara, Hideaki; Ogi, Tomoo; Shibata, Atsushi; Kakarougkas, Andreas; Jeggo, Penny

    2015-09-18

    Recent studies have shown that homologous recombination (HR) requires chromatin repression as well as relaxation at DNA double strand breaks (DSBs). HP1 and SUV39H1/2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localized internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation-induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked. PMID:26206670

  17. The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

    PubMed

    Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

    2014-04-01

    Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation. PMID:24561002

  18. Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.

    PubMed

    Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

    2016-01-01

    Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP(+) myogenic cells were sorted and characterized. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications. PMID:26729410

  19. Rare recombination events generate sequence diversity among balancer chromosomes in Drosophila melanogaster

    PubMed Central

    Miller, Danny E.; Cook, Kevin R.; Yeganeh Kazemi, Nazanin; Smith, Clarissa B.; Cockrell, Alexandria J.; Hawley, R. Scott; Bergman, Casey M.

    2016-01-01

    Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, we sequenced a panel of laboratory stocks containing the most widely used X chromosome balancer, First Multiple 7 (FM7). We mapped the locations of FM7 breakpoints to precise euchromatic coordinates and identified the flanking sequence of breakpoints in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used FM7 stocks. We present evidence that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the singed gene (snX2) on FM7c with a sequence from balanced chromosomes. We propose that although double-crossover events are rare in individual crosses, many FM7c chromosomes in the Bloomington Drosophila Stock Center have lost snX2 by this mechanism on a historical timescale. Finally, we characterize the original allele of the Bar gene (B1) that is carried on FM7, and validate the hypothesis that the origin and subsequent reversion of the B1 duplication are mediated by unequal exchange. Our results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in Drosophila. PMID:26903656

  20. [Intramolecular homologous recombination event occurred in the spider egg case silk gene CySp2 of wasp spider].

    PubMed

    Han, L; Nakagaki, M

    2013-01-01

    To gain further understanding of egg case silk proteins gene family, Zhao et al. isolated two full-length cDNAs for egg case silk proteins, cylindrical silk protein 1 (CySpl) and cylindrical silk protein 2 (CySp2), from the wasp spider, Argiope bruennichi. CySp2 was reported to contain no apparent signal peptide sequences, and the CySp1-CySp2 complex, which would possess a signal peptide, would be transported across the endoplasmic reticulum and secreted to the Golgi. Genomic DNA sequencing is one approach that can be successfully utilized to retrieve 5' ends of silk genes; using this method, we retrieved the 5' end of CySp2. We found that CySp2 contained a typical signal peptide similar to that found in CySp1; thus, due to technical limitations, an artificial error had occurred in the CySp2 sequence reported by Zhao et al. PMID:25509350

  1. The yeast Shu complex utilizes homologous recombination machinery for error-free lesion bypass via physical interaction with a Rad51 paralogue.

    PubMed

    Xu, Xin; Ball, Lindsay; Chen, Wangyang; Tian, Xuelei; Lambrecht, Amanda; Hanna, Michelle; Xiao, Wei

    2013-01-01

    DNA-damage tolerance (DDT) is defined as a mechanism by which eukaryotic cells resume DNA synthesis to fill the single-stranded DNA gaps left by replication-blocking lesions. Eukaryotic cells employ two different means of DDT, namely translesion DNA synthesis (TLS) and template switching, both of which are coordinately regulated through sequential ubiquitination of PCNA at the K164 residue. In the budding yeast Saccharomyces cerevisiae, the same PCNA-K164 residue can also be sumoylated, which recruits the Srs2 helicase to prevent undesired homologous recombination (HR). While the mediation of TLS by PCNA monoubiquitination has been extensively characterized, the method by which K63-linked PCNA polyubiquitination leads to template switching remains unclear. We recently identified a yeast heterotetrameric Shu complex that couples error-free DDT to HR as a critical step of template switching. Here we report that the Csm2 subunit of Shu physically interacts with Rad55, an accessory protein involved in HR. Rad55 and Rad57 are Rad51 paralogues and form a heterodimer to promote Rad51-ssDNA filament formation by antagonizing Srs2 activity. Although Rad55-Rad57 and Shu function in the same pathway and both act to inhibit Srs2 activity, Shu appears to be dedicated to error-free DDT while the Rad55-Rad57 complex is also involved in double-strand break repair. This study reveals the detailed steps of error-free lesion bypass and also brings to light an intrinsic interplay between error-free DDT and Srs2-mediated inhibition of HR. PMID:24339919

  2. Rad51c- and Trp53-double-mutant mouse model reveals common features of homologous recombination-deficient breast cancers.

    PubMed

    Tumiati, M; Munne, P M; Edgren, H; Eldfors, S; Hemmes, A; Kuznetsov, S G

    2016-09-01

    Almost half of all hereditary breast cancers (BCs) are associated with germ-line mutations in homologous recombination (HR) genes. However, the tumor phenotypes associated with different HR genes vary, making it difficult to define the role of HR in BC predisposition. To distinguish between HR-dependent and -independent features of BCs, we generated a mouse model in which an essential HR gene, Rad51c, is knocked-out specifically in epidermal tissues. Rad51c is one of the key mediators of HR and a well-known BC predisposition gene. Here, we demonstrate that deletion of Rad51c invariably requires inactivation of the Trp53 tumor suppressor (TP53 in humans) to produce mammary carcinomas in 63% of female mice. Nonetheless, loss of Rad51c shortens the latency of Trp53-deficient mouse tumors from 11 to 6 months. Remarkably, the histopathological features of Rad51c-deficient mammary carcinomas, such as expression of hormone receptors and luminal epithelial markers, faithfully recapitulate the histopathology of human RAD51C-mutated BCs. Similar to other BC models, Rad51c/p53 double-mutant mouse mammary tumors also reveal a propensity for genomic instability, but lack the focal amplification of the Met locus or distinct mutational signatures reported for other HR genes. Using the human mammary epithelial cell line MCF10A, we show that deletion of TP53 can rescue RAD51C-deficient cells from radiation-induced cellular senescence, whereas it exacerbates their centrosome amplification and nuclear abnormalities. Altogether, our data indicate that a trend for genomic instability and inactivation of Trp53 are common features of HR-mediated BCs, whereas histopathology and somatic mutation patterns are specific for different HR genes. PMID:26820992

  3. Inhibition of Homologous Recombination and Promotion of Mutagenic Repair of DNA Double-Strand Breaks Underpins Arabinoside-Nucleoside Analogue Radiosensitization.

    PubMed

    Magin, Simon; Papaioannou, Maria; Saha, Janapriya; Staudt, Christian; Iliakis, George

    2015-06-01

    In concurrent chemoradiotherapy, drugs are used to sensitize tumors to ionizing radiation. Although a spectrum of indications for simultaneous treatment with drugs and radiation has been defined, the molecular mechanisms underpinning tumor radiosensitization remain incompletely characterized for several such combinations. Here, we investigate the mechanisms of radiosensitization by the arabinoside nucleoside analogue 9-β-D-arabinofuranosyladenine (araA) placing particular emphasis on the repair of DNA double-strand breaks (DSB), and compare the results to those obtained with fludarabine (F-araA) and cytarabine (araC). Postirradiation treatment with araA strongly sensitizes cells to ionizing radiation, but leaves unchanged DSB repair by NHEJ in logarithmically growing cells, in sorted G1 or G2 phase populations, as well as in cells in the plateau phase of growth. Notably, araA strongly inhibits DSB repair by homologous recombination (HRR), as assessed by scoring ionizing radiation-induced RAD51 foci, and in functional assays using integrated reporter constructs. Cells compromised in HRR by RNAi-mediated transient knockdown of RAD51 show markedly reduced radiosensitization after treatment with araA. Remarkably, mutagenic DSB repair compensates for HRR inhibition in araA-treated cells. Compared with araA, F-araA and araC are only modestly radiosensitizing under the conditions examined. We propose that the radiosensitizing potential of nucleoside analogues is linked to their ability to inhibit HRR and concomitantly promote the error-prone processing of DSBs. Our observations pave the way to treatment strategies harnessing the selective inhibitory potential of nucleoside analogues and the development of novel compounds specifically utilizing HRR inhibition as a means of tumor cell radiosensitization. PMID:25840584

  4. Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

    SciTech Connect

    Zheng Zhiming; Wang Ping; Wang Hongyan; Zhang Xiangming; Wang Minli; Cucinotta, Francis A.; Wang Ya

    2013-02-01

    Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials: Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.

  5. Activity of CEP-9722, a poly (ADP-ribose) polymerase inhibitor, in urothelial carcinoma correlates inversely with homologous recombination repair response to DNA damage.

    PubMed

    Jian, Weiguo; Xu, Hua-Guo; Chen, Jianfeng; Xu, Zhi-Xiang; Levitt, Jonathan M; Stanley, Jennifer A; Yang, Eddy S; Lerner, Seth P; Sonpavde, Guru

    2014-09-01

    As loss of DNA-repair proteins is common in urothelial carcinoma (UC), a rationale can be made to evaluate the activity of poly (ADP-ribose) polymerase (PARP) inhibitors to exploit synthetic lethality. We aimed to preclinically evaluate a PARP inhibitor, CEP-9722, and its active metabolite, CEP-8983, in UC. The activity of CEP-8983 was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay against human UC cell lines. Flow cytometry, COMET assay, and western blot were performed to assess apoptosis, DNA damage, and DNA-repair proteins, respectively. RT4 xenografts received placebo or CEP-9722 (100 or 200 mg/kg/day) orally. Xenografts were subjected to immunohistochemistry for apoptosis [cleaved caspase (cc)-3] and angiogenesis (CD31). CEP-8983 (1 μmol/l) reduced the viability of RT4 and T24 cells by 20%, but did not reduce the viability of 5637 and TCC-SUP cells. Apoptosis and necrosis occurred in 9.7 and 9.1% of RT4 and 5637 cells, respectively. RT4 cells showed greater DNA damage compared with 5637 cells. Increased DNA damage occurred with combination versus CEP-8983 or cisplatin alone in RT4 and 5637 cells. T24 and RT4 showed the least RAD51 foci 8 h following radiation, whereas TCC-SUP and 5637 robustly induced RAD51 foci. CEP-9722 showed dose-dependent antitumor activity in RT4 xenografts; 200 mg/kg daily was better than control (P=0.04) and 100 mg/kg was not (P=0.26). Immunohistochemistry of xenografts showed a significant increase in cc-3 and decrease in CD31 with both doses (P<0.05). Biomarker-driven evaluation of PARP inhibitors in UC is justified as the activity of CEP-9722 correlated inversely with homologous recombination repair response to DNA damage. PMID:24714082

  6. High Quality Maize Centromere 10 Sequence Reveals Evidence of Frequent Recombination Events

    PubMed Central

    Wolfgruber, Thomas K.; Nakashima, Megan M.; Schneider, Kevin L.; Sharma, Anupma; Xie, Zidian; Albert, Patrice S.; Xu, Ronghui; Bilinski, Paul; Dawe, R. Kelly; Ross-Ibarra, Jeffrey; Birchler, James A.; Presting, Gernot G.

    2016-01-01

    The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR) has presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here, we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 × 10−6 and 5 × 10−5 for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb from the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length CR from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB) repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. In many cases examined here, DSB repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to efficiently repair frequent DSBs in centromeres. PMID:27047500

  7. High Quality Maize Centromere 10 Sequence Reveals Evidence of Frequent Recombination Events.

    PubMed

    Wolfgruber, Thomas K; Nakashima, Megan M; Schneider, Kevin L; Sharma, Anupma; Xie, Zidian; Albert, Patrice S; Xu, Ronghui; Bilinski, Paul; Dawe, R Kelly; Ross-Ibarra, Jeffrey; Birchler, James A; Presting, Gernot G

    2016-01-01

    The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR) has presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here, we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 × 10(-6) and 5 × 10(-5) for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb from the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length CR from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB) repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. In many cases examined here, DSB repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to efficiently repair frequent DSBs in centromeres. PMID:27047500

  8. Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence.

    PubMed

    Gladyshev, Eugene; Kleckner, Nancy

    2016-05-01

    Haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5'-GAC-3' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous

  9. Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence

    PubMed Central

    Kleckner, Nancy

    2016-01-01

    Haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5'-GAC-3' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous

  10. PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events.

    PubMed

    Mendes, Rui D; Sarmento, Leonor M; Canté-Barrett, Kirsten; Zuurbier, Linda; Buijs-Gladdines, Jessica G C A M; Póvoa, Vanda; Smits, Willem K; Abecasis, Miguel; Yunes, J Andres; Sonneveld, Edwin; Horstmann, Martin A; Pieters, Rob; Barata, João T; Meijerink, Jules P P

    2014-07-24

    Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression. PMID:24904117

  11. Recombinative events of the T cell antigen receptor delta gene in peripheral T cell lymphomas.

    PubMed Central

    Kanavaros, P; Farcet, J P; Gaulard, P; Haioun, C; Divine, M; Le Couedic, J P; Lefranc, M P; Reyes, F

    1991-01-01

    Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation. Images PMID:1991851

  12. Computational analysis of four human adenovirus type 4 genomes reveals molecular evolution through two interspecies recombination events

    PubMed Central

    Dehghan, Shoaleh; Seto, Jason; Liu, Elizabeth B.; Walsh, Michael P.; Dyer, David W.; Chodosh, James; Seto, Donald

    2013-01-01

    Computational analysis of human adenovirus type 4 (HAdV-E4), a pathogen that is the only HAdV member of species E, provides insights into its zoonotic origin and molecular adaptation. Its genome encodes a domain of the major capsid protein, hexon, from HAdV-B16 recombined into the genome chassis of a simian adenovirus. Genomes of two recent field strains provide a clue to its adaptation to the new host: recombination of a NF-I binding site motif, which is required for efficient viral replication, from another HAdV genome. This motif is absent in the chimpanzee adenoviruses and the HAdV-E4 prototype, but is conserved amongst other HAdVs. This is the first report of an interspecies recombination event for HAdVs, and the first documentation of a lateral partial gene transfer from a chimpanzee AdV. The potential for such recombination events are important when considering chimpanzee adenoviruses as candidate gene delivery vectors for human patients. PMID:23763770

  13. Genome Sequence Analysis of New Isolates of the Winona Strain of Plum pox virus and the First Definitive Evidence of Intrastrain Recombination Events.

    PubMed

    James, Delano; Sanderson, Dan; Varga, Aniko; Sheveleva, Anna; Chirkov, Sergei

    2016-04-01

    Plum pox virus (PPV) is genetically diverse with nine different strains identified. Mutations, indel events, and interstrain recombination events are known to contribute to the genetic diversity of PPV. This is the first report of intrastrain recombination events that contribute to PPV's genetic diversity. Fourteen isolates of the PPV strain Winona (W) were analyzed including nine new strain W isolates sequenced completely in this study. Isolates of other strains of PPV with more than one isolate with the complete genome sequence available in GenBank were included also in this study for comparison and analysis. Five intrastrain recombination events were detected among the PPV W isolates, one among PPV C strain isolates, and one among PPV M strain isolates. Four (29%) of the PPV W isolates analyzed are recombinants; one of which (P2-1) is a mosaic, with three recombination events identified. A new interstrain recombinant event was identified between a strain M isolate and a strain Rec isolate, a known recombinant. In silico recombination studies and pairwise distance analyses of PPV strain D isolates indicate that a threshold of genetic diversity exists for the detectability of recombination events, in the range of approximately 0.78×10(-2) to 1.33×10(-2) mean pairwise distance. RDP4 analyses indicate that in the case of PPV Rec isolates there may be a recombinant breakpoint distinct from the obvious transition point of strain sequences. Evidence was obtained that indicates that the frequency of PPV recombination is underestimated, which may be true for other RNA viruses where low genetic diversity exists. PMID:26667187

  14. Inhibition of protein phosphatase 2A radiosensitizes pancreatic cancers by modulating CDC25C/CDK1 and homologous recombination repair

    PubMed Central

    Wei, Dongping; Parsels, Leslie A.; Karnak, David; Davis, Mary A.; Parsels, Joshua D.; Zhao, Lili; Maybaum, Jonathan; Lawrence, Theodore S.; Sun, Yi; Morgan, Meredith A.

