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Sample records for homologous recombination system

  1. The homologous recombination system of Ustilago maydis.

    PubMed

    Holloman, William K; Schirawski, Jan; Holliday, Robin

    2008-08-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of U. maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins.

  2. The homologous recombination system of Ustilago maydis

    PubMed Central

    Holloman, William K.; Schirawski, Jan; Holliday, Robin

    2008-01-01

    Homologous recombination is a high fidelity, template-dependent process that is used in repair of damaged DNA, recovery of broken replication forks, and disjunction of homologous chromosomes in meiosis. Much of what is known about recombination genes and mechanisms comes from studies on baker's yeast. Ustilago maydis, a basidiomycete fungus, is distant evolutionarily from baker's yeast and so offers the possibility of gaining insight into recombination from an alternative perspective. Here we have surveyed the genome of Ustilago maydis to determine the composition of its homologous recombination system. Compared to baker's yeast, there are fundamental differences in the function as well as in the repertoire of dedicated components. These include the use of a BRCA2 homolog and its modifier Dss1 rather than Rad52 as a mediator of Rad51, the presence of only a single Rad51 paralog, and the absence of Dmc1 and auxiliary meiotic proteins. PMID:18502156

  3. 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

  4. Induction of the homologous recombination system by hexavalent chromium in Rhizobium etli.

    PubMed

    Santoyo, Gustavo; Orozco-Mosqueda, Montserrat; Valdez-Martínez, Gabriela; Orozco-Mosqueda, Ma Del Carmen

    2015-01-01

    Induction of homologous recombination in Rhizobium etli to repair the DNA damage caused by hexavalent chromium (Cr) was evaluated. Mutants in recombination genes such as addA, recF, recA, ruvB, recG, and a double mutant ruvBrecG showed different sensitivity levels to Cr. As expected, the recA mutant showed the highest susceptibility, while complementation restored the Cr-resistant phenotype, similar to the wild-type strain. Small plasmid recombination increased up to 30-fold in the presence of Cr (0.05 mM) in the wild-type strain, while no change was observed in the recA mutant. A 20-fold increase in small plasmid recombination was also observed in the addA mutant in the presence of Cr. In addition, the ruvB mutant showed similar increases with Cr exposure to the wild-type strain, suggesting that other genetic elements may substitute its important role during recombination. Interestingly, continuous Cr exposure (0.05 mM) clearly induced the genetic expression of addA, recA, and ruvB genes. Finally, recombination mutants also showed susceptibility to other DNA-damaging agents such as tellurite and selenite. Together, these results confirm the induction and significance of the R. etli homologous recombination system to repair DNA damage caused by hexavalent Cr.

  5. [An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea].

    PubMed

    Xiaoli, Wang; Chuang, Jiang; Jianhua, Liu; Xipeng, Liu

    2015-04-01

    With the development of functional genomics, gene-knockout is becoming an important tool to elucidate gene functions in vivo. As a good model strain for archaeal genetics, Haloferax volcanii has received more attention. Although several genetic manipulation systems have been developed for some halophilic archaea, it is time-consuming because of the low percentage of positive clones during the second-recombination selection. These classical gene knockout methods are based on DNA recombination between the genomic homologous sequence and the circular suicide plasmid, which carries a pyrE selection marker and two DNA fragments homologous to the upstream and downstream fragments of the target gene. Many wild-type clones are obtained through a reverse recombination between the plasmid and genome in the classic gene knockout method. Therefore, it is necessary to develop an efficient gene knockout system to increase the positive clone percentage. Here we report an improved gene knockout method using a linear DNA cassette consisting of upstream and downstream homologous fragments, and the pyrE marker. Gene deletions were subsequently detected by colony PCR analysis. We determined the efficiency of our knockout method by deleting the xpb2 gene from the H. volcanii genome, with the percentage of positive clones higher than 50%. Our method provides an efficient gene knockout strategy for halophilic archaea.

  6. Orientation Dependence in Homologous Recombination

    PubMed Central

    Yamamoto, K.; Takahashi, N.; Fujitani, Y.; Yoshikura, H.; Kobayashi, I.

    1996-01-01

    Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo(+) recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistant with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs. PMID:8722759

  7. Homologous recombination and its regulation

    PubMed Central

    Krejci, Lumir; Altmannova, Veronika; Spirek, Mario; Zhao, Xiaolan

    2012-01-01

    Homologous recombination (HR) is critical both for repairing DNA lesions in mitosis and for chromosomal pairing and exchange during meiosis. However, some forms of HR can also lead to undesirable DNA rearrangements. Multiple regulatory mechanisms have evolved to ensure that HR takes place at the right time, place and manner. Several of these impinge on the control of Rad51 nucleofilaments that play a central role in HR. Some factors promote the formation of these structures while others lead to their disassembly or the use of alternative repair pathways. In this article, we review these mechanisms in both mitotic and meiotic environments and in different eukaryotic taxa, with an emphasis on yeast and mammal systems. Since mutations in several proteins that regulate Rad51 nucleofilaments are associated with cancer and cancer-prone syndromes, we discuss how understanding their functions can lead to the development of better tools for cancer diagnosis and therapy. PMID:22467216

  8. Random-walk model of homologous recombination

    NASA Astrophysics Data System (ADS)

    Fujitani, Youhei; Kobayashi, Ichizo

    1995-12-01

    Interaction between two homologous (i.e., identical or nearly identical) DNA sequences leads to their homologous recombination in the cell. We present the following stochastic model to explain the dependence of the frequency of homologous recombination on the length of the homologous region. The branch point connecting the two DNAs in a reaction intermediate follows the random-walk process along the homology (N base-pairs). If the branch point reaches either of the homology ends, it bounds back to the homologous region at a probability of γ (reflection coefficient) and is destroyed at a probability of 1-γ. When γ is small, the frequency of homologous recombination is found to be proportional to N3 for smaller N and a linear function of N for larger N. The exponent of the nonlinear dependence for smaller N decreases from three as γ increases. When γ=1, only the linear dependence is left. These theoretical results can explain many experimental data in various systems. (c) 1995 The American Physical Society

  9. 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.

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

    PubMed

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

    2014-04-15

    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.

  11. [Establishment of genetic transformation system of Schizochytrium sp. by homologous recombination].

    PubMed

    Zhuang, Xiaoyan; Chen, Shenglan; Ji, Xiaojun; Xu, Xian; Ren, Lujing

    2015-04-04

    Schizochytrium sp. is a marine fungus that can produce DHA efficiently. Genetic engineering has been successfully used in industrial strain improvement and metabolic studies. In order to use genetic engineering to modified Schizochytrium sp., we established an genetic transformation system of Schizochytrium sp. A genetic transformation system of Schizochytrium sp. was established by 18S rDNA-targeted homologous recombination. The targeting vector contained a part of 18S rDNA from Schizochytrium sp. and the ble gene. This targeting vector was transformed into Schizochytrium sp. by electroporation and then selected by Zeocin-containing plates. The incorporation of exogenous ble gene into the genome of Schizochytrium was inspected by PCR amplification. Fermentation results show that the transformants had similar cell dry weight, lipid yield, DHA content, and composition of other fatty acids to the wild type strain. Our results show that the introduction of resistance gene did not affect the cell growth and lipid metabolism. This system could be used to introduce new functional genes into Schizochytrium sp.

  12. Homologous recombination using bacterial artificial chromosomes.

    PubMed

    Lai, Cary; Fischer, Tobias; Munroe, Elizabeth

    2015-02-02

    This protocol introduces the technique of homologous recombination in bacteria to insert a linear DNA fragment into bacterial artificial chromosomes (BACs). Homologous recombination allows the modification of large DNA molecules, in contrast with conventional restriction endonuclease-based strategies, which cleave large DNAs into numerous fragments and are unlikely to permit the precise targeting afforded by recombination-based approaches. The method uses a phage lambda-derived recombination system (using exo, beta, and gam) as well as other enzymatic activities provided by the host (Escherichia coli). In the method described here, a DNA fragment encoding enhanced cyan fluorescent protein is inserted immediately after the start codon of the gene encoding choline acetyltransferase ("ChAT"), the final enzyme in acetylcholine biosynthesis, using homologous recombination between sequences that are present both on the introduced DNA fragment and in the target BAC. The desired recombination products are identified via positive selection for resistance to kanamycin. In principle, the resulting modified BAC could be used to produce transgenic mice that express this fluorescent protein in cholinergic neurons. The approach described here could be used to insert any DNA fragment.

  13. 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

  14. Homologous recombination in plants is organ specific.

    PubMed

    Boyko, Alexander; Filkowski, Jody; Hudson, Darryl; Kovalchuk, Igor

    2006-03-20

    In this paper we analysed the genome stability of various Arabidopsis thaliana plant organs using a transgenic recombination system. The system was based on two copies of non-functional GUS (lines #651 and #11) or LUC (line #15D8) reporter genes serving as a recombination substrate. Both reporter assays showed that recombination in flowers or stems were rare events. Most of the recombination sectors were found in leaves and roots, with leaves having over 2-fold greater number of the recombination events per single cell genome as compared to roots. The recombination events per single genome were 9.7-fold more frequent on the lateral half of the leaves than on the medial halves. This correlated with a 2.5-fold higher metabolic activity in the energy source (lateral) versus energy sink (medial) of leaves. Higher metabolic activity was paralleled by a higher anthocyanin production in lateral halves. The level of double strand break (DSB) occurrence was also different among plant organs; the highest level was observed in roots and the lowest in leaves. High level of DSBs strongly positively correlated with the activity of the key repair enzymes, AtKU70 and AtRAD51. The ratio of AtRAD51 to AtKU70 expression was the highest in leaves, supporting the more active involvement of homologous recombination pathway in the repair of DSBs in this organ. Western blot analysis confirmed the real time PCR expression data for AtKU70 gene.

  15. Efficient system of homologous RNA recombination in brome mosaic virus: sequence and structure requirements and accuracy of crossovers.