    2013-01-01

    Purpose To identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and thus improve survival, we performed an siRNA library screen in pancreatic cancer cells. We investigated PPP2R1A, a scaffolding subunit of protein phosphatase 2A (PP2A) as a lead radiosensitizing target. Experimental Design We determined the effect of PP2A inhibition by genetic (PPP2R1A siRNA) and pharmacological (LB100, a small molecule entering Phase I clinical trials) approaches on radiosensitization of Panc-1 and MiaPaCa-2 pancreatic cancer cells both in vitro and in vivo. Results PPP2R1A depletion by siRNA radiosensitized Panc-1 and MiaPaCa-2 cells, with radiation enhancement ratios of 1.4 (P<0.05). Likewise, LB100 produced similar radiosensitization in pancreatic cancer cells, but minimal radiosensitization in normal small intestinal cells. Mechanistically, PPP2R1A siRNA or LB100 caused aberrant CDK1 activation, likely resulting from accumulation of the active forms of PLK1 (pPLK1 T210) and CDC25C (pCDC25C T130). Furthermore, LB100 inhibited radiation-induced Rad51 focus formation and homologous recombination repair (HRR), ultimately leading to persistent radiation-induced DNA damage, as reflected by γH2AX expression. Finally, we identified CDC25C as a key PP2A substrate involved in LB100-mediated radiosensitization as depletion of CDC25C partially reversed LB100-mediated radiosensitization. In a mouse xenograft model of human pancreatic cancer, LB100 produced significant radiosensitization with minimal weight loss. Conclusions Collectively, our data demonstrate that PP2A inhibition radiosensitizes pancreatic cancer both in vitro and in vivo via activation of CDC25C/CDK1 and inhibition of HRR, and provide proof-of-concept evidence that PP2A is a promising target for the improvement of local therapy in pancreatic cancer. PMID:23780887

  15. Rad18 and Rnf8 facilitate homologous recombination by two distinct mechanisms, promoting Rad51 focus formation and suppressing the toxic effect of nonhomologous end joining.

    PubMed

    Kobayashi, S; Kasaishi, Y; Nakada, S; Takagi, T; Era, S; Motegi, A; Chiu, R K; Takeda, S; Hirota, K

    2015-08-13

    The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at γ-ray-induced DSB sites. To analyze functional interactions between the three enzymes, we created RAD18(-/-), RNF8(-/-), RAD18(-/-)/RNF8(-/-) and UBC13(-/-)clones in chicken DT40 cells. To assess the capability of HR, we measured the cellular sensitivity to camptothecin (topoisomerase I poison) and olaparib (poly(ADP ribose)polymerase inhibitor) because these chemotherapeutic agents induce DSBs during DNA replication, which are repaired exclusively by HR. RAD18(-/-), RNF8(-/-) and RAD18(-/-)/RNF8(-/-) clones showed very similar levels of hypersensitivity, indicating that Rad18 and Rnf8 operate in the same pathway in the promotion of HR. Although these three mutants show less prominent defects in the formation of Rad51 foci than UBC13(-/-)cells, they are more sensitive to camptothecin and olaparib than UBC13(-/-)cells. Thus, Rad18 and Rnf8 promote HR-dependent repair in a manner distinct from Ubc13. Remarkably, deletion of Ku70, a protein essential for nonhomologous end joining (NHEJ) significantly restored tolerance of RAD18(-/-) and RNF8(-/-) cells to camptothecin and olaparib without affecting Rad51 focus formation. Thus, in cellular tolerance to the chemotherapeutic agents, the two enzymes collaboratively promote DSB repair by HR by suppressing the toxic effect of NHEJ on HR rather than enhancing Rad51 focus formation. In contrast, following exposure to γ-rays, RAD18(-/-), RNF8(-/-), RAD18(-/-)/RNF8(-/-) and UBC13(-/-)cells showed close correlation between cellular survival and Rad51 focus formation at DSB sites. In summary, the current study reveals that Rad18 and Rnf8 facilitate HR by two distinct mechanisms: suppression of the toxic effect of NHEJ on HR during DNA replication and the promotion of Rad51 focus formation at radiotherapy

  16. DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

    SciTech Connect

    Urushihara, Yusuke; Kobayashi, Junya; Matsumoto, Yoshihisa; Komatsu, Kenshi; Oda, Shoji; Mitani, Hiroshi

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. Black-Right-Pointing-Pointer A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. Black-Right-Pointing-Pointer DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. Black-Right-Pointing-Pointer DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. Black-Right-Pointing-Pointer DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after {gamma}-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of {gamma}H2AX foci after {gamma}-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of {gamma}H2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after {gamma}-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the

  17. Downregulation of Homologous Recombination DNA Repair Genes by HDAC Inhibition in Prostate Cancer Is Mediated through the E2F1 Transcription Factor

    PubMed Central

    Kachhap, Sushant K.; Rosmus, Nadine; Collis, Spencer J.; Kortenhorst, Madeleine S. Q.; Wissing, Michel D.; Hedayati, Mohammad; Shabbeer, Shabana; Mendonca, Janet; Deangelis, Justin; Marchionni, Luigi; Lin, Jianqing; Höti, Naseruddin; Nortier, Johan W. R.; DeWeese, Theodore L.; Hammers, Hans; Carducci, Michael A.

    2010-01-01

    Background Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. Methodology/Principal Findings Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. Conclusions/Significance Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could

  18. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  19. A novel third type of recurrent NF1 microdeletion mediated by nonallelic homologous recombination between LRRC37B-containing low-copy repeats in 17q11.2.

    PubMed

    Bengesser, Kathrin; Cooper, David N; Steinmann, Katharina; Kluwe, Lan; Chuzhanova, Nadia A; Wimmer, Katharina; Tatagiba, Marcos; Tinschert, Sigrid; Mautner, Victor-Felix; Kehrer-Sawatzki, Hildegard

    2010-06-01

    Large microdeletions encompassing the neurofibromatosis type-1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type-1) span 1.4-Mb and have breakpoints located within NF1-REPs A and C, low-copy repeats (LCRs) containing LRRC37-core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by nonallelic homologous recombination (NAHR) between the highly homologous NF1-REPs B and C. The breakpoints of these approximately 1.0-Mb ("type-3") NF1 deletions were characterized at the DNA sequence level in three unrelated patients. Recombination regions, spanning 275, 180, and 109-bp, respectively, were identified within the LRRC37B-P paralogues of NF1-REPs B and C, and were found to contain sequences capable of non-B DNA formation. Both LCRs contain LRRC37-core duplicons, abundant and highly dynamic sequences in the human genome. NAHR between LRRC37-containing LCRs at 17q21.31 is known to have mediated the 970-kb polymorphic inversions of the MAPT-locus that occurred independently in different primate species, but also underlies the syndromes associated with recurrent 17q21.31 microdeletions and reciprocal microduplications. The novel NF1 microdeletions reported here provide further evidence for the unusually high recombinogenic potential of LRRC37-containing LCRs in the human genome. PMID:20506354

  20. Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure

    PubMed Central

    Yang, Darren; Boyer, Benjamin; Prévost, Chantal; Danilowicz, Claudia; Prentiss, Mara

    2015-01-01

    RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results. PMID:26384422

  1. Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure.

    PubMed

    Yang, Darren; Boyer, Benjamin; Prévost, Chantal; Danilowicz, Claudia; Prentiss, Mara

    2015-12-01

    RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results. PMID:26384422

  2. DNA origami based visualization system for studying site-specific recombination events.

    PubMed

    Suzuki, Yuki; Endo, Masayuki; Katsuda, Yousuke; Ou, Keiyu; Hidaka, Kumi; Sugiyama, Hiroshi

    2014-01-01

    Site-specific recombination involves reciprocal exchange between defined DNA sites. The reaction initiates from the formation of a recombinase-DNA synaptic complex, in which two recombination sites arrange in an appropriate configuration. However, there is incomplete information about how the topological state of the substrate influences the synapsis and outcome of the reaction. Here, we show that Cre-mediated recombination can be regulated by controlling the orientation and topology of the loxP substrate in a DNA frame nanoscaffold. High-speed atomic force microscopy analyses revealed that the loxP-containing substrate strands in the antiparallel orientation can be recombined only through formation of synaptic complexes. By tethering Holliday junction (HJ) intermediates to DNA frames in different connection patterns and using them as a starting substrate, we found that the topological state of the HJ intermediates dictates the outcome of the resolution. Our approach should provide a new platform for structural-functional studies of various DNA targeting enzymes, especially which require formation of synaptic complexes. PMID:24328161

  3. The DNA structure and sequence preferences of WRN underlie its function in telomeric recombination events

    PubMed Central

    Edwards, Deanna N.; Machwe, Amrita; Chen, Li; Bohr, Vilhelm A.; Orren, David K.

    2015-01-01

    Telomeric abnormalities caused by loss of function of the RecQ helicase WRN are linked to the multiple premature ageing phenotypes that characterize Werner syndrome. Here we examine WRN's role in telomeric maintenance, by comparing its action on a variety of DNA structures without or with telomeric sequences. Our results show that WRN clearly prefers to act on strand invasion intermediates in a manner that favours strand invasion and exchange. Moreover, WRN unwinding of these recombination structures is further enhanced when the invading strand contains at least three G-rich single-stranded telomeric repeats. These selectivities are most pronounced at NaCl concentrations within the reported intranuclear monovalent cation concentration range, and are partly conferred by WRN's C-terminal region. Importantly, WRN's specificity for the G-rich telomeric sequence within this precise structural context is particularly relevant to telomere metabolism and strongly suggests a physiological role in telomeric recombination processes, including T-loop dynamics. PMID:26420422

  4. Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event

    SciTech Connect

    Kroeckel, L.; Focht, D.D.

    1987-10-01

    Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-0, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absences of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene of 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant-unlike it parent, R5-3-did not grown on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown in 4-methylbenzoate than in the same cells grown on benzene. Benzene was metabolized through the MP pathway in CB1-9, while chlorobenzene was metabolized through the OP pathway.

  5. Graph Model of Coalescence with Recombinations

    NASA Astrophysics Data System (ADS)

    Parida, Laxmi

    One of the primary genetic events shaping an autosomal chromosome is recombination. This is a process that occurs during meiosis, in eukaryotes, that results in the offsprings having different combinations of (homologous) genes, or chromosomal segments, of the two parents. The presence of these recombination events in the evolutionary history of each chromosome complicates the genetic landscape of a population, and understanding the manifestations of these genetic exchanges in the chromosome sequences has been a subject of intense curiosity (see [Hud83, Gri99, HSW05] and citations therein).

  6. Characterization of RAD51-Independent Break-Induced Replication That Acts Preferentially with Short Homologous Sequences

    PubMed Central

    Ira, Grzegorz; Haber, James E.

    2002-01-01

    Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase. PMID:12192038

  7. A replication-competent retrovirus arising from a split-function packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences.

    PubMed

    Chong, H; Starkey, W; Vile, R G

    1998-04-01

    Previously we reported the presence of a replication-competent retrovirus in supernatant from a vector-producing line derived from a widely used split-function amphotropic packaging cell line. Rigorous routine screening of all retroviral stocks produced in our laboratory has not, previously or since, indicated the presence of such a virus. Replication-competent retroviruses have never previously been used in our laboratory, and stringent screening of all routinely used cell lines has not revealed the presence of any helper viruses. Therefore, it is highly unlikely that this virus represents an adventitious cross-contaminant or had been imported unknowingly with our cell line stocks. PCR studies with DNA from infected cell lines and Northern blot analysis and reverse transcriptase PCR with RNA from infected cells suggest that the helper virus arose by recombination events, at sites of partial homology, between sequences in the vector, one of the packaging constructs, and endogenous retroviral elements. These recombinations were not present in stocks of the packaging cell line or in an initial stock of the vector-producing line, indicating that these events occurred while the vector-producing line was being passaged for harvest of supernatant stocks. PMID:9525583

  8. Genomic Investigation Reveals Highly Conserved, Mosaic, Recombination Events Associated with Capsular Switching among Invasive Neisseria meningitidis Serogroup W Sequence Type (ST)-11 Strains

    PubMed Central

    Mustapha, Mustapha M.; Marsh, Jane W.; Krauland, Mary G.; Fernandez, Jorge O.; de Lemos, Ana Paula S.; Hotopp, Julie C. Dunning; Wang, Xin; Mayer, Leonard W.; Lawrence, Jeffrey G.; Hiller, N. Luisa; Harrison, Lee H.

    2016-01-01

    Neisseria meningitidis is an important cause of meningococcal disease globally. Sequence type (ST)-11 clonal complex (cc11) is a hypervirulent meningococcal lineage historically associated with serogroup C capsule and is believed to have acquired the W capsule through a C to W capsular switching event. We studied the sequence of capsule gene cluster (cps) and adjoining genomic regions of 524 invasive W cc11 strains isolated globally. We identified recombination breakpoints corresponding to two distinct recombination events within W cc11: A 8.4-kb recombinant region likely acquired from W cc22 including the sialic acid/glycosyl-transferase gene, csw resulted in a C→W change in capsular phenotype and a 13.7-kb recombinant segment likely acquired from Y cc23 lineage includes 4.5 kb of cps genes and 8.2 kb downstream of the cps cluster resulting in allelic changes in capsule translocation genes. A vast majority of W cc11 strains (497/524, 94.8%) retain both recombination events as evidenced by sharing identical or very closely related capsular allelic profiles. These data suggest that the W cc11 capsular switch involved two separate recombination events and that current global W cc11 meningococcal disease is caused by strains bearing this mosaic capsular switch. PMID:27289093

  9. Alcohol homologation

    DOEpatents

    Wegman, Richard W.; Moloy, Kenneth G.

    1988-01-01

    A process for the homologation of an alkanol by reaction with synthesis gas in contact with a system containing rhodium atom, ruthenium atom, iodine atom and a bis(diorganophosphino) alkane to selectivity produce the next higher homologue.

  10. Alcohol homologation

    DOEpatents

    Wegman, R.W.; Moloy, K.G.

    1988-02-23

    A process is described for the homologation of an alkanol by reaction with synthesis gas in contact with a system containing rhodium atom, ruthenium atom, iodine atom and a bis(diorganophosphino) alkane to selectivity produce the next higher homologue.

  11. Vaccine protection against lethal homologous and heterologous challenge using recombinant AAV vectors expressing codon-optimized genes from pandemic swine origin influenza virus (SOIV).

    PubMed

    Sipo, Isaac; Knauf, Mathias; Fechner, Henry; Poller, Wolfgang; Planz, Oliver; Kurth, Reinhard; Norley, Stephen

    2011-02-11

    The recent H1N1 influenza pandemic and the inevitable delay between identification of the virus and production of the specific vaccine have highlighted the urgent need for new generation influenza vaccines that can preemptively induce broad immunity to different strains of the virus. In this study we have produced AAV-based vectors expressing the A/Mexico/4603/2009 (H1N1) hemagglutinin (HA), nucleocapsid (NP) and the matrix protein M1 and have evaluated their ability to induce specific immune response and protect mice against homologous and heterologous challenge. Each of the vaccine vectors elicited potent cellular and humoral immune responses in mice. Although immunization with AAV-M1 did not improve survival after challenge with the homologous strain, immunization with the AAV-H1 and AAV-NP vectors resulted in survival of all mice, as did inoculation with a combination of all three vectors. Furthermore, trivalent vaccination also conferred partial protection against challenge with the highly heterologous and virulent A/PR/8/34 strain of H1N1 influenza. PMID:21195079

  12. NRAGE is involved in homologous recombination repair to resist the DNA-damaging chemotherapy and composes a ternary complex with RNF8-BARD1 to promote cell survival in squamous esophageal tumorigenesis.

    PubMed

    Yang, Q; Pan, Q; Li, C; Xu, Y; Wen, C; Sun, F

    2016-08-01

    NRAGE, a neurotrophin receptor-interacting melanoma antigen-encoding gene homolog, is significantly increased in the nucleus of radioresistant esophageal tumor cell lines and is highly upregulated to promote cell proliferation in esophageal carcinomas (ECs). However, whether the overexpressed NRAGE promotes cell growth by participating in DNA-damage response (DDR) is still unclear. Here we show that NRAGE is required for efficient double-strand breaks (DSBs) repair via homologous recombination repair (HRR) and downregulation of NRAGE greatly sensitizes EC cells to DNA-damaging agents both in vitro and in vivo. Moreover, NRAGE not only regulates the stability of DDR factors, RNF8 and BARD1, in a ubiquitin-proteolytic pathway, but also chaperons the interaction between BARD1 and RNF8 via their RING domains to form a novel ternary complex. Additionally, the expression of NRAGE is closely correlated with RNF8 and BARD1 in esophageal tumor tissues. In summary, our findings reveal a novel function of NRAGE that will help to guide personalized esophageal cancer treatments by targeting NRAGE to increase cell sensitivity to DNA-damaging therapeutics in the long run. PMID:27035619

  13. DNA-PK Phosphorylation of RPA32 Ser4/Ser8 Regulates Replication Stress Checkpoint Activation, Fork Restart, Homologous Recombination and Mitotic Catastrophe

    PubMed Central

    Ashley, Amanda K.; Shrivastav, Meena; Nie, Jingyi; Amerin, Courtney; Troksa, Kyle; Glanzer, Jason G.; Liu, Shengqin; Opiyo, Stephen O.; Dimitrova, Diana D.; Le, Phuong; Sishc, Brock; Bailey, Susan M.; Oakley, Greg G.; Nickoloff, Jac A.

    2014-01-01

    Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress. PMID:24819595

  14. The human and rat recombinant receptors for advanced glycation end products have a high degree of homology but different pharmacokinetic properties in rats.

    PubMed

    Renard, C; Chappey, O; Wautier, M P; Nagashima, M; Morser, J; Scherrmann, J M; Wautier, J L

    1999-09-01

    The accelerated formation of advanced glycation end products (AGEs) is implicated in diabetic microvascular and macrovascular complications. The binding of AGEs to their cellular surface receptor (RAGE) induces vascular dysfunction and in particular an increase in vascular permeability. We previously demonstrated that rat recombinant RAGE (rR-RAGE) produced in insect cells corrected the hyperpermeability due to RAGE-AGE interaction and that pharmacokinetic properties of rR-RAGE after i.v. administration in rats were compatible with a potential therapeutic use. In the present study, we showed that recombinant human RAGE (rH-RAGE) had a similar efficacy in inhibiting AGE-induced endothelial alteration and in reducing the hyperpermeability observed in streptozotocin-induced diabetic rats. (125)I-rH-RAGE elimination half-life after i.v. administration was similar in diabetic and normal rats (53.7 +/- 7.6 and 45.3 +/- 4.0 h, respectively). The presence of AGEs is responsible for a higher distribution volume in diabetic rats compared with normal rats (15.3 +/- 2.7 and 7.7 +/- 0. 7 l/kg, respectively). Immunoreactive (125)I-rH-RAGE decreased more rapidly than did immunoreactive (125)I-rR-RAGE. The differences between (125)I-rH-RAGE and (125)I-rR-RAGE pharmacokinetics in rat may be related to differences in potential O-glycosylation and protease cleavage sites between the two RAGE molecules. PMID:10454525

  15. Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination*

    PubMed Central

    Yan, Yi; Pieretti, Joyce; Ju, Zhongliang; Wei, Shiniu; Christin, John R.; Bah, Fatmata; Birshtein, Barbara K.; Eckhardt, Laurel A.

    2011-01-01

    Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3′ regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established “pairs” of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3′ regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR. PMID:21673112

  16. Regulation of recombinant human insulin-induced maturational events in Clarias batrachus (L.) oocytes in vitro.

    PubMed

    Hajra, Sudip; Das, Debabrata; Ghosh, Pritha; Pal, Soumojit; Nath, Poulomi; Maitra, Sudipta

    2016-04-01

    Regulation of insulin-mediated resumption of meiotic maturation in catfish oocytes was investigated. Insulin stimulation of post-vitellogenic oocytes promotes the synthesis of cyclin B, histone H1 kinase activation and a germinal vesicle breakdown (GVBD) response in a dose-dependent and duration-dependent manner. The PI3K inhibitor wortmannin abrogates recombinant human (rh)-insulin action on histone H1 kinase activation and meiotic G2-M1 transition in denuded and follicle-enclosed oocytes in vitro. While the translational inhibitor cycloheximide attenuates rh-insulin action, priming with transcriptional blocker actinomycin D prevents insulin-stimulated maturational response appreciably, albeit in low amounts. Compared with rh-insulin, human chorionic gonadotrophin (hCG) stimulation of follicle-enclosed oocytes in vitro triggers a sharp increase in 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP) secreted in the incubation medium at 12 h. Interestingly, the insulin, but not the hCG-induced, maturational response shows less susceptibility to steroidogenesis inhibitors, trilostane or dl-aminoglutethimide. In addition, priming with phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) or cell-permeable dbcAMP or adenylyl cyclase activator forskolin reverses the action of insulin on meiotic G2-M1 transition. Conversely, the adenylyl cyclase inhibitor, SQ 22536, or PKA inhibitor H89 promotes the resumption of meiosis alone and further potentiates the GVBD response in the presence of rh-insulin. Furthermore, insulin-mediated meiotic maturation involves the down-regulation of endogenous protein kinase A (PKA) activity in a manner sensitive to PI3K activation, suggesting potential involvement of a cross-talk between cAMP/PKA and insulin-mediated signalling cascade in catfish oocytes in vitro. Taken together, these results suggest that rh-insulin regulation of the maturational response in C. batrachus oocytes involves down-regulation of PKA, synthesis of cyclin

  17. High-Resolution Mapping of Crossover and Non-crossover Recombination Events by Whole-Genome Re-sequencing of an Avian Pedigree

    PubMed Central

    Qvarnström, Anna; Ellegren, Hans

    2016-01-01

    Recombination is an engine of genetic diversity and therefore constitutes a key process in evolutionary biology and genetics. While the outcome of crossover recombination can readily be detected as shuffled alleles by following the inheritance of markers in pedigreed families, the more precise location of both crossover and non-crossover recombination events has been difficult to pinpoint. As a consequence, we lack a detailed portrait of the recombination landscape for most organisms and knowledge on how this landscape impacts on sequence evolution at a local scale. To localize recombination events with high resolution in an avian system, we performed whole-genome re-sequencing at high coverage of a complete three-generation collared flycatcher pedigree. We identified 325 crossovers at a median resolution of 1.4 kb, with 86% of the events localized to <10 kb intervals. Observed crossover rates were in excellent agreement with data from linkage mapping, were 52% higher in male (3.56 cM/Mb) than in female meiosis (2.28 cM/Mb), and increased towards chromosome ends in male but not female meiosis. Crossover events were non-randomly distributed in the genome with several distinct hot-spots and a concentration to genic regions, with the highest density in promoters and CpG islands. We further identified 267 non-crossovers, whose location was significantly associated with crossover locations. We detected a significant transmission bias (0.18) in favour of ‘strong’ (G, C) over ‘weak’ (A, T) alleles at non-crossover events, providing direct evidence for the process of GC-biased gene conversion in an avian system. The approach taken in this study should be applicable to any species and would thereby help to provide a more comprehensive portray of the recombination landscape across organism groups. PMID:27219623

  18. High-Resolution Mapping of Crossover and Non-crossover Recombination Events by Whole-Genome Re-sequencing of an Avian Pedigree.

    PubMed

    Smeds, Linnéa; Mugal, Carina F; Qvarnström, Anna; Ellegren, Hans

    2016-05-01

    Recombination is an engine of genetic diversity and therefore constitutes a key process in evolutionary biology and genetics. While the outcome of crossover recombination can readily be detected as shuffled alleles by following the inheritance of markers in pedigreed families, the more precise location of both crossover and non-crossover recombination events has been difficult to pinpoint. As a consequence, we lack a detailed portrait of the recombination landscape for most organisms and knowledge on how this landscape impacts on sequence evolution at a local scale. To localize recombination events with high resolution in an avian system, we performed whole-genome re-sequencing at high coverage of a complete three-generation collared flycatcher pedigree. We identified 325 crossovers at a median resolution of 1.4 kb, with 86% of the events localized to <10 kb intervals. Observed crossover rates were in excellent agreement with data from linkage mapping, were 52% higher in male (3.56 cM/Mb) than in female meiosis (2.28 cM/Mb), and increased towards chromosome ends in male but not female meiosis. Crossover events were non-randomly distributed in the genome with several distinct hot-spots and a concentration to genic regions, with the highest density in promoters and CpG islands. We further identified 267 non-crossovers, whose location was significantly associated with crossover locations. We detected a significant transmission bias (0.18) in favour of 'strong' (G, C) over 'weak' (A, T) alleles at non-crossover events, providing direct evidence for the process of GC-biased gene conversion in an avian system. The approach taken in this study should be applicable to any species and would thereby help to provide a more comprehensive portray of the recombination landscape across organism groups. PMID:27219623

  19. Quantitation of Radiation Induced Deletion and Recombination Events Associated with Repeated DNA Sequences

    NASA Technical Reports Server (NTRS)

    Sinden, Richard R.

    1999-01-01

    Manned exploration of space exposes the explorers to a complex and novel radiation environment. The galactic cosmic ray and trapped belt radiation (predominantly proton) components of this environment are relatively constant, and the variations with the solar cycle are well understood and predictable. The level of radiation encountered in low earth orbits is determined by several factors, including altitude, inclination of orbit with respect to the equator, and spacecraft shielding. At higher altitudes, and on a Mars mission, the level of radiation exposure will increase significantly. A significant fraction of the dose may be delivered by solar particle events which vary dramatically in dose rate and incident particle spectrum. High-LET radiation is of particular concern. High-LET radiation, a component of galactic cosmic rays (GCR), is comprised of a variety of charged particles of various energies (10 MeV/n to 10 GeV/n), including about 87% photons, 12% helium ions, and heavy ions (including iron). These high energy particles can cause significant damage to target cells. The different particle types and energies result in different patterns of energy deposition at the molecular and cellular level in a primary target cell. They can also cause significant damage to other, nearby cells as a result of secondary particles. Protons, for instance produce secondaries that include photons, neutrons, pions, heavy particles, as well as gamma rays. Heavy ions deposit energy in a "track" in which the magnitude of the damage varies as the particle loses energy. Heavy ions produce secondary delta rays, or electrons. The distribution of damage through tissue is described by a Bragg curve which will be characteristic for different energies. Needless to say there are differences in the RBE of protons and a particles. High-LET heavy ions are particularly damaging to cells as they do continual damage throughout their track. Differences in these energy deposition patterns can

  20. The effective expression of xylanase gene in Candida utilis by 18S rDNA targeted homologous recombination in pGLR9K.

    PubMed

    Wei, Wang; Hong-Lan, Yang; HuiFang, Bao; Daoyuan, Zhang; Qi-mu-ge, Shan; Woof, Andrew J

    2010-07-01

    In order to test whether 18S rDNA can influence positively xylanase gene effective expression in the yeast of Candida utilis, a targeting vector pGLR9K-XA was constructed by adding an interested gene xynA from Streptomyces olivaceoviridis into the vector pGLR9K which is constructed by ourselves. pGLR9K contains the 18S rDNA, GAP promoter and CYH resistance gene sequence, all of which is from C. utilis. Then the vector pGLR9K-XA was transformed into C. utilis. To test the vector and transformed system, PCR, Southern blot and DNS methods were used. The results showed that xylanase gene can be detected in the chromosome DNA of recombinant C. utilis and the enzyme activity of xylanase is up to 60 IU ml(-1) in the study. It is suggested that this system can be used to express exogenous genes in C. utilis as a bioreactors. This is the first report that xylanase gene was expressed in C. utilis. PMID:19731075

  1. Recombinant transferrin binding protein A (rTbpA) fragments of Pasteurella multocida serogroup B:2 provide variable protection following homologous challenge in mouse model.

    PubMed

    Shivachandra, Sathish Bhadravati; Yogisharadhya, Revanaiah; Kumar, Abhinendra; Mohanty, Nihar Nalini; Nagaleekar, Viswas Konasagara

    2015-02-01

    Transferrin binding protein A (TbpA), an iron acquisition surface protein that also acts as virulence factor, is widely distributed among strains of Pasteurella multocida. In the present study, a total of seven clones of TbpA fragments (39D to F777; 39D to Q697; 188V to F777; 188V to Q697; 39D to P377; 188V to P377 and 39D to F187) belonging to P. multocida B:2 were constructed, over-expressed and purified as recombinant fusion proteins from Escherichia coli using affinity chromatography. Immunization of mice with rTbpA fragments resulted in a significant (p < 0.05) rise in antigen specific serum total IgG and subtypes (IgG1 and IgG2a) tires. All immunized mice challenged with 8 LD50 of P. multocida B:2 resulted in a variable protective efficacy up to 50%. The study indicated the potential possibilities to incorporate full length TbpA in subunit vaccine formulation composed of synergistic subunit antigens against haemorrhagic septicaemia (HS) in cattle and buffalo. PMID:25544697

  2. Properties of human immunodeficiency virus type 1 reverse transcriptase recombination upon infection.

    PubMed

    Sakuragi, Sayuri; Shioda, Tatsuo; Sakuragi, Jun-ichi

    2015-11-01

    Reverse transcription (RT) is one of the hallmark features of retroviruses. During RT, virus encoded reverse transcriptase (RTase) must transfer from one end to the other end of the viral genome on two separate occasions to complete RT and move on to the production of proviral DNA. In addition, multiple strand-transfer events between homologous regions of the dimerized viral genome by RTase are also observed, and such recombination events serve as one of the driving forces behind human immunodeficiency virus (HIV) genome sequence diversity. Although retroviral recombination is widely considered to be important, several features of its mechanism are still unclear. We constructed an HIV-1 vector system to examine the target sequences required for virus recombination, and elucidated other necessary prerequisites to harbor recombination, such as the length, homology and the stability of neighbouring structures around the target sequences. PMID:26282329

  3. Modulating Mek1 kinase alters outcomes of meiotic recombination and the stringency of the recombination checkpoint response

    PubMed Central

    Hsin-Yen, Wu; Hsuan-Chung, Ho; Burgess, Sean M.