    PubMed Central

    Nagy, P D; Bujarski, J J

    1995-01-01

    Brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants engineered to support intersegment RNA recombination, was used for the determination of sequence and structural requirements of homologous crossovers. A 60-nucleotide (nt) sequence, common between wild-type RNA2 and mutant RNA3, supported efficient repair (90%) of a modified 3' noncoding region in the RNA3 segment by homologous recombination with wild-type RNA2 3' noncoding sequences. Deletions within this sequence in RNA3 demonstrated that a nucleotide identity as short as 15 nt can support efficient homologous recombination events, while shorter (5-nt) sequence identity resulted in reduced recombination frequency (5%) within this region. Three or more mismatches within a downstream portion of the common 60-nt RNA3 sequence affected both the incidence of recombination and the distribution of crossover sites, suggesting that besides the length, the extent of sequence identity between two recombining BMV RNAs is an important factor in homologous recombination. Site-directed mutagenesis of the common sequence in RNA3 did not reveal a clear correlation between the stability of predicted secondary structures and recombination activity. This indicates that homologous recombination does not require similar secondary structures between two recombining RNAs at the sites of crossovers. Nearly 20% of homologous recombinants were imprecise (aberrant), containing either nucleotide mismatches, small deletions, or small insertions within the region of crossovers. This implies that homologous RNA recombination is not as accurate as proposed previously. Our results provide experimental evidence that the requirements and thus the mechanism of homologous recombination in BMV differ from those of previously described heteroduplex-mediated nonhomologous recombination (P. D. Nagy and J. J. Bujarski, Proc. Natl. Acad. Sci. USA 90:6390-6394, 1993). PMID:7983703

  16. Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system.

    PubMed

    Lu, Xiongbin; Lozano, Guillermina; Donehower, Lawrence A

    2003-01-28

    DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo(R) marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53(+/-) mouse fibroblasts show elevated levels of homologous recombination compared to their p53(+/+) counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

  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. DNA Sequence Alignment during Homologous Recombination*

    PubMed Central

    Greene, Eric C.

    2016-01-01

    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

  19. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system.

    PubMed Central

    Jessberger, R; Berg, P

    1991-01-01

    We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed. Images PMID:1986239

  1. 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

  2. Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks.

    PubMed

    Nakano, Toshiaki; Morishita, Soh; Katafuchi, Atsushi; Matsubara, Mayumi; Horikawa, Yusuke; Terato, Hiroaki; Salem, Amir M H; Izumi, Shunsuke; Pack, Seung Pil; Makino, Keisuke; Ide, Hiroshi

    2007-10-12

    DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strand-block the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCD-dependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.

  3. Mismatch repair during homologous and homeologous recombination.

    PubMed

    Spies, Maria; Fishel, Richard

    2015-03-02

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans.

  4. Mismatch Repair during Homologous and Homeologous Recombination

    PubMed Central

    Spies, Maria; Fishel, Richard

    2015-01-01

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans. PMID:25731766

  5. Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

    PubMed Central

    Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G.; Court, Donald L.

    2009-01-01

    Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about a week's time. PMID:19180090

  6. 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.

  7. Reversing Platinum Resistance in High-Grade Serous Ovarian Carcinoma: Targeting BRCA and the Homologous Recombination System.

    PubMed

    Wiedemeyer, W Ruprecht; Beach, Jessica A; Karlan, Beth Y

    2014-01-01

    Resistance to platinum chemotherapy is one of the main factors driving ovarian cancer mortality, and overcoming platinum resistance is considered one of the greatest challenges in ovarian cancer research. Genetic and functional evidence points to the homologous recombination (HR) DNA repair system, and BRCA1 and BRCA2 in particular, as main determinants of response to platinum therapy. BRCA-mutant ovarian cancers are especially sensitive to platinum, associated with better survival, and amenable to poly ADP ribose polymerase inhibitor treatment. Here, we discuss a therapeutic concept that seeks to disrupt HR capacity via targeting of BRCA1 and BRCA2 functionality in order to reverse platinum resistance in BRCA-proficient high-grade serous ovarian cancers (HGSOC). We review the molecular signaling pathways that converge on BRCA1 and BRCA2, their activation status in ovarian cancer, and therapeutic options to modulate BRCA function. Several recent publications demonstrate efficient chemosensitization of BRCA-proficient cancers by combining targeted therapy with standard platinum-based agents. Due to its inherent genomic heterogeneity, molecularly defined subgroups of HGSOC may require different approaches. We seek to provide an overview of available agents and their potential use to reverse platinum resistance by inhibiting the HR system, either directly or indirectly, by targeting oncogenic activators of HR.

  8. Homologous recombination in plants: an antireview.

    PubMed

    Lieberman-Lazarovich, Michal; Levy, Avraham A

    2011-01-01

    Homologous recombination (HR) is a central cellular process involved in many aspects of genome maintenance such as DNA repair, replication, telomere maintenance, and meiotic chromosomal segregation. HR is highly conserved among eukaryotes, contributing to genome stability as well as to the generation of genetic diversity. It has been intensively studied, for almost a century, in plants and in other organisms. In this antireview, rather than reviewing existing knowledge, we wish to underline the many open questions in plant HR. We will discuss the following issues: how do we define homology and how the degree of homology affects HR? Are there any plant-specific HR qualities, how extensive is functional conservation and did HR proteins acquire new functions? How efficient is HR in plants and what are the cis and the trans factors that regulate it? Finally, we will give the prospects for enhancing the rates of gene targeting and meiotic HR for plant breeding purposes.

  9. Recombineering: genetic engineering in bacteria using homologous recombination.

    PubMed

    Thomason, Lynn C; Sawitzke, James A; Li, Xintian; Costantino, Nina; Court, Donald L

    2014-04-14

    The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques. Copyright © 2014 John Wiley & Sons, Inc.

  10. Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development.

    PubMed

    Orii, Kenji E; Lee, Youngsoo; Kondo, Naomi; McKinnon, Peter J

    2006-06-27

    The repair of DNA double-strand breaks (DSBs) occurs via nonhomologous end-joining (NHEJ) or homologous recombination (HR). These mechanistically distinct pathways are critical for maintenance of genomic integrity and organismal survival. Although inactivation of either pathway leads to embryonic lethality, here we show selective requirements for each DNA DSB repair pathway at different stages of mammalian nervous system development. DNA damage-induced apoptosis resulting from inactivation of HR (Xrcc2 deficiency) only occurred in proliferating neural precursor cells, whereas disruption of NHEJ (DNA ligase IV deficiency) mainly affected differentiating cells at later developmental stages. Therefore, these data suggest that NHEJ is dispensable for a substantial portion of early development because DSB repair during this period utilizes HR. Moreover, DNA damage-induced apoptosis required the ataxia telangiectasia mutated (Atm) kinase after disruption of NHEJ, but not HR, during neurogenesis. However, embryonic lethality arising from disruption of either repair pathway was rescued by loss of p53 and resulted in specific tumor types reflective of the particular DSB repair pathway inactivated. Thus, these data reveal distinct tissue- and cell-type requirements for each DNA DSB repair pathway during neural development and provide insights for understanding the contributions of DNA DSB responses to disease.

  11. Recombination between adenovirus type 12 DNA and a hamster preinsertion sequence in a cell-free system. Patch homologies and fractionation of nuclear extracts.

    PubMed

    Tatzelt, J; Scholz, B; Fechteler, K; Jessberger, R; Doerfler, W

    1992-07-05

    We have previously described a cell-free recombination system derived from hamster cell nuclear extracts in which the in vitro recombination between a hamster preinsertion sequence, the cloned 1768 base-pair p7 fragment, and adenovirus type 12 (Ad12) DNA has been demonstrated. The nuclear extracts have now been subfractionated by gel filtration on a Sephacryl S-300 column. The activity promoting cell-free recombination elutes from the Sephacryl S-300 matrix with the shoulder and not the peak fractions of the absorbancy profile. By using these protein subfractions, in vitro recombinants have been generated between the p7 preinsertion sequence and the 60 to 70 map unit fragment of Ad12 DNA, which has previously shown high recombination frequency. In all of the analyzed recombinants thus produced in vitro, striking patchy homologies have been observed between the p7 and Ad12 junction sequences, and between Ad12 DNA or p7 DNA and pBR322 DNA. The patchy homologies are similar to those found earlier during the analyses of some of the junction sequences in integrated Ad12 genomes in Ad12-induced hamster tumor cell lines. Proteins in the shoulder fractions of the gel-filtration experiment can form specific complexes with double-stranded synthetic oligodeoxyribonucleotides corresponding to several p7 and Ad12 DNA sequences. These sequences participate in the recombination reactions catalyzed by the same column fractions in the shoulder of the absorbancy profile. Such proteins have not been found in the peak fractions. Further work will be required to ascertain that the cell-free recombination system mimics certain elements of the mechanisms of integrative recombination and to purify the cellular components essential for recombination.

  12. Homologous recombination catalyzed by a nuclear extract from Xenopus oocytes.

    PubMed Central

    Lehman, C W; Carroll, D

    1991-01-01

    Xenopus laevis oocytes efficiently recombine linear DNA injected into their nuclei (germinal vesicles). This process requires homologous sequences at or near the molecular ends. Here we report that a cell-free extract made from germinal vesicles is capable of accomplishing the complete recombination reaction in vitro. Like the in vivo process, the extract converts the overlapping ends of linear substrate molecules into covalently closed products. Establishment of this cell-free system has allowed examination of the cofactors required for recombination. The first step involves a 5'----3' exonuclease activity that requires a divalent cation but not NTPs. Completion of recombination requires a hydrolyzable NTP; maximal product formation occurs in the presence of millimolar levels of ATP or dATP. At submillimolar levels of all four dNTPs, homologous recombination is inefficient, and a side reaction produces end-joined products. This cell-free system should facilitate a step-by-step understanding of an homologous recombination pathway that operates not only in Xenopus laevis oocytes but also in cells from a wide variety of organisms. Images PMID:1961753

  13. Homologous recombination prevents methylation-induced toxicity in Escherichia coli.

    PubMed

    Nowosielska, Anetta; Smith, Stephen A; Engelward, Bevin P; Marinus, M G

    2006-01-01

    Methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.