    2010-01-01

    Summary Background During meiosis, recombination between homologous chromosomes promotes their proper segregation. In budding yeast, programmed double-strand breaks (DSBs) promote recombination between homologs versus sister chromatids by dimerizing and activating Mek1, a chromosome axis-associated kinase. Mek1 is also a proposed effector kinase in the recombination checkpoint that arrests exit from pachytene in response to aberrant DNA/axis structures. Elucidating a role for Mek1 in the recombination checkpoint has been difficult since in mek1 loss-of-function mutants DSBs are rapidly repaired using a sister chromatid thereby bypassing formation of checkpoint-activating lesions. Here we tested the hypothesis that a MEK1 gain-of-function allele would enhance interhomolog bias and the recombination checkpoint response. Results When Mek1 activation was artificially maintained through GST-mediated dimerization, there was an enhanced skew toward interhomolog recombination and reduction of intersister events including multi-chromatid joint molecules. Increased interhomolog events were specifically repaired as noncrossovers rather than crossovers. Ectopic Mek1 dimerization was also sufficient to impose interhomolog bias in the absence of recombination checkpoint functions, thereby uncoupling these two processes. Finally, the stringency of the recombination checkpoint was enhanced in weak meiotic recombination mutants by blocking prophase exit in a subset of cells where arrest is not absolute. Conclusions We propose that Mek1 plays dual roles during meiotic prophase I by phosphorylating targets directly involved in the recombination checkpoint as well as targets involved in sister chromatid recombination. We discuss how regulation of pachytene exit by Mek1 or similar kinases could influence checkpoint stringency, which may differ among species and between sexes. PMID:20888230

  4. Mutation and Recombination in the Upstream Homology Box-Flanked ospE-Related Genes of the Lyme Disease Spirochetes Result in the Development of New Antigenic Variants during Infection

    PubMed Central

    Sung, Shian Ying; McDowell, John V.; Carlyon, Jason A.; Marconi, Richard T.

    2000-01-01

    The ospE gene family of the Lyme disease spirochetes encodes a polymorphic group of immunogenic lipoproteins. The ospE genes are one of several gene families that are flanked by a highly conserved upstream sequence called the upstream homology box, or UHB, element. Earlier analyses in our lab demonstrated that ospE-related genes are characterized by defined hypervariable domains (domains 1 and 2) that are predicted to be hydrophilic, surface exposed, and antigenic. The flanking of hypervariable domain 1 by DNA repeats may indicate that recombination contributes to ospE diversity and thus ultimately to antigenic variation. Using an isogeneic clone of Borrelia burgdorferi B31G (designated B31Gc1), we demonstrate that the ospE-related genes undergo mutation and rearrangement during infection in mice. The mutations that develop during infection resulted in the generation of OspE proteins with altered antigenic characteristics. The data support the hypothesized role of OspE-related proteins in immune system evasion. PMID:10678944

  5. ROLE OF THE HOMOLOGOUS RECOMBINATION GENES RAD51 and RAD59 IN THE RESISTANCE OF Candida albicans TO UV LIGHT, RADIOMIMETIC AND ANTI-TUMOR COMPOUNDS AND OXIDIZING AGENTS

    PubMed Central

    García-Prieto, Fátima; Gómez-Raja, Jonathan; Andaluz, Encarnación; Calderone, Richard; Larriba, Germán

    2010-01-01

    We have cloned and characterized the RAD51 and RAD59 orthologues of the pathogenic fungus Candida albicans. CaRad51 exhibited more than 50% identity with several other eukaryotes and the conserved the catalytic domain of a bacterial RecA. As compared to the parental strain, null strains of rad51 exhibited a filamentous morphology, had a decreased grow rate and exhibited a moderate sensitivity to UV light, oxidizing agents, and compounds that cause double-strand breaks (DSB), indicating a role in DNA repair. By comparison, the rad52 null had a higher percentage of filaments, a more severe growth defect and a greater sensitivity to DNA-damaging compounds. Null strains of rad59 showed a UV-sensitive phenotype but behaved similarly to the parental strain in the rest of the assays. As compared to S. cerevisiae, C. albicans was much more resistant to bleomycin and the same was true for their respective homologous recombination (HR) mutants. These results indicate that, as described in S. cerevisiae, RAD52 plays a more prominent role than RAD51 in the repair of DSBs in C. albicans and suggest the existence of at least two Rad52-dependent HR pathways, one dependent and one independent of Rad51. PMID:20206282

  6. Meiotic recombination and genome evolution in plants.

    PubMed

    Melamed-Bessudo, Cathy; Shilo, Shay; Levy, Avraham A

    2016-04-01

    Homologous recombination affects genome evolution through crossover, gene conversion and point mutations. Whole genome sequencing together with a detailed epigenome analysis have shed new light on our understanding of how meiotic recombination shapes plant genes and genome structure. Crossover events are associated with DNA sequence motifs, together with an open chromatin signature (hypomethylated CpGs, low nucleosome occupancy or specific histone modifications). The crossover landscape may differ between male and female meiocytes and between species. At the gene level, crossovers occur preferentially in promoter regions in Arabidopsis. In recent years, there is rising support suggesting that biased mismatch repair during meiotic recombination may increase GC content genome-wide and may be responsible for the GC content gradient found in many plant genes. PMID:26939088

  7. Single Nucleotide Polymorphisms (SNPs) of RAD51-G172T and XRCC2-41657C/T Homologous Recombination Repair Genes and the Risk of Triple- Negative Breast Cancer in Polish Women.

    PubMed

    Michalska, Magdalena M; Samulak, Dariusz; Romanowicz, Hanna; Smolarz, Beata

    2015-09-01

    Double strand DNA breaks are the most dangerous DNA damage which, if non-repaired or misrepaired, may result in genomic instability, cancer transformation or cell death. RAD51 and XRCC2 encode proteins that are important for the repair of double-strand DNA breaks by homologous recombination. Therefore, genetic variability in these genes may contribute to the occurrence and progression of triple-negative breast cancer. The polymorphisms of the XRCC2 gene -41657C/T (rs718282) and of the RAD51 gene, -172G/T (rs1801321), were investigated by PCR-RFLP in 70 patients with triple-negative breast cancer and 70 age- and sex matched non-cancer controls. The obtained results demonstrated a significant positive association between the RAD51 T/T genotype and TNBC, with an adjusted odds ratio (OR) of 4.94 (p = 0.001). The homozygous T/T genotype was found in 60 % of TNBC cases and in 14 % of the used controls. Variant 172 T allele of RAD51 increased cancer risk (OR = 2.81 (1.72-4.58), p < .0001). No significant associations were observed between -41657C/T genotype of XRCC2 and the incidence of TNBC. There were no significant differences between the distribution of XRCC2 -41657C/T genotypes in the subgroups assigned to histological grades. The obtained results indicate that the polymorphism of RAD51, but not of XRCC2 gene, may be positively associated with the incidence of triple-negative breast carcinoma in the population of Polish women. PMID:25743260

  8. Chromosomally-retained RNA mediates homologous pairing.

    PubMed

    Ding, Da-Qiao; Haraguchi, Tokuko; Hiraoka, Yasushi

    2012-01-01

    Pairing and recombination of homologous chromosomes are essential for ensuring correct segregation of chromosomes in meiosis. In S. pombe, chromosomes are first bundled at the telomeres (forming a telomere bouquet) and then aligned by oscillatory movement of the elongated "horsetail" nucleus. Telomere clustering and subsequent chromosome alignment promote pairing of homologous chromosomes. However, this telomere-bundled alignment of chromosomes cannot be responsible for the specificity of chromosome pairing. Thus, there must be some mechanism to facilitate recognition of homologous partners after telomere clustering. Recent studies in S. pombe have shown that RNA transcripts retained on the chromosome, or RNA bodies, may play a role in recognition of homologous chromosomes for pairing. Acting as fiducial markers of homologous loci they would abrogate the need for direct DNA sequence homology searching. PMID:23117617

  9. Phylogenetic evidence for intratypic recombinant events in a novel human adenovirus C that causes severe acute respiratory infection in children

    PubMed Central

    Wang, Yanqun; Li, Yamin; Lu, Roujian; Zhao, Yanjie; Xie, Zhengde; Shen, Jun; Tan, Wenjie

    2016-01-01

    Human adenoviruses (HAdVs) are prevalent in hospitalized children with severe acute respiratory infection (SARI). Here, we report a unique recombinant HAdV strain (CBJ113) isolated from a HAdV-positive child with SARI. The whole-genome sequence was determined using Sanger sequencing and high-throughput sequencing. A phylogenetic analysis of the complete genome indicated that the CBJ113 strain shares a common origin with HAdV-C2, HAdV-C6, HAdV-C1, HAdV-C5, and HAdV-C57 and formed a novel subclade on the same branch as other HAdV-C subtypes. BootScan and single nucleotide polymorphism analyses showed that the CBJ113 genome has an intra-subtype recombinant structure and comprises gene regions mainly originating from two circulating viral strains: HAdV-1 and HAdV-2. The parental penton base, pVI, and DBP genes of the recombinant strain clustered with the HAdV-1 prototype strain, and the E1B, hexon, fiber, and 100 K genes of the recombinant clustered within the HAdV-2 subtype, meanwhile the E4orf1 and DNA polymerase genes of the recombinant shared the greatest similarity with those of HAdV-5 and HAdV-6, respectively. All of these findings provide insight into our understanding of the dynamics of the complexity of the HAdV-C epidemic. More extensive studies should address the pathogenicity and clinical characteristics of the novel recombinant. PMID:26960434

  10. Phylogenetic evidence for intratypic recombinant events in a novel human adenovirus C that causes severe acute respiratory infection in children.

    PubMed

    Wang, Yanqun; Li, Yamin; Lu, Roujian; Zhao, Yanjie; Xie, Zhengde; Shen, Jun; Tan, Wenjie

    2016-01-01

    Human adenoviruses (HAdVs) are prevalent in hospitalized children with severe acute respiratory infection (SARI). Here, we report a unique recombinant HAdV strain (CBJ113) isolated from a HAdV-positive child with SARI. The whole-genome sequence was determined using Sanger sequencing and high-throughput sequencing. A phylogenetic analysis of the complete genome indicated that the CBJ113 strain shares a common origin with HAdV-C2, HAdV-C6, HAdV-C1, HAdV-C5, and HAdV-C57 and formed a novel subclade on the same branch as other HAdV-C subtypes. BootScan and single nucleotide polymorphism analyses showed that the CBJ113 genome has an intra-subtype recombinant structure and comprises gene regions mainly originating from two circulating viral strains: HAdV-1 and HAdV-2. The parental penton base, pVI, and DBP genes of the recombinant strain clustered with the HAdV-1 prototype strain, and the E1B, hexon, fiber, and 100 K genes of the recombinant clustered within the HAdV-2 subtype, meanwhile the E4orf1 and DNA polymerase genes of the recombinant shared the greatest similarity with those of HAdV-5 and HAdV-6, respectively. All of these findings provide insight into our understanding of the dynamics of the complexity of the HAdV-C epidemic. More extensive studies should address the pathogenicity and clinical characteristics of the novel recombinant. PMID:26960434

  11. Evidence and Consequence of Porcine Endogenous Retrovirus Recombination

    PubMed Central

    Bartosch, Birke; Stefanidis, Dimitrios; Myers, Richard; Weiss, Robin; Patience, Clive; Takeuchi, Yasuhiro

    2004-01-01

    The genetic nature and biological effects of recombination between porcine endogenous retroviruses (PERV) were studied. An infectious molecular clone was generated from a high-titer, human-tropic PERV isolate, PERV-A 14/220 (B. A. Oldmixon, et al. J. Virol. 76:3045-3048, 2002; T. A. Ericsson et al. Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). To analyze this sequence and 15 available full-length PERV nucleotide sequences, we developed a sequence comparison program, LOHATM to calculate local sequence homology between two sequences. This analysis determined that PERV-A 14/220 arose by homologous recombination of a PERV-C genome replacing an 850-bp region around the pol-env junction with that of a PERV-A sequence. This 850-bp PERV-A sequence encompasses the env receptor binding domain, thereby conferring a wide host range including human cells. In addition, we determined that multiple regions derived from PERV-C are responsible for the increased infectious titer of PERV-A 14/220. Thus, a single recombination event may be a fast and effective way to generate high-titer, potentially harmful PERV. Further, local homology and phylogenetic analyses between 16 full-length sequences revealed evidence for other recombination events in the past that give rise to other PERV genomes that possess the PERV-A, but not the PERV-B, env gene. These results indicate that PERV-A env is more prone to recombination with heterogeneous backbone genomes than PERV-B env. Such recombination events that generate more active PERV-A appear to occur in pigs rather frequently, which increases the potential risk of zoonotic PERV transmission. In this context, pigs lacking non-human-tropic PERV-C would be more suitable as donor animals for clinical xenotransplantation. PMID:15564496

  12. Biochemistry of Meiotic Recombination: Formation, Processing, and Resolution of Recombination Intermediates

    PubMed Central

    Ehmsen, Kirk T.

    2009-01-01

    Meiotic recombination ensures accurate chromosome segregation during the first meiotic division and provides a mechanism to increase genetic heterogeneity among the meiotic products. Unlike homologous recombination in somatic (vegetative) cells, where sister chromatid interactions prevail and crossover formation is avoided, meiotic recombination is targeted to involve homologs, resulting in crossovers to connect the homologs before anaphase of the first meiotic division. The mechanisms responsible for homolog choice and crossover control are poorly understood, but likely involve meiosis-specific recombination proteins, as well as meiosis-specific chromosome organization and architecture. Much progress has been made to identify and biochemically characterize many of the proteins acting during meiotic recombination. This review will focus on the proteins that generate and process heteroduplex DNA, as well as those that process DNA junctions during meiotic recombination, with particular attention to how recombination activities promote crossover resolution between homologs. PMID:20098639

  13. Evidence of Localized Prophage-Host Recombination in the lytA Gene, Encoding the Major Pneumococcal Autolysin ▿

    PubMed Central

    Morales, María; García, Pedro; de la Campa, Adela G.; Liñares, Josefina; Ardanuy, Carmen; García, Ernesto

    2010-01-01

    According to a highly polymorphic region in the lytA gene, encoding the major autolysin of Streptococcus pneumoniae, two different families of alleles can be differentiated by PCR and restriction digestion. Here, we provide evidence that this polymorphic region arose from recombination events with homologous genes of pneumococcal temperate phages. PMID:20304992

  14. Vaccination of Rhesus Macaques with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Env V3 Elicits Neutralizing Antibody-Mediated Protection against Simian-Human Immunodeficiency Virus with a Homologous but Not a Heterologous V3 Motif

    PubMed Central

    Someya, Kenji; Cecilia, Dayaraj; Ami, Yasushi; Nakasone, Tadashi; Matsuo, Kazuhiro; Burda, Sherri; Yamamoto, Hiroshi; Yoshino, Naoto; Kaizu, Masahiko; Ando, Shuji; Okuda, Kenji; Zolla-Pazner, Susan; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

    2005-01-01

    Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations. PMID:15650171

  15. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    NASA Astrophysics Data System (ADS)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  16. Oligo Recombination in Gram Negative Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homologous recombination is important for bacterial survival because it simultaneously provides genomic stability as well as genomic plasticity. Of the mechanistic pathways for homologous recombination, those mediated by RecA are the most thoroughly characterized and are understood to be structural...

  17. Homology, Analogy, and Ethology.

    ERIC Educational Resources Information Center

    Beer, Colin G.