  14. Rad54, the Motor of Homologous Recombination

    PubMed Central

    Mazin, Alexander V.; Mazina, Olga M.; Bugreev, Dmitry V.; Rossi, Matthew J.

    2009-01-01

    Homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. The invasion produces a cross-stranded structure, known as the Holliday junction. Here, we describe the properties of Rad54, an important and versatile HR protein that is evolutionarily conserved in eukaryotes. Rad54 is a motor protein that translocates along dsDNA and performs several important functions in HR. The current review focuses on the recently identified Rad54 activities which contribute to the late phase of HR, especially the branch migration of Holliday junctions. PMID:20089461

  15. Histone deacetylases 9 and 10 are required for homologous recombination.

    PubMed

    Kotian, Shweta; Liyanarachchi, Sandhya; Zelent, Arthur; Parvin, Jeffrey D

    2011-03-11

    We tested the role of histone deacetylases (HDACs) in the homologous recombination process. A tissue-culture based homology-directed repair assay was used in which repair of a double-stranded break by homologous recombination results in gene conversion of an inactive GFP allele to an active GFP gene. Our rationale was that hyperacetylation caused by HDAC inhibitor treatment would increase chromatin accessibility to repair factors, thereby increasing homologous recombination. Contrary to expectation, treatment of cells with the inhibitors significantly reduced homologous recombination activity. Using RNA interference to deplete each HDAC, we found that depletion of either HDAC9 or HDAC10 specifically inhibited homologous recombination. By assaying for sensitivity of cells to the interstrand cross-linker mitomycin C, we found that treatment of cells with HDAC inhibitors or depletion of HDAC9 or HDAC10 resulted in increased sensitivity to mitomycin C. Our data reveal an unanticipated function of HDAC9 and HDAC10 in the homologous recombination process.

  16. [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.

  17. New insights into the mechanism of homologous recombination in yeast.

    PubMed

    Aylon, Yael; Kupiec, Martin

    2004-05-01

    Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. Repair of DSBs by homologous recombination provides an efficient and fruitful pathway to restore chromosomal integrity. Exciting new work in yeast has lately provided insights into this complex process. Many of the proteins involved in recombination have been isolated and the details of the repair mechanism are now being unraveled at the molecular level. In this review, we focus on recent studies which dissect the recombinational repair of a single broken chromosome. After DSB formation, a decision is made regarding the mechanism of repair (recombination or non-homologous end-joining). This decision is under genetic control. Once committed to the recombination pathway, the broken chromosomal ends are resected by a still unclear mechanism in which the DNA damage checkpoint protein Rad24 participates. At this stage several proteins are recruited to the broken ends, including Rad51p, Rad52p, Rad55p, Rad57p, and possibly Rad54p. A genomic search for homology ensues, followed by strand invasion, promoted by the Rad51 filament with the participation of Rad55p, Rad57p and Rad54p. DNA synthesis then takes place, restoring the resected ends. Crossing-over formation depends on the length of the homologous recombining sequences, and is usually counteracted by the activity of the mismatch repair system. Given the conservation of the repair mechanisms and genes throughout evolution, these studies have profound implications for other eukaryotic organisms.

  18. How homologous recombination maintains telomere integrity.

    PubMed

    Tacconi, Eliana M C; Tarsounas, Madalena

    2015-06-01

    Telomeres protect the ends of linear chromosomes against loss of genetic information and inappropriate processing as damaged DNA and are therefore crucial to the maintenance of chromosome integrity. In addition to providing a pathway for genome-wide DNA repair, homologous recombination (HR) plays a key role in telomere replication and capping. Consistent with this, the genomic instability characteristic of HR-deficient cells and tumours is driven in part by telomere dysfunction. Here, we discuss the mechanisms by which HR modulates the response to intrinsic cellular challenges that arise during telomere replication, as well as its impact on the assembly of telomere protective structures. How normal and tumour cells differ in their ability to maintain telomeres is deeply relevant to the search for treatments that would selectively eliminate cells whose capacity for HR-mediated repair has been compromised.

  19. High-frequency homologous recombination in plants mediated by zinc-finger nucleases.

    PubMed

    Wright, David A; Townsend, Jeffrey A; Winfrey, Ronnie Joe; Irwin, Phillip A; Rajagopal, Jyothi; Lonosky, Patricia M; Hall, Bradford D; Jondle, Michael D; Voytas, Daniel F

    2005-11-01

    Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.

  20. Increased chromosome mobility facilitates homology search during recombination.

    PubMed

    Miné-Hattab, Judith; Rothstein, Rodney

    2012-04-08

    Homologous recombination, an essential process for preserving genomic integrity, uses intact homologous sequences to repair broken chromosomes. To explore the mechanism of homologous pairing in vivo, we tagged two homologous loci in diploid yeast Saccharomyces cerevisiae cells and investigated their dynamic organization in the absence and presence of DNA damage. When neither locus is damaged, homologous loci occupy largely separate regions, exploring only 2.7% of the nuclear volume. Following the induction of a double-strand break, homologous loci co-localize ten times more often. The mobility of the cut chromosome markedly increases, allowing it to explore a nuclear volume that is more than ten times larger. Interestingly, the mobility of uncut chromosomes also increases, allowing them to explore a four times larger volume. We propose a model for homology search in which increased chromosome mobility facilitates homologous pairing. Finally, we find that the increase in DNA dynamics is dependent on early steps of homologous recombination.

  1. Homologous Recombination between Autonomously Replicating Plasmids in Mammalian Cells

    PubMed Central

    Ayares, David; Spencer, James; Schwartz, Faina; Morse, Brian; Kucherlapati, Raju

    1985-01-01

    The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neo R colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis. PMID:2996980

  2. Precise genome editing by homologous recombination

    PubMed Central

    Hoshijima, K.; Jurynec, M.J.; Grunwald, D.J.

    2016-01-01

    Simple and efficient methods are presented for creating precise modifications of the zebrafish genome. Edited alleles are generated by homologous recombination between the host genome and double-stranded DNA (dsDNA) donor molecules, stimulated by the induction of double-strand breaks at targeted loci in the host genome. Because several kilobase-long tracts of sequence can be exchanged, multiple genome modifications can be generated simultaneously at a single locus. Methods are described for creating: (1) alleles with simple sequence changes or in-frame additions, (2) knockin/knockout alleles that express a reporter protein from an endogenous locus, and (3) conditional alleles in which exons are flanked by recombinogenic loxP sites. Significantly, our approach to genome editing allows the incorporation of a linked reporter gene into the donor sequences so that successfully edited alleles can be identified by virtue of expression of the reporter. Factors affecting the efficiency of genome editing are discussed, including the finding that dsDNA products of I-SceI meganuclease enzyme digestion are particularly effective as donor molecules for gene-editing events. Reagents and procedures are described for accomplishing efficient genome editing in the zebrafish. PMID:27443923

  3. DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination.

    PubMed

    Kuzminov, A

    2001-07-17

    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.

  4. Recombination, Pairing, and Synapsis of Homologs during Meiosis

    PubMed Central

    Zickler, Denise; Kleckner, Nancy

    2015-01-01

    Recombination is a prominent feature of meiosis in which it plays an important role in increasing genetic diversity during inheritance. Additionally, in most organisms, recombination also plays mechanical roles in chromosomal processes, most notably to mediate pairing of homologous chromosomes during prophase and, ultimately, to ensure regular segregation of homologous chromosomes when they separate at the first meiotic division. Recombinational interactions are also subject to important spatial patterning at both early and late stages. Recombination-mediated processes occur in physical and functional linkage with meiotic axial chromosome structure, with interplay in both directions, before, during, and after formation and dissolution of the synaptonemal complex (SC), a highly conserved meiosis-specific structure that links homolog axes along their lengths. These diverse processes also are integrated with recombination-independent interactions between homologous chromosomes, nonhomology-based chromosome couplings/clusterings, and diverse types of chromosome movement. This review provides an overview of these diverse processes and their interrelationships. PMID:25986558

  5. Recombination, Pairing, and Synapsis of Homologs during Meiosis.

    PubMed

    Zickler, Denise; Kleckner, Nancy

    2015-05-18

    Recombination is a prominent feature of meiosis in which it plays an important role in increasing genetic diversity during inheritance. Additionally, in most organisms, recombination also plays mechanical roles in chromosomal processes, most notably to mediate pairing of homologous chromosomes during prophase and, ultimately, to ensure regular segregation of homologous chromosomes when they separate at the first meiotic division. Recombinational interactions are also subject to important spatial patterning at both early and late stages. Recombination-mediated processes occur in physical and functional linkage with meiotic axial chromosome structure, with interplay in both directions, before, during, and after formation and dissolution of the synaptonemal complex (SC), a highly conserved meiosis-specific structure that links homolog axes along their lengths. These diverse processes also are integrated with recombination-independent interactions between homologous chromosomes, nonhomology-based chromosome couplings/clusterings, and diverse types of chromosome movement. This review provides an overview of these diverse processes and their interrelationships.

  6. A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination.

    PubMed

    Johnson, R A; Hellens, R P; Love, D R

    2011-09-16

    Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidate genes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.