    1984-01-01

    Because the main criterion of structural homology (the principle of connections) does not exist for behavioral homology, the utility of the ethological concept of homology has been questioned. The confidence with which behavioral homologies can be claimed varies inversely with taxonomic distance. Thus, conjectures about long-range phylogenetic…

  18. Serotype IV Streptococcus agalactiae ST-452 has arisen from large genomic recombination events between CC23 and the hypervirulent CC17 lineages

    PubMed Central

    Campisi, Edmondo; Rinaudo, C. Daniela; Donati, Claudio; Barucco, Mara; Torricelli, Giulia; Edwards, Morven S.; Baker, Carol J.; Margarit, Imma; Rosini, Roberto

    2016-01-01

    Streptococcus agalactiae (Group B Streptococcus, GBS) causes life-threatening infections in newborns and adults with chronic medical conditions. Serotype IV strains are emerging both among carriers and as cause of invasive disease and recent studies revealed two main Sequence Types (STs), ST-452 and ST-459 assigned to Clonal Complexes CC23 and CC1, respectively. Whole genome sequencing of 70 type IV GBS and subsequent phylogenetic analysis elucidated the localization of type IV isolates in a SNP-based phylogenetic tree and suggested that ST-452 could have originated through genetic recombination. SNPs density analysis of the core genome confirmed that the founder strain of this lineage originated from a single large horizontal gene transfer event between CC23 and the hypervirulent CC17. Indeed, ST-452 genomes are composed by two parts that are nearly identical to corresponding regions in ST-24 (CC23) and ST-291 (CC17). Chromosome mapping of the major GBS virulence factors showed that ST-452 strains have an intermediate yet unique profile among CC23 and CC17 strains. We described unreported large recombination events, involving the cps IV operon and resulting in the expansion of serotype IV to CC23. This work sheds further light on the evolution of GBS providing new insights on the recent emergence of serotype IV. PMID:27411639

  19. Serotype IV Streptococcus agalactiae ST-452 has arisen from large genomic recombination events between CC23 and the hypervirulent CC17 lineages.

    PubMed

    Campisi, Edmondo; Rinaudo, C Daniela; Donati, Claudio; Barucco, Mara; Torricelli, Giulia; Edwards, Morven S; Baker, Carol J; Margarit, Imma; Rosini, Roberto

    2016-01-01

    Streptococcus agalactiae (Group B Streptococcus, GBS) causes life-threatening infections in newborns and adults with chronic medical conditions. Serotype IV strains are emerging both among carriers and as cause of invasive disease and recent studies revealed two main Sequence Types (STs), ST-452 and ST-459 assigned to Clonal Complexes CC23 and CC1, respectively. Whole genome sequencing of 70 type IV GBS and subsequent phylogenetic analysis elucidated the localization of type IV isolates in a SNP-based phylogenetic tree and suggested that ST-452 could have originated through genetic recombination. SNPs density analysis of the core genome confirmed that the founder strain of this lineage originated from a single large horizontal gene transfer event between CC23 and the hypervirulent CC17. Indeed, ST-452 genomes are composed by two parts that are nearly identical to corresponding regions in ST-24 (CC23) and ST-291 (CC17). Chromosome mapping of the major GBS virulence factors showed that ST-452 strains have an intermediate yet unique profile among CC23 and CC17 strains. We described unreported large recombination events, involving the cps IV operon and resulting in the expansion of serotype IV to CC23. This work sheds further light on the evolution of GBS providing new insights on the recent emergence of serotype IV. PMID:27411639

  20. Ends-in Vs. Ends-Out Recombination in Yeast

    PubMed Central

    Hastings, P. J.; McGill, C.; Shafer, B.; Strathern, J. N.

    1993-01-01

    Integration of linearized plasmids into yeast chromosomes has been used as a model system for the study of recombination initiated by double-strand breaks. The linearized plasmid DNA recombines efficiently into sequences homologous to the ends of the DNA. This efficient recombination occurs both for the configuration in which the break is in a contiguous region of homology (herein called the ends-in configuration) and for ``omega'' insertions in which plasmid sequences interrupt a linear region of homology (herein called the ends-out configuration). The requirements for integration of these two configurations are expected to be different. We compared these two processes in a yeast strain containing an ends-in target and an ends-out target for the same cut plasmid. Recovery of ends-in events exceeds ends-out events by two- to threefold. Possible causes for the origin of this small bias are discussed. The lack of an extreme difference in frequency implies that cooperativity between the two ends does not contribute to the efficiency with which cut circular plasmids are integrated. This may also be true for the repair of chromosomal double-strand breaks. PMID:8307337

  1. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  2. Recombination can partially substitute for SPO13 in regulating meiosis I in budding yeast.

    PubMed Central

    Rutkowski, L H; Esposito, R E

    2000-01-01

    Recombination and chromosome synapsis bring homologous chromosomes together, creating chiasmata that ensure accurate disjunction during reductional division. SPO13 is a key gene required for meiosis I (MI) reductional segregation, but dispensable for recombination, in Saccharomyces cerevisiae. Absence of SPO13 leads to single-division meiosis where reductional segregation is largely eliminated, but other meiotic events occur relatively normally. This phenotype allows haploids to produce viable meiotic products. Spo13p is thought to act by delaying nuclear division until sister centromeres/chromatids undergo proper cohesion for segregation to the same pole at MI. In the present study, a search for new spo13-like mutations that allow haploid meiosis recovered only new spo13 alleles. Unexpectedly, an unusual reduced-expression allele (spo13-23) was recovered that behaves similarly to a null mutant in haploids but to a wild-type allele in diploids, dependent on the presence of recombining homologs rather than on a diploid genome. This finding demonstrates that in addition to promoting accurate homolog disjunction, recombination can also function to partially substitute for SPO13 in promoting sister cohesion. Analysis of various recombination-defective mutants indicates that this contribution of recombination to reductional segregation requires full levels of crossing over. The implications of these results regarding SPO13 function are discussed. PMID:10924460

  3. Third Chromosome Balancer Inversions Disrupt Protein-Coding Genes and Influence Distal Recombination Events in Drosophila melanogaster.

    PubMed

    Miller, Danny E; Cook, Kevin R; Arvanitakis, Alexandra V; Hawley, R Scott

    2016-01-01

    Balancer chromosomes are multiply inverted chromosomes that suppress meiotic crossing over and prevent the recovery of crossover products. Balancers are commonly used in Drosophila melanogaster to maintain deleterious alleles and in stock construction. They exist for all three major chromosomes, yet the molecular location of the breakpoints and the exact nature of many of the mutations carried by the second and third chromosome balancers has not been available. Here, we precisely locate eight of 10 of the breakpoints on the third chromosome balancer TM3, six of eight on TM6, and nine of 11 breakpoints on TM6B We find that one of the inversion breakpoints on TM3 bisects the highly conserved tumor suppressor gene p53-a finding that may have important consequences for a wide range of studies in Drosophila We also identify evidence of single and double crossovers between several TM3 and TM6B balancers and their normal-sequence homologs that have created genetic diversity among these chromosomes. Overall, this work demonstrates the practical importance of precisely identifying the position of inversion breakpoints of balancer chromosomes and characterizing the mutant alleles carried by them. PMID:27172211

  4. Third Chromosome Balancer Inversions Disrupt Protein-Coding Genes and Influence Distal Recombination Events in Drosophila melanogaster

    PubMed Central

    Miller, Danny E.; Cook, Kevin R.; Arvanitakis, Alexandra V.; Hawley, R. Scott

    2016-01-01

    Balancer chromosomes are multiply inverted chromosomes that suppress meiotic crossing over and prevent the recovery of crossover products. Balancers are commonly used in Drosophila melanogaster to maintain deleterious alleles and in stock construction. They exist for all three major chromosomes, yet the molecular location of the breakpoints and the exact nature of many of the mutations carried by the second and third chromosome balancers has not been available. Here, we precisely locate eight of 10 of the breakpoints on the third chromosome balancer TM3, six of eight on TM6, and nine of 11 breakpoints on TM6B. We find that one of the inversion breakpoints on TM3 bisects the highly conserved tumor suppressor gene p53—a finding that may have important consequences for a wide range of studies in Drosophila. We also identify evidence of single and double crossovers between several TM3 and TM6B balancers and their normal-sequence homologs that have created genetic diversity among these chromosomes. Overall, this work demonstrates the practical importance of precisely identifying the position of inversion breakpoints of balancer chromosomes and characterizing the mutant alleles carried by them. PMID:27172211

  5. Illegitimate RAG-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia.

    PubMed

    Dong, Y; Liu, F; Wu, C; Li, S; Zhao, X; Zhang, P; Jiao, J; Yu, X; Ji, Y; Zhang, M

    2016-09-01

    Breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1), encoded by the Philadelphia (Ph) chromosome, is the characteristic of chronic myeloid leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). We demonstrated that expression of the Ik6 transcript, which lacked exons 3-6, was observed exclusively in BCR-ABL1(+) B ALL and lymphoid blast crisis CML (BC-CML) patients harbouring the IKZF1 Δ3-6 deletion. To confirm the hypothesis that illegitimate recombination activating gene protein (RAG)-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia, we first demonstrated that the expression rates of RAG1 and RAG2, collectively called RAG, were higher in ALL and BC-CML (lymphoid). Notably, analysis of relationships among RAG, BCR-ABL1 and Ikaros 6 (Ik6) showed that Ik6 can be generated only if RAG and BCR-ABL1 are co-existing. The sequencing data showed that the deleted segments of introns 2 and 6 contained cryptic recombination signal sequences (cRSSs) and frequently had non-template nucleotides inserted between breakpoints. Furthermore, we used chromatin immunoprecipitation (ChIP) technology and demonstrated that the sequences directly flanking IKZF1 Δ3-6 deletion breakpoints have significantly higher levels of histone H3 lysine 4 trimethylation (H3K4me3) modifications. Overall, RAG expression, good-quality cRSS and a specific chromatin modification, H3K4me3, satisfy the conditions of RAG's off-target effects on IKZF1. Our work provides evidence for RAG-mediated IKZF1 Δ3-6 deletion. Our results raise the prospect that RAG is a valuable biomarker in disease surveillance. Dissecting the contribution of RAG should not only provide valuable mechanistic insights, but will also lead to a new therapeutic direction. PMID:27198500

  6. Polyomavirus JC in the context of immunosuppression: a series of adaptive, DNA replication-driven recombination events in the development of progressive multifocal leukoencephalopathy.

    PubMed

    Johnson, Edward M; Wortman, Margaret J; Dagdanova, Ayuna V; Lundberg, Patric S; Daniel, Dianne C

    2013-01-01

    Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating infection of oligodendrocytes in the brain. PML, a frequently fatal opportunistic infection in AIDS, has also emerged as a consequence of treatment with several new immunosuppressive therapeutic agents. Although nearly 80% of adults are seropositive, JCV attains an ability to infect glial cells in only a minority of people. Data suggest that JCV undergoes sequence alterations that accompany this ability, and these changes can be derived from an archetype strain by mutation, deletion, and duplication. While the introductory source and primary tissue reservoir of JCV remain unknown, lymphoid cells have been identified as potential intermediaries in progression of JCV to the brain. This review is focused on sequence changes in the noncoding control region (NCCR) of the virus. We propose an adaptive mechanism that involves a sequential series of DNA replication-driven NCCR recombination events involving stalled DNA replication forks at NCCR palindromic secondary structures. We shall describe how the NCCR sequence changes point to a model in which viral DNA replication drives NCCR recombination, allowing JCV adaptation to different cell types in its progression to neurovirulence. PMID:23690820

  7. Meiotic recombination in normal and cloned bulls and their offspring

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homologous chromosome pairing and recombination are essential components of meiosis and sexual reproduction. The reshuffling of genetic material through breakage and reunion of chromatids ensure proper segregation of homologous chromosomes in reduction division and genetic diversity in the progeny....

  8. D-region electron density and effective recombination coefficients during twilight - experimental data and modelling during solar proton events

    NASA Astrophysics Data System (ADS)

    Osepian, A.; Kirkwood, S.; Dalin, P.; Tereschenko, V.

    2009-10-01

    Accurate measurements of electron density in the lower D-region (below 70 km altitude) are rarely made. This applies both with regard to measurements by ground-based facilities and by sounding rockets, and during both quiet conditions and conditions of energetic electron precipitation. Deep penetration into the atmosphere of high-energy solar proton fluxes (during solar proton events, SPE) produces extra ionisation in the whole D-region, including the lower altitudes, which gives favourable conditions for accurate measurements using ground-based facilities. In this study we show that electron densities measured with two ground-based facilities at almost the same latitude but slightly different longitudes, provide a valuable tool for validation of model computations. The two techniques used are incoherent scatter of radio waves (by the EISCAT 224 MHz radar in Tromsø, Norway, 69.6° N, 19.3° E), and partial reflection of radio-waves (by the 2.8 MHz radar near Murmansk, Russia, 69.0° N, 35.7° E). Both radars give accurate electron density values during SPE, from heights 57-60 km and upward with the EISCAT radar and between 55-70 km with the partial reflection technique. Near noon, there is little difference in the solar zenith angle between the two locations and both methods give approximately the same values of electron density at the overlapping heights. During twilight, when the difference in solar zenith angles increases, electron density values diverge. When both radars are in night conditions (solar zenith angle >99°) electron densities at the overlapping altitudes again become equal. We use the joint measurements to validate model computations of the ionospheric parameters f+, λ, αeff and their variations during solar proton events. These parameters are important characteristics of the lower ionosphere structure which cannot be determined by other methods.

  9. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels.

    PubMed

    Joshi, Neeraj; Brown, M Scott; Bishop, Douglas K; Börner, G Valentin

    2015-03-01

    During meiosis, Spo11-induced double-strand breaks (DSBs) are processed into crossovers, ensuring segregation of homologous chromosomes (homologs). Meiotic DSB processing entails 5' end resection and preferred strand exchange with the homolog rather than the sister chromatid (homolog bias). In many organisms, DSBs appear gradually along the genome. Here we report unexpected effects of global DSB levels on local recombination events. Early-occurring, low-abundance "scout" DSBs lack homolog bias. Their resection and interhomolog processing are controlled by the conserved checkpoint proteins Tel1(ATM) kinase and Pch2(TRIP13) ATPase. Processing pathways controlled by Mec1(ATR) kinase take over these functions only above a distinct DSB threshold, resulting in progressive strengthening of the homolog bias. We conclude that Tel1(ATM)/Pch2 and Mec1(ATR) DNA damage response pathways are sequentially activated during wild-type meiosis because of their distinct sensitivities to global DSB levels. Moreover, relative DSB order controls the DSB repair pathway choice and, ultimately, recombination outcome. PMID:25661491

  10. Progression of meiotic recombination requires structural maturation of the central element of the synaptonemal complex.