  7. MutS2 Promotes Homologous Recombination in Bacillus subtilis.

    PubMed

    Burby, Peter E; Simmons, Lyle A

    2017-01-15

    Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair (MMR) pathway. In contrast to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Gram-positive bacterium Bacillus subtilis In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B. subtilis We found that deletion of mutS2 renders B. subtilis sensitive to the natural antibiotic mitomycin C (MMC), which requires homologous recombination for repair. We demonstrate that the C-terminal small MutS-related (Smr) domain is necessary but not sufficient for tolerance to MMC. Further, we developed a CRISPR/Cas9 genome editing system to test if the inducible prophage PBSX was the underlying cause of the observed MMC sensitivity. Genetic analysis revealed that MMC sensitivity was dependent on recombination and not on nucleotide excision repair or a symptom of prophage PBSX replication and cell lysis. We found that deletion of mutS2 resulted in decreased transformation efficiency using both plasmid and chromosomal DNA. Further, deletion of mutS2 in a strain lacking the Holliday junction endonuclease gene recU resulted in increased MMC sensitivity and decreased transformation efficiency, suggesting that MutS2 could function redundantly with RecU. Together, our results support a model where B. subtilis MutS2 helps to promote homologous recombination, demonstrating a new function for bacterial MutS2.

  8. Regulation of DNA strand exchange in homologous recombination.

    PubMed

    Holthausen, J Thomas; Wyman, Claire; Kanaar, Roland

    2010-12-10

    Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors.

    PubMed

    Kelso, Andrew A; Waldvogel, Sarah M; Luthman, Adam J; Sehorn, Michael G

    2017-01-01

    Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity.

  10. Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors

    PubMed Central

    Kelso, Andrew A.; Waldvogel, Sarah M.; Luthman, Adam J.; Sehorn, Michael G.

    2017-01-01

    Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity. PMID:28936205

  11. Transformation of Mycoplasma pulmonis: demonstration of homologous recombination, introduction of cloned genes, and preliminary description of an integrating shuttle system.

    PubMed Central

    Mahairas, G G; Minion, F C

    1989-01-01

    The transposons Tn916 and Tn4001 and a series of integrating plasmids derived from their antibiotic resistance genes were used to examine polyethylene glycol-mediated transformation of Mycoplasma pulmonis. Under optimal conditions, Tn916 and Tn4001 could be introduced into M. pulmonis at frequencies of 1 x 10(-6) and 5 x 10(-5) per CFU, respectively. Integrating plasmids were constructed with the cloned antibiotic resistance determinants of Tn916 and Tn4001, a pMB1-derived plasmid replicon, and mycoplasmal chromosomal DNA and were used to examine recombinational events after transformation into M. pulmonis. Under optimal conditions, chromosomal integrations could be recovered at a frequency of 1 x 10(-4) to 1 x 10(-6) per CFU, depending on the size and nature of the chromosomal insert and the parental plasmid. Integrated plasmids were stable in the absence of selection and could be rescued in Escherichia coli along with adjacent mycoplasma DNA. These studies provide the first direct evidence of a recombination system in the Mollicutes and describe the first E. coli-M. pulmonis shuttle vectors. Images PMID:2539351

  12. 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

  13. Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

    PubMed Central

    Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

    2014-01-01

    Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

  14. Efficient Assembly of DNA Using Yeast Homologous Recombination (YHR).

    PubMed

    Chandran, Sunil; Shapland, Elaine

    2017-01-01

    The assembly of multiple DNA parts into a larger DNA construct is a requirement in most synthetic biology laboratories. Here we describe a method for the efficient, high-throughput, assembly of DNA utilizing the yeast homologous recombination (YHR). The YHR method utilizes overlapping DNA parts that are assembled together by Saccharomyces cerevisiae via homologous recombination between designed overlapping regions. Using this method, we have successfully assembled up to 12 DNA parts in a single reaction.

  15. A strand invasion 3' polymerization intermediate of mammalian homologous recombination.

    PubMed

    Si, Weiduo; Mundia, Maureen M; Magwood, Alissa C; Mark, Adam L; McCulloch, Richard D; Baker, Mark D

    2010-06-01

    Initial events in double-strand break repair by homologous recombination in vivo involve homology searching, 3' strand invasion, and new DNA synthesis. While studies in yeast have contributed much to our knowledge of these processes, in comparison, little is known of the early events in the integrated mammalian system. In this study, a sensitive PCR procedure was developed to detect the new DNA synthesis that accompanies mammalian homologous recombination. The test system exploits a well-characterized gene targeting assay in which the transfected vector bears a gap in the region of homology to the single-copy chromosomal immunoglobulin mu heavy chain gene in mouse hybridoma cells. New DNA synthesis primed by invading 3' vector ends copies chromosomal mu-gene template sequences excluded by the vector-borne double-stranded gap. Following electroporation, specific 3' extension products from each vector end are detected with rapid kinetics: they appear after 0.5 hr, peak at 3-6 hr, and then decline, likely as a result of the combined effects of susceptibility to degradation and cell division. New DNA synthesis from each vector 3' end extends at least approximately 1000 nucleotides into the gapped region, but the efficiency declines markedly within the first approximately 200 nucleotides. Over this short distance, an average frequency of 3' extension for the two invading vector ends is approximately 0.007 events/vector backbone. DNA sequencing reveals precise copying of the cognate chromosomal mu-gene template. In unsynchronized cells, 3' extension is sensitive to aphidicolin supporting involvement of a replicative polymerase. Analysis suggests that the vast majority of 3' extensions reside on linear plasmid molecules.

  16. Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein.

    PubMed

    Buerstedde, Jean-Marie; Lowndes, Noel; Schatz, David G

    2014-07-08

    The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells.DOI: http://dx.doi.org/10.7554/eLife.03110.001.

  17. Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.

    PubMed

    Scheel, Troels K H; Galli, Andrea; Li, Yi-Ping; Mikkelsen, Lotte S; Gottwein, Judith M; Bukh, Jens

    2013-03-01

    Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13-36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5' end to the NS2-NS3 region followed by JFH1 sequence from Core to the 3' end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and

  18. Productive Homologous and Non-homologous Recombination of Hepatitis C Virus in Cell Culture

    PubMed Central

    Li, Yi-Ping; Mikkelsen, Lotte S.; Gottwein, Judith M.; Bukh, Jens

    2013-01-01

    Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13–36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5′ end to the NS2–NS3 region followed by JFH1 sequence from Core to the 3′ end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants

  19. Genome destabilization by homologous recombination in the germline

    PubMed Central

    Sasaki, Mariko; Lange, Julian; Keeney, Scott

    2011-01-01

    Meiotic recombination, which promotes proper homologous chromosome segregation at the first meiotic division, normally occurs between allelic sequences on homologues. However, recombination can also take place between non-allelic DNA segments that share high sequence identity. Such non-allelic homologous recombination (NAHR) can markedly alter genome architecture during gametogenesis by generating chromosomal rearrangements. Indeed, NAHR-mediated deletions, duplications, inversions and other alterations have been implicated in numerous human genetic disorders. Studies in yeast have revealed insights into the molecular mechanisms of meiotic NAHR as well as the cellular strategies that limit NAHR. PMID:20164840

  20. Homologous recombination in bovine pestiviruses. Phylogenetic and statistic evidence.

    PubMed

    Jones, Leandro Roberto; Weber, E Laura

    2004-12-01

    Bovine pestiviruses (Bovine Viral Diarrea Virus 1 (BVDV 1) and Bovine Viral Diarrea Virus 2 (BVDV 2)) belong to the genus Pestivirus (Flaviviridae), which is composed of positive stranded RNA viruses causing significant economic losses world-wide. We used phylogenetic and bootstrap analyses to systematically scan alignments of previously sequenced genomes in order to explore further the evolutionary mechanisms responsible for variation in the virus. Previously published data suggested that homologous crossover might be one of the mechanisms responsible for the genomic rearrangements observed in cytopathic (cp) strains of bovine pestiviruses. Nevertheless, homologous recombination involves not just homologous crossovers, but also replacement of a homologous region of the acceptor RNA. Furthermore, cytopathic strains represent dead paths in evolution, since they are isolated exclusively from the fatal cases of mucosal disease. Herein, we report evidence of homologous inter-genotype recombination in the genome of a non-cytopathic (ncp) strain of Bovine Viral Diarrea Virus 1, the type species of the genus Pestivirus. We also show that intra-genotype homologous recombination might be a common phenomenon in both species of Pestivirus. This evidence demonstrates that homologous recombination contribute to the diversification of bovine pestiviruses in nature. Implications for virus evolution, taxonomy and phylogenetics are discussed.

  1. Single-Stranded DNA Curtains for Studying Homologous Recombination.

    PubMed

    Ma, C J; Steinfeld, J B; Greene, E C

    2017-01-01

    Homologous recombination is an important pathway involved in the repair of double-stranded DNA breaks. Genetic studies form the foundation of our knowledge on homologous recombination. Significant progress has also been made toward understanding the biochemical and biophysical properties of the proteins, complexes, and reaction intermediates involved in this essential DNA repair pathway. However, heterogeneous or transient recombination intermediates remain extremely difficult to assess through traditional ensemble methods, leaving an incomplete mechanistic picture of many steps that take place during homologous recombination. To help overcome some of these limitations, we have established DNA curtain methodologies as an experimental platform for studying homologous DNA recombination in real-time at the single-molecule level. Here, we present a detailed overview describing the preparation and use of single-stranded DNA curtains in applications related to the study of homologous DNA recombination with emphasis on recent work related to the study of the eukaryotic recombinase Rad51. © 2017 Elsevier Inc. All rights reserved.

  2. 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.

  3. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

    PubMed Central

    2010-01-01

    Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms. PMID:21129202

  4. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery.

    PubMed

    Liu, Jie; Morrical, Scott W

    2010-12-03

    Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.

  5. Hotspots of homologous recombination in the human genome: not all homologous sequences are equal.

    PubMed

    Lupski, James R

    2004-01-01

    Homologous recombination between alleles or non-allelic paralogous sequences does not occur uniformly but is concentrated in 'hotspots' with high recombination rates. Recent studies of these hotspots show that they do not share common sequence motifs, but they do have other features in common.