    PubMed

    Hamer, Geert; Wang, Hong; Bolcun-Filas, Ewelina; Cooke, Howard J; Benavente, Ricardo; Höög, Christer

    2008-08-01

    The synaptonemal complex is an elaborate meiosis-specific supramolecular protein assembly that promotes chromosome synapsis and meiotic recombination. We inactivated the meiosis-specific gene Tex12 and found that TEX12 is essential for progression of meiosis in both male and female germ cells. Structural analysis of the synaptonemal complex in Tex12-/- meiocytes revealed a disrupted central element structure, a dense structure residing between the synapsed homologous chromosomes. Chromosome synapsis is initiated at multiple positions along the paired homologous chromosomes in Tex12-/- meiotic cells, but fails to propagate along the chromosomes. Furthermore, although meiotic recombination is initiated in Tex12-/- meiotic cells, these early recombination events do not develop into meiotic crossovers. Hence, the mere initiation of synapsis is not sufficient to support meiotic crossing-over. Our results show that TEX12 is a component of the central element structure of the synaptonemal complex required for propagation of synapsis along the paired homologous chromosomes and maturation of early recombination events into crossovers. PMID:18611960

  11. Telomeric IGH Losses Detectable by Fluorescence in Situ Hybridization in Chronic Lymphocytic Leukemia Reflect Somatic VH Recombination Events

    PubMed Central

    Wlodarska, Iwona; Matthews, Christine; Veyt, Ellen; Pospisilova, Helena; Catherwood, Mark A.; Poulsen, Tim S.; Vanhentenrijk, Vera; Ibbotson, Rachel; Vandenberghe, Peter; Morris, T.C.M. “Curly”; Alexander, H. Denis

    2007-01-01

    Routine interphase fluorescence in situ hybridization (FISH) analysis of chronic lymphocytic leukemia (CLL) with LSI IGH/CCND1 assay, applied to differentiate CLL from leukemic mantle cell lymphoma, identified a subset of cases (42/174) with translocation-like IGH signal pattern. To unravel the underlying 14q32/IGH aberrations, 14 of these cases were subjected to cytogenetic, detailed FISH, and VH mutation analyses. FISH identified cryptic losses of various portions of the IGHV region in all 14 cases. Fine mapping of these VH deletions revealed a strict correlation between their distal border and localization of the used VH gene, suggesting that they are not oncogenic but reflect physiological events accompanying somatic V-D-J assembly. This hypothesis was further supported by FISH analysis of 20 CLL and hairy cell leukemia cases with the known VH usage showing a constant loss of sequences proximal to the used gene, identification of VH deletions in normal B cells, and their exclusive demonstration in B cell malignancies, but not of T cell and myeloid linage. Given that these cryptic physiological VH losses in B cells may seriously complicate analysis of B cell leukemia/lymphoma and lead to false conclusions, FISH users should take them into consideration when interpreting IGH aberrations in these malignancies. PMID:17251335

  12. Homologs of Breast Cancer Genes in Plants

    PubMed Central

    Trapp, Oliver; Seeliger, Katharina; Puchta, Holger

    2011-01-01

    Since the initial discovery of genes involved in hereditary breast cancer in humans, a vast wealth of information has been published. Breast cancer proteins were shown to work as tumor suppressors primarily through their involvement in DNA-damage repair. Surprisingly, homologs of these genes can be found in plant genomes, as well. Here, we want to give an overview of the identification and characterization of the biological roles of these proteins, in plants. In addition to the conservation of their function in DNA repair, new plant-specific characteristics have been revealed. BRCA1 is required for the efficient repair of double strand breaks (DSB) by homologous recombination in somatic cells of the model plant Arabidopsis thaliana. Bioinformatic analysis indicates that, whereas most homologs of key components of the different mammalian BRCA1 complexes are present in plant genomes, homologs of most factors involved in the recruitment of BRCA1 to the DSB cannot be identified. Thus, it is not clear at the moment whether differences exist between plants and animals at this important step. The most conserved region of BRCA1 and BARD1 homologs in plants is a PHD domain which is absent in mammals and which, in AtBARD1, might be involved in the transcriptional regulation of plant development. The presence of a plant-specific domain prompted us to reevaluate the current model for the evolution of BRCA1 homologs and to suggest a new hypothesis, in which we postulate that plant BRCA1 and BARD1 have one common predecessor that gained a PHD domain before duplication. Furthermore, work in Arabidopsis demonstrates that – as in animals – BRCA2 homologs are important for meiotic DNA recombination. Surprisingly, recent research has revealed that AtBRCA2 also has an important role in systemic acquired resistance. In Arabidopsis, BRCA2 is involved in the transcriptional regulation of pathogenesis-related (PR) genes via its interaction with the strand exchange protein RAD51. PMID

  13. OsDMC1 Is Not Required for Homologous Pairing in Rice Meiosis1[OPEN

    PubMed Central

    Tang, Ding; Liu, Xiaofei; Du, Guijie; Shen, Yi; Li, Yafei; Cheng, Zhukuan

    2016-01-01

    Meiotic homologous recombination is pivotal to sexual reproduction. DMC1, a conserved recombinase, is involved in directing single-end invasion between interhomologs during meiotic recombination. In this study, we identified OsDMC1A and OsDMC1B, two closely related proteins in rice (Oryza sativa) with high sequence similarity to DMC1 proteins from other species. Analysis of Osdmc1a and Osdmc1b Tos17 insertion mutants indicated that these genes are functionally redundant. Immunolocalization analysis revealed OsDMC1 foci occurred at leptotene, which disappeared from late pachytene chromosomes in wild-type meiocytes. According to cytological analyses, homologous pairing is accomplished in the Osdmc1a Osdmc1b double mutant, but synapsis is seriously disrupted. The reduced number of bivalents and abnormal OsHEI10 foci in Osdmc1a Osdmc1b establishes an essential role for OsDMC1 in crossover formation. In the absence of OsDMC1, early recombination events probably occur normally, leading to normal localization of γH2AX, PAIR3, OsMRE11, OsCOM1, and OsRAD51C. Moreover, OsDMC1 was not detected in pairing-defective mutants, such as pair2, pair3, Oscom1, and Osrad51c, while it was loaded onto meiotic chromosomes in zep1, Osmer3, Oszip4, and Oshei10. Taken together, these results suggest that during meiosis, OsDMC1 is dispensable for homologous pairing in rice, which is quite different from the DMC1 homologs identified so far in other organisms. PMID:26960731

  14. OsDMC1 Is Not Required for Homologous Pairing in Rice Meiosis.

    PubMed

    Wang, Hongjun; Hu, Qing; Tang, Ding; Liu, Xiaofei; Du, Guijie; Shen, Yi; Li, Yafei; Cheng, Zhukuan

    2016-05-01

    Meiotic homologous recombination is pivotal to sexual reproduction. DMC1, a conserved recombinase, is involved in directing single-end invasion between interhomologs during meiotic recombination. In this study, we identified OsDMC1A and OsDMC1B, two closely related proteins in rice (Oryza sativa) with high sequence similarity to DMC1 proteins from other species. Analysis of Osdmc1a and Osdmc1b Tos17 insertion mutants indicated that these genes are functionally redundant. Immunolocalization analysis revealed OsDMC1 foci occurred at leptotene, which disappeared from late pachytene chromosomes in wild-type meiocytes. According to cytological analyses, homologous pairing is accomplished in the Osdmc1a Osdmc1b double mutant, but synapsis is seriously disrupted. The reduced number of bivalents and abnormal OsHEI10 foci in Osdmc1a Osdmc1b establishes an essential role for OsDMC1 in crossover formation. In the absence of OsDMC1, early recombination events probably occur normally, leading to normal localization of γH2AX, PAIR3, OsMRE11, OsCOM1, and OsRAD51C. Moreover, OsDMC1 was not detected in pairing-defective mutants, such as pair2, pair3, Oscom1, and Osrad51c, while it was loaded onto meiotic chromosomes in zep1, Osmer3, Oszip4, and Oshei10 Taken together, these results suggest that during meiosis, OsDMC1 is dispensable for homologous pairing in rice, which is quite different from the DMC1 homologs identified so far in other organisms. PMID:26960731

  15. Oligonucleotide recombination enabled site-specific mutagenesis in bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligo recombination is one type of recombineering that uses ssDNA olig...

  16. Nitrogenase and Homologs

    PubMed Central

    2014-01-01

    Nitrogenase catalyzes biological nitrogen fixation, a key step in the global nitrogen cycle. Three homologous nitrogenases have been identified to date, along with several structural and/or functional homologs of this enzyme that are involved in nitrogenase assembly, bacteriochlorophyll biosynthesis and methanogenic process, respectively. In this article, we provide an overview of the structures and functions of nitrogenase and its homologs, which highlights the similarity and disparity of this uniquely versatile group of enzymes. PMID:25491285

  17. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  18. Nucleotide Substitution and Recombination at Orthologous Loci in Staphylococcus aureus†

    PubMed Central

    Hughes, Austin L.; Friedman, Robert

    2005-01-01

    The pattern of nucleotide substitution was examined at 2,129 orthologous loci among five genomes of Staphylococcus aureus, which included two sister pairs of closely related genomes (MW2/MSSA476 and Mu50/N315) and the more distantly related MRSA252. A total of 108 loci were unusual in lacking any synonymous differences among the five genomes; most of these were short genes encoding proteins highly conserved at the amino acid sequence level (including many ribosomal proteins) or unknown predicted genes. In contrast, 45 genes were identified that showed anomalously high divergence at synonymous sites. The latter genes were evidently introduced by homologous recombination from distantly related genomes, and in many cases, the pattern of nucleotide substitution made it possible to reconstruct the most probable recombination event involved. These recombination events introduced genes encoding proteins that differed in amino acid sequence and thus potentially in function. Several of the proteins are known or likely to be involved in pathogenesis (e.g., staphylocoagulase, exotoxin, Ser-Asp fibrinogen-binding bone sialoprotein-binding protein, fibrinogen and keratin-10 binding surface-anchored protein, fibrinogen-binding protein ClfA, and enterotoxin P). Therefore, the results support the hypothesis that exchange of homologous genes among S. aureus genomes can play a role in the evolution of pathogenesis in this species. PMID:15805516

  19. Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

    PubMed Central

    Laureau, Raphaëlle; Loeillet, Sophie; Salinas, Francisco; Bergström, Anders; Legoix-Né, Patricia; Liti, Gianni; Nicolas, Alain

    2016-01-01

    In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction. PMID:26828862

  20. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  1. The Contribution of Genetic Recombination to CRISPR Array Evolution

    PubMed Central

    Kupczok, Anne; Landan, Giddy; Dagan, Tal

    2015-01-01

    CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and

  2. Homological stabilizer codes

    SciTech Connect

    Anderson, Jonas T.

    2013-03-15

    In this paper we define homological stabilizer codes on qubits which encompass codes such as Kitaev's toric code and the topological color codes. These codes are defined solely by the graphs they reside on. This feature allows us to use properties of topological graph theory to determine the graphs which are suitable as homological stabilizer codes. We then show that all toric codes are equivalent to homological stabilizer codes on 4-valent graphs. We show that the topological color codes and toric codes correspond to two distinct classes of graphs. We define the notion of label set equivalencies and show that under a small set of constraints the only homological stabilizer codes without local logical operators are equivalent to Kitaev's toric code or to the topological color codes. - Highlights: Black-Right-Pointing-Pointer We show that Kitaev's toric codes are equivalent to homological stabilizer codes on 4-valent graphs. Black-Right-Pointing-Pointer We show that toric codes and color codes correspond to homological stabilizer codes on distinct graphs. Black-Right-Pointing-Pointer We find and classify all 2D homological stabilizer codes. Black-Right-Pointing-Pointer We find optimal codes among the homological stabilizer codes.

  3. Recombination induced by triple-helix-targeted DNA damage in mammalian cells.

    PubMed Central

    Faruqi, A F; Seidman, M M; Segal, D J; Carroll, D; Glazer, P M

    1996-01-01

    Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also observed. The variety of products suggests that multiple cellular pathways can act on the targeted damage, and data showing that the triple helix can influence these pathways are presented. The ability to specifically induce recombination or gene conversion within mammalian cells by using TFOs may provide a new research tool and may eventually lead to novel applications in gene therapy. PMID:8943337

  4. Chimeric Rhinoviruses Obtained via Genetic Engineering or Artificially Induced Recombination Are Viable Only if the Polyprotein Coding Sequence Derives from the Same Species

    PubMed Central

    Schibler, Manuel; Piuz, Isabelle; Hao, Weidong

    2015-01-01

    ABSTRACT Recombination is a widespread phenomenon that ensures both the stability and variation of RNA viruses. This phenomenon occurs with different frequencies within species of the Enterovirus genus. Intraspecies recombination is described frequently among non-rhinovirus enteroviruses but appears to be sporadic in rhinoviruses. Interspecies recombination is even rarer for rhinoviruses and mostly is related to ancient events which contributed to the speciation of these viruses. We reported that artificially engineered 5′ untranslated region (UTR) interspecies rhinovirus/rhinovirus or rhinovirus/non-rhinovirus enterovirus recombinants are fully viable. Using a similar approach, we demonstrated in this study that exchanges of the P1-2A polyprotein region between members of the same rhinovirus species, but not between members of different species, give rise to competent chimeras. To further assess the rhinovirus intra- and interspecies recombination potential, we used artificially induced recombination by cotransfection of 5′-end-deleted and 3′-end-deleted and replication-deficient genomes. In this system, intraspecies recombination also resulted in viable viruses with high frequency, whereas no interspecies rhinovirus recombinants could be recovered. Mapping intraspecies recombination sites within the polyprotein highlighted recombinant hotspots in nonstructural genes and at gene boundaries. Notably, all recombinants occurring at gene junctions presented in-frame sequence duplications, whereas most intragenic recombinants were homologous. Taken together, our results suggest that only intraspecies recombination gives rise to viable rhinovirus chimeras in the polyprotein coding region and that recombination hotspots map to nonstructural genes with in-frame duplications at gene boundaries. These data provide new insights regarding the mechanism and limitations of rhinovirus recombination. IMPORTANCE Recombination represents a means to ensure both the stability

  5. Molecular analysis of a deletion polymorphism in alpha satellite of human chromosome 17: evidence for homologous unequal crossing-over and subsequent fixation.

    PubMed Central

    Waye, J S; Willard, H F

    1986-01-01

    The human alpha satellite DNA family is organized into chromosome-specific subsets characterized by distinct higher-order repeats based on a approximately 171 basepair monomer unit. On human chromosome 17, the predominant form of alpha satellite is a 16-monomer (16-mer) higher-order repeat present in 500-1000 copies per chromosome 17. In addition, less abundant 15-monomer and 14-monomer repeats are also found constitutively on chromosome 17. Polymorphisms in the form of different higher-order repeat lengths have been described for this subset, the most prominent polymorphism being a 13-monomer (13-mer) higher-order repeat present on approximately 35% of all chromosomes 17. To investigate the nature of this polymorphism, we have cloned, sequenced and compared the relevant regions of the 13-mer to the previously characterized 16-mer repeat. The results show that the repeats are virtually identical, with the principal difference being the exclusion of three monomers from the 13-mer repeat. We propose that the 13-mer is the product of an isolated homologous recombination event between two monomers of the 16-mer repeat. Sequence comparisons reveal the approximate site of recombination and flanking regions of homology. This recombination site corresponds to a position within the alphoid monomer which has been previously implicated in an independent homologous recombination event, suggesting that there may exist a preferred register for recombination in alphoid DNA. We suggest that these events are representative of an ongoing process capable of reorganizing the satellite subset of a given chromosome, thereby contributing to the establishment of chromosome-specific alpha satellite subsets. Images PMID:3763396

  6. Homologous gene targeting in Caenorhabditis elegans by biolistic transformation

    PubMed Central

    Berezikov, Eugene; Bargmann, Cornelia I.; Plasterk, Ronald H. A.