  6. Homologous recombination is required for recovery from oxidative DNA damage.

    PubMed

    Hayashi, Michio; Umezu, Keiko

    2017-04-03

    We have been studying the genetic events, including chromosome loss, chromosome rearrangements and intragenic point mutations, that are responsible for the deletion of a URA3 marker in a loss of heterozygosity (LOH) assay in the yeast Saccharomycess cerevisiae. With this assay, we previously showed that homologous recombination plays an important role in genome maintenance in response to DNA lesions that occur spontaneously in normally growing cells. Here, to investigate DNA lesions capable of triggering homologous recombination, we examined the effects of oxidative stress, a prominent cause of endogenous DNA damage, on LOH events. Treatment of log-phase cells with H2O2 first caused growth arrest and then, during the subsequent recovery, chromosome loss and various chromosome rearrangements were induced more than 10-fold. Further analysis of the rearrangements showed that gene conversion was strongly induced, approximately 100 times more frequently than in untreated cells. Consistent with these results, two diploid strains deficient for homologous recombination, rad52Δ/rad52Δ and rad51Δ/rad51Δ, were sensitive to H2O2 treatment. In addition, chromosome DNA breaks were detected in H2O2-treated cells using pulsed-field gel electrophoresis. Altogether, these results suggest that oxidative stress induced recombinogenic lesions on chromosomes, which then triggered homologous recombination leading to chromosome rearrangements, and that this response contributed to the survival of cells afflicted by oxidative DNA damage. We therefore conclude that homologous recombination is required for the recovery of cells from oxidative stress.

  7. Control of the endonuclease activity of type I restriction-modification systems is required to maintain chromosome integrity following homologous recombination.

    PubMed

    Blakely, Garry W; Murray, Noreen E

    2006-05-01

    A type I restriction-modification enzyme will bind to an unmethylated target sequence in DNA and, while still bound to the target, translocate DNA through the protein complex in both directions. DNA breakage occurs when two translocating complexes collide. However, if type I restriction-modification systems bind to unmodified target sequences within the resident bacterial chromosome, as opposed to incoming 'foreign' DNA, their activity is curtailed; a process known as restriction alleviation (RA). We have identified two genes in Escherichia coli, rnhA and recG, mutations in which lead to the alleviation of restriction. Induction of RA in response to these mutations is consistent with the production of unmodified target sequences following DNA synthesis associated with both homologous recombination and R-loop formation. This implies that a normal function of RA is to protect the bacterial chromosome when recombination generates unmodified products. For EcoKI, our experiments demonstrate the contribution of two pathways that serve to protect unmodified DNA in the bacterial chromosome: the primary pathway in which ClpXP degrades the restriction endonuclease and a mechanism dependent on the lar gene within Rac, a resident, defective prophage of E. coli K-12. Previously, the potential of the second pathway has only been demonstrated when expression of lar has been elevated. Our data identify the effect of lar from the repressed prophage.

  8. Artificial restriction DNA cutters to promote homologous recombination in human cells.

    PubMed

    Katada, Hitoshi; Komiyama, Makoto

    2011-02-01

    Homologous recombination is almost the only way to modify the genome in a predetermined fashion, despite its quite low frequency in mammalian cells. It has been already reported that the frequency of this biological process can be notably increased by inducing a double strand break (DSB) at target site. This article presents completely chemistry-based artificial restriction DNA cutter (ARCUT) for the promotion of homologous recombination in human cells. This cutter is composed of Ce(IV)/EDTA complex (molecular scissors) and two strands of peptide nucleic acid (PNA), and contains no proteins. Its scission site in the genome is determined simply by Watson-Crick rule so that ARCUT for desired homologous recombination is easily and straightforwardly designed and synthesized. The site-specificity of the scission is high enough to cut human genome at one target site. The DSB induced by this cutter is satisfactorily recognized by the repair system in human cells and promotes the targeted homologous recombination.

  9. Homologous recombination in Sulfolobus acidocaldarius: genetic assays and functional properties.

    PubMed

    Grogan, Dennis W

    2009-02-01

    HR (homologous recombination) is expected to play important roles in the molecular biology and genetics of archaea, but, so far, few functional properties of archaeal HR have been measured in vivo. In the extreme thermoacidophile Sulfolobus acidocaldarius, a conjugational mechanism of DNA transfer enables quantitative analysis of HR between chromosomal markers. Early studies of this system indicated that HR occurred frequently between closely spaced mutations within the pyrE gene, and this result was later supported by various analyses involving defined point mutations and deletions. These properties of intragenic HR suggested a non-reciprocal mechanism in which donor sequences become incorporated into the recipient genome as short segments. Because fragmentation of donor DNA during cell-to-cell transfer could not be excluded from contributing to this result, subsequent analyses have focused on electroporation of selectable donor DNA directly into recipient strains. For example, S. acidocaldarius was found to incorporate synthetic ssDNA (single-stranded DNA) of more than approximately 20 nt readily into its genome. With respect to various molecular properties of the ssDNA substrates, the process resembled bacteriophage lambdaRed-mediated 'recombineering' in Escherichia coli. Another approach used electroporation of a multiply marked pyrE gene to measure donor sequence tracts transferred to the recipient genome in individual recombination events. Initial results indicate multiple discontinuous tracts in the majority of recombinants, representing a relatively broad distribution of tract lengths. This pattern suggests that properties of the HR process could, in principle, account for many of the apparent peculiarities of intragenic recombination initiated by S. acidocaldarius conjugation.

  10. Homologous recombination drives both sequence diversity and gene content variation in Neisseria meningitidis.

    PubMed

    Kong, Ying; Ma, Jennifer H; Warren, Keisha; Tsang, Raymond S W; Low, Donald E; Jamieson, Frances B; Alexander, David C; Hao, Weilong

    2013-01-01

    The study of genetic and phenotypic variation is fundamental for understanding the dynamics of bacterial genome evolution and untangling the evolution and epidemiology of bacterial pathogens. Neisseria meningitidis (Nm) is among the most intriguing bacterial pathogens in genomic studies due to its dynamic population structure and complex forms of pathogenicity. Extensive genomic variation within identical clonal complexes (CCs) in Nm has been recently reported and suggested to be the result of homologous recombination, but the extent to which recombination contributes to genomic variation within identical CCs has remained unclear. In this study, we sequenced two Nm strains of identical serogroup (C) and multi-locus sequence type (ST60), and conducted a systematic analysis with an additional 34 Nm genomes. Our results revealed that all gene content variation between the two ST60 genomes was introduced by homologous recombination at the conserved flanking genes, and 94.25% or more of sequence divergence was caused by homologous recombination. Recombination was found in genes associated with virulence factors, antigenic outer membrane proteins, and vaccine targets, suggesting an important role of homologous recombination in rapidly altering the pathogenicity and antigenicity of Nm. Recombination was also evident in genes of the restriction and modification systems, which may undermine barriers to DNA exchange. In conclusion, homologous recombination can drive both gene content variation and sequence divergence in Nm. These findings shed new light on the understanding of the rapid pathoadaptive evolution of Nm and other recombinogenic bacterial pathogens.

  11. Evidence for homologous recombination in Chikungunya Virus.

    PubMed

    Casal, Pablo E; Chouhy, Diego; Bolatti, Elisa M; Perez, Germán R; Stella, Emma J; Giri, Adriana A

    2015-04-01

    Chikungunya Virus (CHIKV), a mosquito-transmitted alphavirus, causes acute fever and joint pain in humans. Recently, endemic CHIKV infection outbreaks have jeopardized public health in wider geographical regions. Here, we analyze the phylogenetic associations of CHIKV and explore the potential recombination events on 152 genomic isolates deposited in GenBank database. The CHIKV genotypes [West African, Asian, East/Central/South African (ECSA)], and a clear division of ECSA clade into three sub-groups (I-II-III), were defined by Bayesian analysis; similar results were obtained using E1 gene sequences. A nucleotide identity-based approach is provided to facilitate CHIKV classification within ECSA clade. Using seven methods to detect recombination, we found a statistically significant event (p-values range: 1.14×10(-7)-4.45×10(-24)) located within the nsP3 coding region. This finding was further confirmed by phylogenetic networks (PHI Test, p=0.004) and phylogenetic tree incongruence analysis. The recombinant strain, KJ679578/India/2011 (ECSA III), derives from viruses of ECSA III and ECSA I. Our study demonstrates that recombination is an additional mechanism of genetic diversity in CHIKV that might assist in the cross-species transmission process.

  12. General method for plasmid construction using homologous recombination.

    PubMed

    Raymond, C K; Pownder, T A; Sexson, S L

    1999-01-01

    We describe a general method for plasmid assembly that uses yeast and extends beyond yeast-specific research applications. This technology exploits the homologous recombination, double-stranded break repair pathway in Saccharomyces cerevisiae to join DNA fragments. Synthetic, double-stranded "recombination linkers" were used to "subclone" a DNA fragment into a plasmid with > 80% efficiency. Quantitative data on the influence of DNA concentration and overlap length on the efficiency of recombination are presented. Using a simple procedure, plasmids were shuttled from yeast into E. coli for subsequent screening and large-scale plasmid preps. This simple method for plasmid construction has several advantages. (i) It bypasses the need for extensive PCR amplification and for purification, modification and/or ligation techniques routinely used for plasmid constructions. (ii) The method does not rely on available restriction sites, thus fragment and vector DNA can be joined within any DNA sequence. This enables the use of multifunctional cloning vectors for protein expression in mammalian cells, other yeast species, E. coli and other expression systems as discussed. (iii) Finally, the technology exploits yeast strains, plasmids and microbial techniques that are inexpensive and readily available.