    2004-01-01

    Targeted homologous recombination is a powerful approach for genome manipulation that is widely used for gene alteration and knockouts in mouse and yeast. In Caenorhabditis elegans, several methods of target-selected mutagenesis have been implemented but none of them provides the opportunity of introducing exact predefined changes into the genome. Although anecdotal cases of homologous gene targeting in C.elegans have been reported, no practical technique of gene targeting has been developed so far. In this work we demonstrate that transformation of C.elegans by microparticle bombardment (biolistic transformation) can result in homologous recombination between introduced DNA and the chromosomal locus. We describe a scaled up version of biolistic transformation that can be used as a method for homologous gene targeting in the worm. PMID:14982959

  7. Homology, convergence and parallelism.

    PubMed

    Ghiselin, Michael T

    2016-01-01

    Homology is a relation of correspondence between parts of parts of larger wholes. It is used when tracking objects of interest through space and time and in the context of explanatory historical narratives. Homologues can be traced through a genealogical nexus back to a common ancestral precursor. Homology being a transitive relation, homologues remain homologous however much they may come to differ. Analogy is a relationship of correspondence between parts of members of classes having no relationship of common ancestry. Although homology is often treated as an alternative to convergence, the latter is not a kind of correspondence: rather, it is one of a class of processes that also includes divergence and parallelism. These often give rise to misleading appearances (homoplasies). Parallelism can be particularly hard to detect, especially when not accompanied by divergences in some parts of the body. PMID:26598721

  8. Recombination analysis of Maize dwarf mosaic virus (MDMV) in the Sugarcane mosaic virus (SCMV) subgroup of potyviruses.

    PubMed

    Gell, Gyöngyvér; Sebestyén, Endre; Balázs, Ervin

    2015-02-01

    Recombination among RNA viruses is a natural phenomenon that appears to have played a significant role in the species development and the evolution of many strains. It also has particular significance for the risk assessment of plants which have been genetically modified for disease resistance by incorporating viral sequences into their genomes. However, the exact recombination events taking place in viral genomes are not investigated in detail for many virus groups. In this analysis, different single-stranded positive-sense RNA potyviruses were compared using various in silico recombination detection methods and new recombination events in the Sugarcane mosaic virus (SCMV) subgroup were detected. For an extended in silico recombination analysis, two of the analyzed Maize dwarf mosaic virus full-length genomes were sequenced additionally during this work. These results strengthen the evidence that recombination is a major driving force in virus evolution, and the emergence of new virus variants in the SCMV subgroup, paired with mutations, could generate viruses with altered biological properties. The intra- and interspecific homolog recombinations seem to be a general trait in this virus group, causing little or no changes to the amino acid of the progenies. However, we found a few breakpoints between the members of SCMV subgroup and the weed-infecting distant relatives, but only a few methods of the RDP3 package predicted these events with low significance level. PMID:25392089

  9. Oligonucleotide recombination in gram negative bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...

  10. Unraveling recombination rate evolution using ancestral recombination maps

    PubMed Central

    Munch, Kasper; Schierup, Mikkel H; Mailund, Thomas

    2014-01-01

    Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it. PMID:25043668

  11. Braid Floer homology

    NASA Astrophysics Data System (ADS)

    van den Berg, J. B.; Ghrist, R.; Vandervorst, R. C.; Wójcik, W.

    2015-09-01

    Area-preserving diffeomorphisms of a 2-disc can be regarded as time-1 maps of (non-autonomous) Hamiltonian flows on R / Z ×D2. The periodic flow-lines define braid (conjugacy) classes, up to full twists. We examine the dynamics relative to such braid classes and define a new invariant for such classes, the BRAID FLOER HOMOLOGY. This refinement of Floer homology, originally used for the Arnol'd Conjecture, yields a Morse-type forcing theory for periodic points of area-preserving diffeomorphisms of the 2-disc based on braiding. Contributions of this paper include (1) a monotonicity lemma for the behavior of the nonlinear Cauchy-Riemann equations with respect to algebraic lengths of braids; (2) establishment of the topological invariance of the resulting braid Floer homology; (3) a shift theorem describing the effect of twisting braids in terms of shifting the braid Floer homology; (4) computation of examples; and (5) a forcing theorem for the dynamics of Hamiltonian disc maps based on braid Floer homology.

  12. The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

    PubMed Central

    George, Carolyn M.; Lyndaker, Amy M.; Alani, Eric

    2011-01-01

    In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9- dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences. PMID:21978436

  13. The role of UvrD in RecET-mediated illegitimate recombination in Escherichia coli.

    PubMed

    Shiraishi, Kouya; Imai, Yukiho; Yoshizaki, Shinji; Tadaki, Toshimasa; Ogata, Yasuyuki; Ikeda, Hideo

    2006-08-01

    To study the mechanism of RecET-mediated illegitimate recombination, we examined the formation of lambdabio-transducing phage in Escherichia coli in the presence or absence of UV irradiation. We have previously reported that coexpression of RecE and RecT enhances the frequency of recA-independent illegitimate recombination. RecJOR proteins are required for this RecET-mediated illegitimate recombination, and RecQ suppresses it. Here, we showed that the frequencies of both spontaneous and UV-induced RecET-mediated illegitimate recombination events are reduced by a uvrD mutation. It should be noted that UvrD is required for illegitimate recombination only in the presence, but not in the absence, of RecET. In contrast, frequencies of RecET-mediated illegitimate recombination were not affected by ruvAB, ruvC, recG, and recN mutations. The frequency of spontaneous and UV-induced illegitimate recombination in the uvrD recR double mutant was comparable to that of the uvrD single mutant, suggesting that UvrD works at the same step as RecR in the RecET-mediated recombination pathway. Nucleotide sequence analyses of the recombination junctions showed that RecET-mediated illegitimate recombination detected in UvrD-deficient strain is short-homology-dependent. Based on these and previous results, we propose a model for the role of UvrD on RecET-mediated illegitimate recombination. PMID:17038801

  14. Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

    PubMed Central

    Clark, S. H.; Hilliker, A. J.; Chovnick, A.

    1988-01-01

    This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry(301) and ry(2) utilizing DNA restriction site polymorphisms as genetic markers. Both ry(301) and ry(2) are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen. PMID:2834266

  15. Interspecific Recombination between Two Ruminant Alphaherpesviruses, Bovine Herpesviruses 1 and 5

    PubMed Central

    Meurens, François; Keil, Günther M.; Muylkens, Benoît; Gogev, Sacha; Schynts, Frédéric; Negro, Sandra; Wiggers, Laetitia; Thiry, Etienne

    2004-01-01

    Homologous recombination between different species of alphaherpesviruses has been described between herpes simplex viruses 1 and 2 but has not yet been observed between other alphaherpesviruses. In the present study we chose to assess to what extent in vitro recombination can occur between members of a well-defined group of closely related viruses such as ruminant alphaherpesviruses. At 24 h after infection of epithelial bovine kidney cells with a double-deleted mutant of bovine herpesvirus 1 (BoHV-1) (containing green fluorescent protein and red fluorescent protein genes) and different ruminant alphaherpesviruses, four types of progeny viruses were detected and distinguished according to their phenotype. Frequent recombination events between identical or different strains of BoHV-1 were observed (up to 30%), whereas only two BoHV-1/BoHV-5 recombinants were identified, and no recombinants between BoHV-1 and less closely related caprine and cervine herpesviruses were detected. Restriction analysis of the genomes of the two BoHV-1/BoHV-5 recombinants showed different genetic backgrounds. One possessed a restriction pattern close to BoHV-1, whereas the other one was close to BoHV-5. This exhaustive analysis of each combination of coinfection in a unique situation of five closely related alphaherpesviruses revealed the importance of a high degree of genetic relatedness and similar parental virus growth kinetics for successful interspecific recombination. PMID:15331717

  16. Genetic control of recombination partner preference in yeast meiosis. Isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination.

    PubMed Central

    Thompson, D A; Stahl, F W

    1999-01-01

    Meiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids. To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR). The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability. Analysis of the msc mutants according to these criteria defines three classes. Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6. The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint. Meiotic unequal SCR was also elevated in a rad17 mutant. Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues. The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3. These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues. We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway. Class III mutants may be elevated for rates of both SCR and homologue exchange. PMID:10511544

  17. Homolog interaction during meiotic prophase I in Arabidopsis requires the SOLO DANCERS gene encoding a novel cyclin-like protein

    PubMed Central

    Azumi, Yoshitaka; Liu, Dehua; Zhao, Dazhong; Li, Wuxing; Wang, Guanfang; Hu, Yi; Ma, Hong

    2002-01-01

    Interactions between homologs in meiotic prophase I, such as recombination and synapsis, are critical for proper homolog segregation and involve the coordination of several parallel events. However, few regulatory genes have been identified; in particular, it is not clear what roles the proteins similar to the mitotic cell cycle regulators might play during meiotic prophase I. We describe here the isolation and characterization of a new Arabidopsis mutant called solo dancers that exhibits a severe defect in homolog synapsis, recombination and bivalent formation in meiotic prophase I, subsequently resulting in seemingly random chromosome distribution and formation of abnormal meiotic products. We further demonstrate that the mutation affects a meiosis-specific gene encoding a novel protein of 578 amino acid residues with up to 31% amino acid sequence identity to known cyclins in the C-terminal portion. These results argue strongly that homolog interactions during meiotic prophase I require a novel meiosis-specific cyclin in Arabidopsis. PMID:12065421

  18. Bacteriophage recombination systems and biotechnical applications.

    PubMed

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

  19. Identification of Recombination and Positively Selected Genes in Brucella.

    PubMed

    Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2015-12-01

    Brucella is a facultative intracellular bacterium belongs to the class alpha proteobacteria. It causes zoonotic disease brucellosis to wide range of animals. Brucella species are highly conserved in nucleotide level. Here, we employed a comparative genomics approach to examine the role of homologous recombination and positive selection in the evolution of Brucella. For the analysis, we have selected 19 complete genomes from 8 species of Brucella. Among the 1599 core genome predicted, 24 genes were showing signals of recombination but no significant breakpoint was found. The analysis revealed that recombination events are less frequent and the impact of recombination occurred is negligible on the evolution of Brucella. This leads to the view that Brucella is clonally evolved. On other hand, 56 genes (3.5 % of core genome) were showing signals of positive selection. Results suggest that natural selection plays an important role in the evolution of Brucella. Some of the genes that are responsible for the pathogenesis of Brucella were found positively selected, presumably due to their role in avoidance of the host immune system. PMID:26543263

  20. The kinetochore prevents centromere-proximal crossover recombination during meiosis.

    PubMed

    Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel; Oke, Ashwini; Kerr, Alastair Rw; Hochwagen, Andreas; Fung, Jennifer; Keeney, Scott; Vader, Gerben; Marston, Adèle L

    2015-01-01

    During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. PMID:26653857

  1. The kinetochore prevents centromere-proximal crossover recombination during meiosis

    PubMed Central

    Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel; Oke, Ashwini; Kerr, Alastair RW; Hochwagen, Andreas; Fung, Jennifer; Keeney, Scott; Vader, Gerben; Marston, Adèle L

    2015-01-01

    During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. DOI: http://dx.doi.org/10.7554/eLife.10850.001 PMID:26653857

  2. High Degree of HIV-1 Group M (HIV-1M) Genetic Diversity within Circulating Recombinant Forms: Insight into the Early Events of HIV-1M Evolution.

    PubMed

    Tongo, Marcel; Dorfman, Jeffrey R; Martin, Darren P

    2016-03-01

    The existence of various highly divergent HIV-1 lineages and of recombination-derived sequence tracts of indeterminate origin within established circulating recombinant forms (CRFs) strongly suggests that HIV-1 group M (HIV-1M) diversity is not fully represented under the current classification system. Here we used a fully exploratory screen for recombination on a set of 480 near-full-length genomes representing the full known diversity of HIV-1M. We decomposed recombinant sequences into their constituent parts and then used maximum-likelihood phylogenetic analyses of this mostly recombination-free data set to identify rare divergent sequence lineages that fall outside the major named HIV-1M taxonomic groupings. We found that many of the sequence fragments occurring within CRFs (including CRF04_cpx, CRF06_cpx, CRF11_cpx, CRF18_cpx, CRF25_cpx, CRF27_cpx, and CRF49_cpx) are in fact likely derived from divergent unclassified parental lineages that may predate the current subtypes, even though they are presently identified as derived from currently defined HIV-1M subtypes. Our evidence suggests that some of these CRFs are descended predominantly from what were or are major previously unidentified HIV-1M lineages that were likely epidemiologically relevant during the early stages of the HIV-1M epidemic. The restriction of these divergent lineages to the Congo basin suggests that they were less infectious and/or simply not present at the time and place of the initial migratory wave that triggered the global epidemic.IMPORTANCE HIV-1 group M (HIV-1M) likely spread to the rest of the world from the Congo basin in the mid-1900s (N. R. Faria et al., Science 346:56-61, 2014, http://dx.doi.org/10.1126/science.1256739) and is today the principal cause of the AIDS pandemic. Here, we show that large sequence fragments from several HIV-1M circulating recombinant forms (CRFs) are derived from divergent parental lineages that cannot reasonably be classified within the nine

  3. Class-switch recombination: after the dawn of AID

    PubMed Central

    Kenter, Amy L

    2015-01-01

    Isotype class switching is central to the humoral immune response. The discovery that mutations in the activation-induced deaminase (AID) gene inhibit class-switch recombination, somatic hypermutation and gene conversion is a major step forward in defining the underlying mechanisms of these gene modification events. The propensity of mutations to occur at dC/dG nucleotides during somatic hypermutation and the homology between AID and cytidine deaminase has resulted in studies demonstrating that AID has the properties of a cytidine-specific mutator and also that elements of the base-excision repair pathway play a central role in class switching and hypermutation. AID is not a promiscuous mutator in the B cell, suggesting that there are specific molecular targeting mechanisms that regulate the accessibility of DNA to AID and differentially regulate class-switch recombination and somatic hypermutation. During class switching, isotype-specific targeting occurs independently of AID and provides another level of specificity to this recombination event. PMID:12633669

  4. Insertion DNA Accelerates Meiotic Interchromosomal Recombination in Arabidopsis thaliana.

    PubMed

    Sun, Xiao-Qin; Li, Ding-Hong; Xue, Jia-Yu; Yang, Si-Hai; Zhang, Yan-Mei; Li, Mi-Mi; Hang, Yue-Yu

    2016-08-01

    Nucleotide insertions/deletions are ubiquitous in eukaryotic genomes, and the resulting hemizygous (unpaired) DNA has significant, heritable effects on adjacent DNA. However, little is known about the genetic behavior of insertion DNA. Here, we describe a binary transgenic system to study the behavior of insertion DNA during meiosis. Transgenic Arabidopsis lines were generated to carry two different defective reporter genes on nonhomologous chromosomes, designated as "recipient" and "donor" lines. Double hemizygous plants (harboring unpaired DNA) were produced by crossing between the recipient and the donor, and double homozygous lines (harboring paired DNA) via self-pollination. The transfer of the donor's unmutated sequence to the recipient generated a functional β-glucuronidase gene, which could be visualized by histochemical staining and corroborated by polymerase chain reaction amplification and sequencing. More than 673 million seedlings were screened, and the results showed that meiotic ectopic recombination in the hemizygous lines occurred at a frequency  >6.49-fold higher than that in the homozygous lines. Gene conversion might have been exclusively or predominantly responsible for the gene correction events. The direct measurement of ectopic recombination events provided evidence that an insertion, in the absence of an allelic counterpart, could scan the entire genome for homologous counterparts with which to pair. Furthermore, the unpaired (hemizygous) architectures could accelerate ectopic recombination between itself and interchromosomal counterparts. We suggest that the ectopic recombination accelerated by hemizygous architectures may be a general mechanism for interchromosomal recombination through ubiquitously dispersed repeat sequences in plants, ultimately contributing to genetic renovation and eukaryotic evolution. PMID:27189569

  5. Contributions of ancestral inter-species recombination to the genetic diversity of extant Streptomyces lineages.

    PubMed

    Andam, Cheryl P; Choudoir, Mallory J; Vinh Nguyen, Anh; Sol Park, Han; Buckley, Daniel H

    2016-07-01

    Streptomyces species produce many important antibiotics and have a crucial role in soil nutrient cycling. However, their evolutionary history remains poorly characterized. We have evaluated the impact of homologous recombination on the evolution of Streptomyces using multi-locus sequence analysis of 234 strains that represent at least 11 species clusters. Evidence of inter-species recombination is widespread but not uniform within the genus and levels of mosaicism vary between species clusters. Most phylogenetically incongruent loci are monophyletic at the scale of species clusters and their subclades, suggesting that these recombination events occurred in shared ancestral lineages. Further investigation of two mosaic species clusters suggests that genes acquired by inter-species recombination may have become fixed in these lineages during periods of demographic expansion; implicating a role for phylogeography in determining contemporary patterns of genetic diversity. Only by examining the phylogeny at the scale of the genus is apparent that widespread phylogenetically incongruent loci in Streptomyces are derived from a far smaller number of ancestral inter-species recombination events. PMID:26849310

  6. CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells

    PubMed Central

    Zhu, Li; Mon, Hiroaki; Xu, Jian; Lee, Jae Man; Kusakabe, Takahiro

    2015-01-01

    Gene targeting can be achieved by precise genetic modifications through homology-directed repair (HDR) after DNA breaks introduced by genome editing tools such as CRISPR/Cas9 system. The most common form of HDR is homologous recombination (HR). Binding to the DNA breaks by HR factors is thought to compete with non-homologous end joining (NHEJ), an alternative DNA repair pathway. Here, we knocked out the factors in NHEJ by CRISPR/Cas9 system in silkworm cells, so that increased the activities of HR up to 7-fold. Also efficient HR-mediated genome editing events occurred between the chromosomal BmTUDOR-SN gene and donor DNA sequences with an EGFP gene in the middle of two homologous arms for the target gene. Utilizing the NHEJ-deficient silkworm cells, we found that homologous arms as short as 100 bp in donor DNA could be designed to perform precise genome editing. These studies should greatly accelerate investigations into genome editing of silkworm. PMID:26657947

  7. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  8. Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination.