  13. Widespread homologous recombination within and between Streptomyces species.

    PubMed

    Doroghazi, James R; Buckley, Daniel H

    2010-09-01

    Horizontal gene transfer (HGT) is widespread in the microbial world, but its impact on the origin and persistence of microbial species remains poorly defined. HGT can result in either acquisition of new genetic material or homologous replacement of existing genes. The evolutionary significance of homologous recombination in a population can be quantified by examining the relative rates at which polymorphisms are introduced from recombination (rho) and mutation (theta(w)). We used multilocus sequence analysis (MLSA) to quantify both intraspecies and interspecies homologous recombination among streptomycetes, multicellular Gram-positive bacteria ubiquitous in soil, which are an important source of antibiotics and bioactive compounds. Intraspecies recombination was examined using strains of Streptomyces flavogriseus isolated from soils at five locations spanning 1000 km. The strains had >99.8% nucleotide identity across the loci examined. We found remarkable levels of gene exchange within S. flavogriseus (rho/theta(w)=27.9), and found that the population was in linkage equilibrium (standardized index of association=0.0018), providing evidence for a freely recombining sexual population structure. We also examined interspecies homologous recombination among different Streptomyces species in an MLSA data set and found that 40% of the species had housekeeping genes acquired through HGT. The recombination rate between these named species (rho/theta(w)=0.21) exceeds that observed within many species of bacteria. Despite widespread gene exchange in the genus, the intraspecies recombination rate exceeded the interspecies rate by two orders of magnitude suggesting that patterns of gene exchange and recombination may shape the evolution of streptomycetes.

  14. Rapid one-step construction of a Middle East Respiratory Syndrome (MERS-CoV) infectious clone system by homologous recombination.

    PubMed

    Nikiforuk, Aidan M; Leung, Anders; Cook, Bradley W M; Court, Deborah A; Kobasa, Darwyn; Theriault, Steven S

    2016-10-01

    Viral Infectious clone systems serve as robust platforms to study viral gene or replicative function by reverse genetics, formulate vaccines and adapt a wild type-virus to an animal host. Since the development of the first viral infectious clone system for the poliovirus, novel strategies of viral genome construction have allowed for the assembly of viral genomes across the identified viral families. However, the molecular profiles of some viruses make their genome more difficult to construct than others. Two factors that affect the difficulty of infectious clone construction are genome length and genome complexity. This work examines the available strategies for overcoming the obstacles of assembling the long and complex RNA genomes of coronaviruses and reports one-step construction of an infectious clone system for the Middle East Respiratory Syndrome coronavirus (MERS-CoV) by homologous recombination in S. cerevisiae. Future use of this methodology will shorten the time between emergence of a novel viral pathogen and construction of an infectious clone system. Completion of a viral infectious clone system facilitates further study of a virus's biology, improvement of diagnostic tests, vaccine production and the screening of antiviral compounds. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  15. Modulation of meiotic homologous recombination by DNA helicases.

    PubMed

    Lorenz, Alexander

    2017-05-01

    DNA helicases are ATP-driven motor proteins which translocate along DNA capable of dismantling DNA-DNA interactions and/or removing proteins bound to DNA. These biochemical capabilities make DNA helicases main regulators of crucial DNA metabolic processes, including DNA replication, DNA repair, and genetic recombination. This budding topic will focus on reviewing the function of DNA helicases important for homologous recombination during meiosis, and discuss recent advances in how these modulators of meiotic recombination are themselves regulated. The emphasis is placed on work in the two model yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, which has vastly expanded our understanding of meiotic homologous recombination, a process whose correct execution is instrumental for healthy gamete formation, and thus functioning sexual reproduction. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  16. High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication.

    PubMed

    Fu, Xinping; Wang, Hua; Zhang, Xiaoliu

    2002-06-01

    Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.

  17. The Functions of BRCA2 in Homologous Recombinational Repair

    DTIC Science & Technology

    2004-07-01

    February 2004. 2. A manuscript related to this project entitled "Human Rad51C deficiency destabilizes XRCC3 , impairs recombination and radiosensitizes...Cell Biol 15: 1968-1973 15. Brenneman MA et al. (2000) XRCC3 is required for efficient repair of chromosome breaks by homologous recombination. Mutat...JBiol Chem 278: 2469-2478 APPENDICES 1. Lio, Y-C, Schild, D, Brenneman, MA, Redpath, JL, and Chen, DJ (2004) Human RadS1C deficiency destabilizes XRCC3

  18. Guidelines for identifying homologous recombination events in influenza A virus.

    PubMed

    Boni, Maciej F; de Jong, Menno D; van Doorn, H Rogier; Holmes, Edward C

    2010-05-03

    The rapid evolution of influenza viruses occurs both clonally and non-clonally through a variety of genetic mechanisms and selection pressures. The non-clonal evolution of influenza viruses comprises relatively frequent reassortment among gene segments and a more rarely reported process of non-homologous RNA recombination. Homologous RNA recombination within segments has been proposed as a third such mechanism, but to date the evidence for the existence of this process among influenza viruses has been both weak and controversial. As homologous recombination has not yet been demonstrated in the laboratory, supporting evidence, if it exists, may come primarily from patterns of phylogenetic incongruence observed in gene sequence data. Here, we review the necessary criteria related to laboratory procedures and sample handling, bioinformatic analysis, and the known ecology and evolution of influenza viruses that need to be met in order to confirm that a homologous recombination event occurred in the history of a set of sequences. To determine if these criteria have an effect on recombination analysis, we gathered 8307 publicly available full-length sequences of influenza A segments and divided them into those that were sequenced via the National Institutes of Health Influenza Genome Sequencing Project (IGSP) and those that were not. As sample handling and sequencing are executed to a very high standard in the IGSP, these sequences should be less likely to be exposed to contamination by other samples or by laboratory strains, and thus should not exhibit laboratory-generated signals of homologous recombination. Our analysis shows that the IGSP data set contains only two phylogenetically-supported single recombinant sequences and no recombinant clades. In marked contrast, the non-IGSP data show a very large amount of potential recombination. We conclude that the presence of false positive signals in the non-IGSP data is more likely than false negatives in the IGSP data

  19. Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate.

    PubMed

    Kaiser, Gitte S; Germann, Susanne M; Westergaard, Tine; Lisby, Michael

    2011-08-01

    Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted. 2011 Elsevier B.V. All rights reserved.

  20. 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,

  1. DNA-protein crosslinks processed by nucleotide excision repair and homologous recombination with base and strand preference in E. coli model system.

    PubMed

    Fang, Qingming

    2013-01-01

    Bis-electrophiles including dibromoethane and epibromohydrin can react with O(6)-alkylguanine-DNA alkyltransferase (AGT) and form AGT-DNA crosslinks in vitro and in vivo. The presence of human AGT (hAGT) paradoxically increases the mutagenicity and cytotoxicity of bis-electrophiles in cells. Here we establish a bacterial system to study the repair mechanism and cellular responses to DNA-protein crosslinks (DPCs) in vivo. Results show that both nucleotide excision repair (NER) and homologous recombination (HR) pathways can process hAGT-DNA crosslinks with HR playing a dominant role. Mutation spectra show that HR has no strand preference but NER favors processing of the DPCs in the transcribed strand; UvrA, UvrB and Mfd can interfere with small size DPCs but only UvrA can interfere with large size DPCs in the transcribed strand processed by HR. Further, we found that DPCs at TA deoxynucleotide sites are very inefficiently processed by NER and the presence of NER can interfere with these DNA lesions processed by HR. These data indicate that NER and HR can process DPCs cooperatively and competitively and NER processes DPCs with base and strand preference. Therefore, the formation of hAGT-DNA crosslinks can be a plausible and specific system to study the repair mechanism and effects of DPCs precisely in vivo.

  2. DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

    PubMed Central

    Morrical, Scott W.

    2015-01-01

    The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways—DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species. PMID:25646379

  3. Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli.

    PubMed

    Didelot, Xavier; Méric, Guillaume; Falush, Daniel; Darling, Aaron E

    2012-06-19

    Escherichia coli is an important species of bacteria that can live as a harmless inhabitant of the guts of many animals, as a pathogen causing life-threatening conditions or freely in the non-host environment. This diversity of lifestyles has made it a particular focus of interest for studies of genetic variation, mainly with the aim to understand how a commensal can become a deadly pathogen. Many whole genomes of E. coli have been fully sequenced in the past few years, which offer helpful data to help understand how this important species evolved. We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A, B1, B2 and E). From the core-genome we established the clonal relationships between the isolates as well as the role played by homologous recombination during their evolution from a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E), which could be explained by the ecological structuring of E. coli and may represent on-going speciation. We identified three hotspots of homologous recombination, one of which had not been previously described and contains the aroC gene, involved in the essential shikimate metabolic pathway. We also described the role played by non-homologous recombination in the pan-genome, and showed that this process was highly heterogeneous. Our analyses revealed in particular that the genomes of three enterohaemorrhagic (EHEC) strains within phylogroup B1 have converged from originally separate backgrounds as a result of both homologous and non-homologous recombination. Recombination is an important force shaping the genomic evolution and diversification of E. coli, both by replacing fragments of genes with an homologous sequence and also by introducing new genes. In this study, several non-random patterns of these events were identified which correlated with important changes in the lifestyle of the bacteria, and therefore provide additional evidence to explain the

  4. 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

  5. DNA sequence alignment by microhomology sampling during homologous recombination.

    PubMed

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

    2015-02-26

    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 single-strand DNA (ssDNA) with a homologous double-strand DNA (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 ninth 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Detection of homologous recombination between yeast artificial chromosomes with overlapping inserts.

    PubMed Central

    Cellini, A; Lacatena, R M; Tocchini-Valentini, G P

    1991-01-01

    We have developed a system which facilitates the detection of recombination between Yeast Artificial Chromosomes (YAC's) carrying homologous inserts. The system consists of a classical YAC vector, a new YAC vector and two appropriately labelled yeast strains of opposite mating type. The new YAC vector differs in markers from the canonical YAC vector. To test whether homologous recombination takes place, phage lambda DNA was cloned in the two vectors to provide a region of homology. The two constructs were then introduced into yeast strains of opposite mating type in which the endogenous genes for the selective markers present in the vectors are not expressed. Artificial chromosomes obtained by meiotic recombination are detected in the spores resulting from the mating. PMID:1826951

  7. Generation of hybrid human immunodeficiency virus by homologous recombination.

    PubMed Central

    Srinivasan, A; York, D; Jannoun-Nasr, R; Kalyanaraman, S; Swan, D; Benson, J; Bohan, C; Luciw, P A; Schnoll, S; Robinson, R A

    1989-01-01

    Human immunodeficiency virus (HIV) type 1, isolated from diverse sources, exhibits genomic diversity. The mechanisms by which the genomic diversity takes place in individuals exposed to multiple virus isolates is yet to be elucidated. Genetic variation, in general, might result from mutagenic events such as point mutations, rearrangements (insertions and deletions), and recombination. In an attempt to evaluate the process of genetic diversity, we designed experiments to analyze recombination between HIV DNAs by using DNA transfection in cell cultures. Here we report the successful recombination between truncated HIV proviral DNAs with an overlap homology of 53 base pairs that leads to the formation of viable hybrid virus. Recombination was also seen between exogenous DNA introduced into cells and homologous HIV sequences resident in the cells. These results indicate that recombination among various HIV isolates may play a significant role in the generation of genetic diversity of HIV. Further, the method used here enables the construction of hybrid HIV genomes to identify the viral determinants responsible for tropism, replication, and cytopathic effects. Images PMID:2474834

  8. Evidence for the murine IgH mu locus acting as a hot spot for intrachromosomal homologous recombination.

    PubMed

    Raynard, Steven J; Read, Leah R; Baker, Mark D

    2002-03-01

    Homologous recombination accomplishes the exchange of genetic information between two similar or identical DNA duplexes. It can occur either by gene conversion, a process of unidirectional genetic exchange, or by reciprocal crossing over. Homologous recombination is well known for its role in generating genetic diversity in meiosis and, in mitosis, as a DNA repair mechanism. In the immune system, the evidence suggests a role for homologous recombination in Ig gene evolution and in the diversification of Ab function. Previously, we reported the occurrence of homologous recombination between repeated, donor and recipient alleles of the Ig H chain mu gene C (Cmu) region residing at the Ig mu locus in mouse hybridoma cells. In this study, we constructed mouse hybridoma cell lines bearing Cmu region heteroalleles to learn more about the intrachromosomal homologous recombination process. A high frequency of homologous recombination (gene conversion) was observed for markers spanning the entire recipient Cmu region, suggesting that recombination might initiate at random sites within the Cmu region. The Cmu region heteroalleles were equally proficient as either conversion donors or recipients. Remarkably, when the same Cmu heteroalleles were tested for recombination in ectopic genomic positions, the mean frequency of gene conversion was reduced by at least 65-fold. These results are consistent with the murine IgH mu locus behaving as a hot spot for intrachromosomal homologous recombination.

  9. Accelerated homologous recombination and subsequent genome modification in Drosophila

    PubMed Central

    Baena-Lopez, Luis Alberto; Alexandre, Cyrille; Mitchell, Alice; Pasakarnis, Laurynas; Vincent, Jean-Paul

    2013-01-01

    Gene targeting by ‘ends-out’ homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR. PMID:24154526

  10. Accelerated homologous recombination and subsequent genome modification in Drosophila.

    PubMed

    Baena-Lopez, Luis Alberto; Alexandre, Cyrille; Mitchell, Alice; Pasakarnis, Laurynas; Vincent, Jean-Paul

    2013-12-01

    Gene targeting by 'ends-out' homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR.

  11. Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli

    PubMed Central

    2012-01-01

    Background Escherichia coli is an important species of bacteria that can live as a harmless inhabitant of the guts of many animals, as a pathogen causing life-threatening conditions or freely in the non-host environment. This diversity of lifestyles has made it a particular focus of interest for studies of genetic variation, mainly with the aim to understand how a commensal can become a deadly pathogen. Many whole genomes of E. coli have been fully sequenced in the past few years, which offer helpful data to help understand how this important species evolved. Results We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A, B1, B2 and E). From the core-genome we established the clonal relationships between the isolates as well as the role played by homologous recombination during their evolution from a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E), which could be explained by the ecological structuring of E. coli and may represent on-going speciation. We identified three hotspots of homologous recombination, one of which had not been previously described and contains the aroC gene, involved in the essential shikimate metabolic pathway. We also described the role played by non-homologous recombination in the pan-genome, and showed that this process was highly heterogeneous. Our analyses revealed in particular that the genomes of three enterohaemorrhagic (EHEC) strains within phylogroup B1 have converged from originally separate backgrounds as a result of both homologous and non-homologous recombination. Conclusions Recombination is an important force shaping the genomic evolution and diversification of E. coli, both by replacing fragments of genes with an homologous sequence and also by introducing new genes. In this study, several non-random patterns of these events were identified which correlated with important changes in the lifestyle of the bacteria, and therefore provide additional

  12. The Landscape of Realized Homologous Recombination in Pathogenic Bacteria.

    PubMed

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

    2016-02-01

    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. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  13. Detection of homologous recombination in closely related strains.

    PubMed

    Kalinina, Anastasia S; Suvorikova, Alexandra L; Spokoiny, Vladimir G; Gelfand, Mikhail S

    2016-04-01

    Detection of recombination events in a bacterial genome is both important from the evolutionary point of view, and of practical interest. Indeed, homologous recombination (HR) plays a major role in the exchange of antigenic determinants between strains. There exist statistical methods to detect recently recombined segments in whole-genome sequences that use a high local density of substitutions as a signal of HR events with a source outside considered strains. However, it is difficult to detect the HR events within a set of strains, which represent whole species diversity, due to a low number of substitutions in recombined segments and high level of diversity of strains. Here, we analyzed HR in 20 Escherichia coli (E. coli) strains to define what fraction of segments with a high substitution rate were introduced in a genome by HR. For detection of HR, we used the segmentation, performed by the adaptive weights smoothing (AWS) algorithm. It detects sharp changes in the structure of observed data analyzing only qualitative structural information. We validated the approach on simulated data, applied it to the analysis of E. coli strains, and determined the recombination rates between phylogroups.

  14. Effect of monovalent cations and G-quadruplex structures on the outcome of intramolecular homologous recombination.

    PubMed

    Barros, Paula; Boán, Francisco; Blanco, Miguel G; Gómez-Márquez, Jaime

    2009-06-01

    Homologous recombination is a very important cellular process, as it provides a major pathway for the repair of DNA double-strand breaks. This complex process is affected by many factors within cells. Here, we have studied the effect of monovalent cations (K+, Na+, and NH4+) on the outcome of recombination events, as their presence affects the biochemical activities of the proteins involved in recombination as well as the structure of DNA. For this purpose, we used an in vitro recombination system that includes a protein nuclear extract, as a source of recombination machinery, and two plasmids as substrates for intramolecular homologous recombination, each with two copies of different alleles of the human minisatellite MsH43. We found that the presence of monovalent cations induced a decrease in the recombination frequency, accompanied by an increase in the fidelity of the recombination. Moreover, there is an emerging consensus that secondary structures of DNA have the potential to induce genomic instability. Therefore, we analyzed the effect of the sequences capable of forming G-quadruplex on the production of recombinant molecules, taking advantage of the capacity of some MsH43 alleles to generate these kinds of structure in the presence of K+. We observed that the MsH43 recombinants containing duplications, generated in the presence of K+, did not include the repeats located towards the 5'-side of the G-quadruplex motif, suggesting that this structure may be involved in the recombination events leading to duplications. Our results provide new insights into the molecular mechanisms underlying the recombination of repetitive sequences.

  15. Investigations of homologous recombination pathways and their regulation.

    PubMed

    Daley, James M; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick

    2013-12-13

    The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.

  16. 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

  17. 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

  18. P53 Suppression of Homologous Recombination and Tumorigenesis

    DTIC Science & Technology

    2011-01-01

    eEF2 kinase activity in a cell cycle- and amino acid -dependent manner. Embo J 27: 1005–1016. 40. Astanehe A, Arenillas D, Wasserman WW, Leung PC...followed by 433 amino acids of the duplicated exons 8 to 19. After deletion of one of the internal repeats by HR, a functional protein can be...homologous recombination events in mice in vivo. Nucleic Acids Res 38:7538–7545 - Ravi D, Chen Y, Karia B, Brown A, Gu TT, Li J, Carey MS, Hennessy BT

  19. Homologous recombination evidence in human and swine influenza A viruses.

    PubMed

    He, Cheng-Qiang; Han, Guan-Zhu; Wang, Dong; Liu, Wei; Li, Guo-Rong; Liu, Xi-Ping; Ding, Nai-Zheng

    2008-10-10

    Dynamic gene mutation and the reassortment of genes have been considered as the key factors responsible for influenza A virus virulence and host tropism change. This study reports several significant evidence demonstrating that homologous recombination also takes place between influenza A viruses in human and swine lineages. Moreover, in a mosaic descended from swine H1N1 subtype and human H2N2, we found that its minor putative parent might be a derivative from the human cold-adapted vaccine lineage, which suggests that live vaccine is capable of playing a role in genetic change of influenza A virus via recombination with circulating viruses. These results would be important for knowing the molecular mechanism of mammal influenza A virus heredity and evolution.

  20. The many facets of homologous recombination at telomeres

    PubMed Central

    Claussin, Clémence; Chang, Michael

    2015-01-01

    The ends of linear chromosomes are capped by nucleoprotein structures called telomeres. A dysfunctional telomere may resemble a DNA double-strand break (DSB), which is a severe form of DNA damage. The presence of one DSB is sufficient to drive cell cycle arrest and cell death. Therefore cells have evolved mechanisms to repair DSBs such as homologous recombination (HR). HR-mediated repair of telomeres can lead to genome instability, a hallmark of cancer cells, which is why such repair is normally inhibited. However, some HR-mediated processes are required for proper telomere function. The need for some recombination activities at telomeres but not others necessitates careful and complex regulation, defects in which can lead to catastrophic consequences. Furthermore, some cell types can maintain telomeres via telomerase-independent, recombination-mediated mechanisms. In humans, these mechanisms are called alternative lengthening of telomeres (ALT) and are used in a subset of human cancer cells. In this review, we summarize the different recombination activities occurring at telomeres and discuss how they are regulated. Much of the current knowledge is derived from work using yeast models, which is the focus of this review, but relevant studies in mammals are also included.