    PubMed

    Chemler, Joseph A; Tripathi, Ashootosh; Hansen, Douglas A; O'Neil-Johnson, Mark; Williams, Russell B; Starks, Courtney; Park, Sung Ryeol; Sherman, David H

    2015-08-26

    The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as biosynthetic assembly lines. The modular nature of these mega-enzymes presents an opportunity to construct custom biocatalysts built in a lego-like fashion by inserting, deleting, or exchanging native or foreign domains to produce targeted variants of natural polyketides. However, previously engineered PKS enzymes are often impaired resulting in limited production compared to native systems. Here, we show a versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways were hybridized in Saccharomyces cerevisiae to generate hybrid libraries. We used a 96-well plate format for plasmid purification, transformations, sequencing, protein expression, in vitro reactions and analysis of metabolite formation. Active chimeric enzymes were identified with new functionality. Streptomyces venezuelae strains that expressed these PKS chimeras were capable of producing engineered macrolactones. Furthermore, a macrolactone generated from selected PKS chimeras was fully functionalized into a novel macrolide analogue. This method permits the engineering of PKS pathways as modular building blocks for the production of new antibiotic-like molecules. PMID:26230368

  9. Quantifying Homologous Replacement of Loci between Haloarchaeal Species

    PubMed Central

    Williams, David; Gogarten, J. Peter; Papke, R. Thane

    2012-01-01

    In vitro studies of the haloarchaeal genus Haloferax have demonstrated their ability to frequently exchange DNA between species, whereas rates of homologous recombination estimated from natural populations in the genus Halorubrum are high enough to maintain random association of alleles between five loci. To quantify the effects of gene transfer and recombination of commonly held (relaxed core) genes during the evolution of the class Halobacteria (haloarchaea), we reconstructed the history of 21 genomes representing all major groups. Using a novel algorithm and a concatenated ribosomal protein phylogeny as a reference, we created a directed horizontal genetic transfer (HGT) network of contemporary and ancestral genomes. Gene order analysis revealed that 90% of testable HGTs were by direct homologous replacement, rather than nonhomologous integration followed by a loss. Network analysis revealed an inverse log-linear relationship between HGT frequency and ribosomal protein evolutionary distance that is maintained across the deepest divergences in Halobacteria. We use this mathematical relationship to estimate the total transfers and amino acid substitutions delivered by HGTs in each genome, providing a measure of chimerism. For the relaxed core genes of each genome, we conservatively estimate that 11–20% of their evolution occurred in other haloarchaea. Our findings are unexpected, because the transfer and homologous recombination of relaxed core genes between members of the class Halobacteria disrupts the coevolution of genes; however, the generation of new combinations of divergent but functionally related genes may lead to adaptive phenotypes not available through cumulative mutations and recombination within a single population. PMID:23160063

  10. Classifications and comparisons of multilocus recombination distributions

    PubMed Central

    Karlin, Samuel; Liberman, Uri

    1978-01-01

    Various classifications and representations of multilocus recombination structures are delineated based on generalized notions of linkage values and recombination rates. An important class of recombination distributions (called the count-location chiasma process) is parameterized by a distribution of the number of crossover events and, for each such crossover count, by a conditional distribution of crossover locations. A number of properties of this recombination structure are developed. A multilocus definition of a “natural” recombination range is set forth. Orderings among recombination distributions in the multilocus setting are also discussed. Comparisons are made in terms of complete linkage, free assortment and noninterference schemes serving as standards. PMID:16592601

  11. Homology, homoplasy, novelty, and behavior.

    PubMed

    Hall, Brian K

    2013-01-01

    Richard Owen coined the modern definition of homology in 1843. Owen's conception of homology was pre-evolutionary, nontransformative (homology maintained basic plans or archetypes), and applied to the fully formed structures of animals. I sketch out the transition to an evolutionary approach to homology in which all classes of similarity are interpreted against the single branching tree of life, and outline the evidence for the application of homology across all levels and features of the biological hierarchy, including behavior. Owen contrasted homology with analogy. While this is not incorrect it is a pre-evolutionary contrast. Lankester [Lankester [1870] Journal of Natural History, 6 (31), 34-43] proposed homoplasy as the class of homology applicable to features formed by independent evolution. Today we identify homology, convergence, parallelism, and novelties as patterns of evolutionary change. A central issue in homology [Owen [1843] Lectures on comparative anatomy and physiology of the invertebrate animals, delivered at the Royal College of Surgeons in 1843. London: Longman, Brown, Green & Longmans] has been whether homology of features-the "same" portion of the brain in different species, for example-depends upon those features sharing common developmental pathways. Owen did not require this criterion, although he observed that homologues often do share developmental pathways (and we now know, often share gene pathways). A similar situation has been explored in the study of behavior, especially whether behaviors must share a common structural, developmental, neural, or genetic basis to be classified as homologous. However, and importantly, development and genes evolve. As shown with both theory and examples, morphological and behavioral features of the phenotype can be homologized as structural or behavioral homologues, respectively, even when their developmental or genetic bases differ (are not homologous). PMID:22711423

  12. Cytological Studies of Human Meiosis: Sex-Specific Differences in Recombination Originate at, or Prior to, Establishment of Double-Strand Breaks

    PubMed Central

    Gruhn, Jennifer R.; Rubio, Carmen; Broman, Karl W.; Hunt, Patricia A.; Hassold, Terry

    2013-01-01

    Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway. PMID:24376867

  13. Differential Requirements of Singleplex and Multiplex Recombineering of Large DNA Constructs

    PubMed Central

    Reddy, Thimma R.; Kelsall, Emma J.; Fevat, Léna M. S.; Munson, Sarah E.; Cowley, Shaun M.

    2015-01-01

    Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency. PMID:25954970

  14. Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Alani, E.; Reenan, RAG.; Kolodner, R. D.

    1994-01-01

    The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA. PMID:8056309

  15. Cosmological Recombination

    NASA Astrophysics Data System (ADS)

    Wong, Wan Yan

    2008-11-01

    In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

  16. Manipulating or Superseding Host Recombination Functions: A Dilemma That Shapes Phage Evolvability

    PubMed Central

    Bobay, Louis-Marie; Touchon, Marie; Rocha, Eduardo P. C.

    2013-01-01

    Phages, like many parasites, tend to have small genomes and may encode autonomous functions or manipulate those of their hosts'. Recombination functions are essential for phage replication and diversification. They are also nearly ubiquitous in bacteria. The E. coli genome encodes many copies of an octamer (Chi) motif that upon recognition by RecBCD favors repair of double strand breaks by homologous recombination. This might allow self from non-self discrimination because RecBCD degrades DNA lacking Chi. Bacteriophage Lambda, an E. coli parasite, lacks Chi motifs, but escapes degradation by inhibiting RecBCD and encoding its own autonomous recombination machinery. We found that only half of 275 lambdoid genomes encode recombinases, the remaining relying on the host's machinery. Unexpectedly, we found that some lambdoid phages contain extremely high numbers of Chi motifs concentrated between the phage origin of replication and the packaging site. This suggests a tight association between replication, packaging and RecBCD-mediated recombination in these phages. Indeed, phages lacking recombinases strongly over-represent Chi motifs. Conversely, phages encoding recombinases and inhibiting host recombination machinery select for the absence of Chi motifs. Host and phage recombinases use different mechanisms and the latter are more tolerant to sequence divergence. Accordingly, we show that phages encoding their own recombination machinery have more mosaic genomes resulting from recent recombination events and have more diverse gene repertoires, i.e. larger pan genomes. We discuss the costs and benefits of superseding or manipulating host recombination functions and how this decision shapes phage genome structure and evolvability. PMID:24086157

  17. Bacterial genome remodeling through bacteriophage recombination.

    PubMed

    Menouni, Rachid; Hutinet, Geoffrey; Petit, Marie-Agnès; Ansaldi, Mireille

    2015-01-01

    Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes. PMID:25790500

  18. Mouse Pachytene Checkpoint 2 (Trip13) Is Required for Completing Meiotic Recombination but Not Synapsis

    PubMed Central

    Li, Xin; Schimenti, John C

    2007-01-01

    In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice. PMID:17696610

  19. Evolving the Concept of Homology

    ERIC Educational Resources Information Center

    Naples, Virginia L.; Miller, Jon S.

    2009-01-01

    Understanding homology is fundamental to learning about evolution. The present study shows an exercise that can be varied in complexity, for which students compile research illustrating the fate of homologous fish skull elements, and assemble a mural to serve as a learning aid. The skull of the most primitive living Actinopterygian (bony fish),…

  20. Structure and mechanism of the phage T4 recombination mediator protein UvsY

    PubMed Central

    Gajewski, Stefan; Waddell, Michael Brett; Vaithiyalingam, Sivaraja; Nourse, Amanda; Li, Zhenmei; Woetzel, Nils; Alexander, Nathan; Meiler, Jens; White, Stephen W.

    2016-01-01

    The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY–ssDNA interaction occurs within the assembly via two distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNA–gp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNA-binding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA–UvsX filament. PMID:26951671

  1. Recombination between linear double-stranded DNA substrates in vivo

    PubMed Central

    Narayanan, Kumaran; Sim, Edmund Ui-Hang; Ravin, Nikolai V.; Lee, Choon-Weng

    2009-01-01

    Recombineering technology in E. coli enables targeting of linear donor DNA to circular recipient DNA using short shared homology sequences. In this work, we demonstrate that recombineering is also able to support recombination between a pair of linear DNA substrates (linear/linear recombineering) in vivo in E. coli. Linear DNA up to 100 kb is accurately modified and remains intact without undergoing rearrangements after recombination. This system will be valuable for direct in vivo manipulation of large linear DNA including the N15 and PY54 prophages and linear animal viruses, and for assembly of linear constructs as artificial chromosome vectors. PMID:19454252

  2. Effect of vanillin on toxicant-induced mutation and mitotic recombination in proliferating somatic cells of Drosophila melanogaster.

    PubMed

    Sinigaglia, Marialva; Reguly, Maria Luíza; de Andrade, Heloísa Helena Rodrigues

    2004-01-01

    Vanillin (VA; C8H8O3) is a flavoring agent that in previous studies has both increased and decreased the genotoxicity of chemical agents, depending on the nature of both the agent and the genetic event measured. The ability of VA to modulate the mutagenicity and recombinogenicity of three different monoalkylating agents, N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), and ethyl methanesulfonate (EMS), and the intercalating agent bleomycin (BLEO) was examined using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. While neither the mutagenicity nor the recombinagenicity of ENU or MNU was modified by posttreatment with VA, EMS-induced genetic toxicity was enhanced by as much as 30%. This overall enhancement included a synergistic increase in mitotic recombination and a lesser decrease in mutation. Posttreatment with VA also produced an increase in the genotoxicity of BLEO, which was characterized by increases of 120% and 180% for 0.5% and 1% VA, respectively. This enhancement was restricted to an increase in recombinational events, since no alteration in BLEO-induced mutation was observed. The data suggest that the major VA-modulatory action on genotoxicity in D. melanogaster is related to its synergistic effects on somatic recombination, which has a greater consequence on overall genotoxicity than its antimutagenic effects. Since the SMART assay is specifically sensitive to mitotic crossing-over, our data suggest that VA promotes toxicant-induced homologous recombination, at least in the proliferative cells of Drosophila. PMID:15515154

  3. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  4. Coalescent Simulation of Intracodon Recombination

    PubMed Central

    Arenas, Miguel; Posada, David

    2010-01-01

    The coalescent with recombination is a very useful tool in molecular population genetics. Under this framework, genealogies often represent the evolution of the substitution unit, and because of this, the few coalescent algorithms implemented for the simulation of coding sequences force recombination to occur only between codons. However, it is clear that recombination is expected to occur most often within codons. Here we have developed an algorithm that can evolve coding sequences under an ancestral recombination graph that represents the genealogies at each nucleotide site, thereby allowing for intracodon recombination. The algorithm is a modification of Hudson's coalescent in which, in addition to keeping track of events occurring in the ancestral material that reaches the sample, we need to keep track of events occurring in ancestral material that does not reach the sample but that is produced by intracodon recombination. We are able to show that at typical substitution rates the number of nonsynonymous changes induced by intracodon recombination is small and that intracodon recombination does not generally result in inflated estimates of the overall nonsynonymous/synonymous substitution ratio (ω). On the other hand, recombination can bias the estimation of ω at particular codons, resulting in apparent rate variation among sites and in the spurious identification of positively selected sites. Importantly, in this case, allowing for variable synonymous rates across sites greatly reduces the false-positive rate and recovers statistical power. Finally, coalescent simulations with intracodon recombination could be used to better represent the evolution of nuclear coding genes or fast-evolving pathogens such as HIV-1.We have implemented this algorithm in a computer program called NetRecodon, freely available at http://darwin.uvigo.es. PMID:19933876

  5. Combinatorics in Recombinational Population Genomics

    NASA Astrophysics Data System (ADS)

    Parida, Laxmi

    The work that I will discuss is motivated by the need for understanding, and processing, the manifestations of recombination events in chromosome sequences. In this talk, we focus on two related problems. First, we explore the very general problem of reconstructability of pedigree history. How plausible is it to unravel the history of a complete unit (chromosome) of inheritance? The second problem deals with reconstructing the recombinational history of a collection of chromosomes.

  6. An Overview of the Molecular Mechanisms of Recombinational DNA Repair.

    PubMed

    Kowalczykowski, Stephen C

    2015-11-01

    Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of th