  1. Enhancement of extra chromosomal recombination in somatic cells by affecting the ratio of homologous recombination (HR) to non-homologous end joining (NHEJ).

    PubMed

    Zaunbrecher, Gretchen M; Dunne, Patrick W; Mir, Bashir; Breen, Matthew; Piedrahita, Jorge A

    2008-01-01

    Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.

  2. Homologous recombination promoted by reverse transcriptase during copying of two distinct RNA templates.

    PubMed Central

    Negroni, M; Ricchetti, M; Nouvel, P; Buc, H

    1995-01-01

    Retroviruses are known to mutate at high rates. An important source of genetic variability is recombination taking place during reverse transcription of internal regions of the two genomic RNAs. We have designed an in vitro model system, involving genetic markers carried on two RNA templates, to allow a search for individual recombination events and to score their frequency of occurrence. We show that Moloney murine leukemia virus reverse transcriptase alone promotes homologous recombination efficiently. While RNA concentration has little effect on recombination frequency, there is a clear correlation between the amount of reverse transcriptase used in the assay and the extent of recombination observed. Under conditions mimicking the in vivo situation, a rate compatible with ex vivo estimates has been obtained. PMID:7542781

  3. 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.

  4. 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

  5. Reappearance from Obscurity: Mammalian Rad52 in Homologous Recombination.

    PubMed

    Hanamshet, Kritika; Mazina, Olga M; Mazin, Alexander V

    2016-09-14

    Homologous recombination (HR) plays an important role in maintaining genomic integrity. It is responsible for repair of the most harmful DNA lesions, DNA double-strand breaks and inter-strand DNA cross-links. HR function is also essential for proper segregation of homologous chromosomes in meiosis, maintenance of telomeres, and resolving stalled replication forks. Defects in HR often lead to genetic diseases and cancer. Rad52 is one of the key HR proteins, which is evolutionarily conserved from yeast to humans. In yeast, Rad52 is important for most HR events; Rad52 mutations disrupt repair of DNA double-strand breaks and targeted DNA integration. Surprisingly, in mammals, Rad52 knockouts showed no significant DNA repair or recombination phenotype. However, recent work demonstrated that mutations in human RAD52 are synthetically lethal with mutations in several other HR proteins including BRCA1 and BRCA2. These new findings indicate an important backup role for Rad52, which complements the main HR mechanism in mammals. In this review, we focus on the Rad52 activities and functions in HR and the possibility of using human RAD52 as therapeutic target in BRCA1 and BRCA2-deficient familial breast cancer and ovarian cancer.

  6. Reappearance from Obscurity: Mammalian Rad52 in Homologous Recombination

    PubMed Central

    Hanamshet, Kritika; Mazina, Olga M.; Mazin, Alexander V.

    2016-01-01

    Homologous recombination (HR) plays an important role in maintaining genomic integrity. It is responsible for repair of the most harmful DNA lesions, DNA double-strand breaks and inter-strand DNA cross-links. HR function is also essential for proper segregation of homologous chromosomes in meiosis, maintenance of telomeres, and resolving stalled replication forks. Defects in HR often lead to genetic diseases and cancer. Rad52 is one of the key HR proteins, which is evolutionarily conserved from yeast to humans. In yeast, Rad52 is important for most HR events; Rad52 mutations disrupt repair of DNA double-strand breaks and targeted DNA integration. Surprisingly, in mammals, Rad52 knockouts showed no significant DNA repair or recombination phenotype. However, recent work demonstrated that mutations in human RAD52 are synthetically lethal with mutations in several other HR proteins including BRCA1 and BRCA2. These new findings indicate an important backup role for Rad52, which complements the main HR mechanism in mammals. In this review, we focus on the Rad52 activities and functions in HR and the possibility of using human RAD52 as therapeutic target in BRCA1 and BRCA2-deficient familial breast cancer and ovarian cancer. PMID:27649245

  7. Cell cycle-dependent control of homologous recombination.

    PubMed

    Zhao, Xin; Wei, Chengwen; Li, Jingjing; Xing, Poyuan; Li, Jingyao; Zheng, Sihao; Chen, Xuefeng

    2017-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious type of DNA lesions threatening genome integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are two major pathways to repair DSBs. HR requires a homologous template to direct DNA repair, and is generally recognized as a high-fidelity pathway. In contrast, NHEJ directly seals broken ends, but the repair product is often accompanied by sequence alterations. The choice of repair pathways is strictly controlled by the cell cycle. The occurrence of HR is restricted to late S to G2 phases while NHEJ operates predominantly in G1 phase, although it can act throughout most of the cell cycle. Deregulation of repair pathway choice can result in genotoxic consequences associated with cancers. How the cell cycle regulates the choice of HR and NHEJ has been extensively studied in the past decade. In this review, we will focus on the current progresses on how HR is controlled by the cell cycle in both Saccharomyces cerevisiae and mammals. Particular attention will be given to how cyclin-dependent kinases modulate DSB end resection, DNA damage checkpoint signaling, repair and processing of recombination intermediates. In addition, we will discuss recent findings on how HR is repressed in G1 and M phases by the cell cycle. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Correlated Mutations and Homologous Recombination Within Bacterial Populations.

    PubMed

    Lin, Mingzhi; Kussell, Edo

    2017-02-01

    Inferring the rate of homologous recombination within a bacterial population remains a key challenge in quantifying the basic parameters of bacterial evolution. Due to the high sequence similarity within a clonal population, and unique aspects of bacterial DNA transfer processes, detecting recombination events based on phylogenetic reconstruction is often difficult, and estimating recombination rates using coalescent model-based methods is computationally expensive, and often infeasible for large sequencing data sets. Here, we present an efficient solution by introducing a set of mutational correlation functions computed using pairwise sequence comparison, which characterize various facets of bacterial recombination. We provide analytical expressions for these functions, which precisely recapitulate simulation results of neutral and adapting populations under different coalescent models. We used these to fit correlation functions measured at synonymous substitutions using whole-genome data on Escherichia coli and Streptococcus pneumoniae populations. We calculated and corrected for the effect of sample selection bias, i.e., the uneven sampling of individuals from natural microbial populations that exists in most datasets. Our method is fast and efficient, and does not employ phylogenetic inference or other computationally intensive numerics. By simply fitting analytical forms to measurements from sequence data, we show that recombination rates can be inferred, and the relative ages of different samples can be estimated. Our approach, which is based on population genetic modeling, is broadly applicable to a wide variety of data, and its computational efficiency makes it particularly attractive for use in the analysis of large sequencing datasets. Copyright © 2017 by the Genetics Society of America.

  9. Homologous recombination efficiency enhanced by inhibition of MEK and GSK3β.

    PubMed

    Lin, Zhaoyu; Zhang, Yanli; Gao, Tianyun; Wang, Liudi; Zhang, Qing; Zhou, Juan; Zhao, Jing

    2014-11-01

    Homologous recombination in embryonic stem cells (ESCs) is widely utilized in genome engineering, particularly in the generation of gene targeted mice. However, genome engineering is often plagued by the problem of low homologous recombination efficiency. In this study, we developed a novel method to increase the efficiency of homologous recombination in ESCs by changing its culture conditions. By comparing the efficiency of different ESCs in various culture conditions, we determined that chemicals that inhibit the MEK and GSK3β pathways (2i condition) enhance homologous recombination and eliminate differences in efficiencies among cell lines. Analysis of gene expression patterns in ESCs maintained in different culture conditions has identified several homologous recombination-related candidates, including the pluripotent markers Eras and Tbx3. The results of this study suggest that homologous recombination is associated with ESC pluripotency. © 2014 Wiley Periodicals, Inc.

  10. 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.

  11. Cohesin Is limiting for the suppression of DNA damage-induced recombination between homologous chromosomes.

    PubMed

    Covo, Shay; Westmoreland, James W; Gordenin, Dmitry A; Resnick, Michael A

    2010-07-01

    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 G(2)/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G(2)/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 G(2)/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.

  12. Eukaryotic DNA damage responses: Homologous recombination factors and ubiquitin modification.

    PubMed

    Lee, Nam Soo; Kim, Soomi; Jung, Yong Woo; Kim, Hongtae

    2017-05-06

    To prevent genomic instability disorders, cells have developed a DNA damage response. The response involves various proteins that sense damaged DNA, transduce damage signals, and effect DNA repair. In addition, ubiquitin modifications modulate the signaling pathway depending on cellular context. Among various types of DNA damage, double-stranded breaks are highly toxic to genomic integrity. Homologous recombination (HR) repair is an essential mechanism that fixes DNA damage because of its high level of accuracy. Although factors in the repair pathway are well established, pinpointing the exact mechanisms of repair and devising therapeutic applications requires more studies. Moreover, essential functions of ubiquitin modification in the DNA damage signaling pathway have emerged. In this review, to explore the eukaryotic DNA damage response, we will mention the functions of main factors in the HR repair pathway and ubiquitin modification. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. DNA end resection controls the balance between homologous and illegitimate recombination in Escherichia coli.

    PubMed

    Ivanković, Siniša; Đermić, Damir

    2012-01-01

    Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD's RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3' tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3' ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human

  14. Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

    PubMed Central

    Kogoma, T

    1997-01-01

    Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored. PMID:9184011

  15. A new type of illegitimate recombination is dependent on restriction and homologous interaction.

    PubMed Central

    Kusano, K; Sakagami, K; Yokochi, T; Naito, T; Tokinaga, Y; Ueda, E; Kobayashi, I

    1997-01-01

    Illegitimate (nonhomologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a delta rac recBC recG ruvC strain. Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination. The recombining sites themselves shared, at most, a few base pairs of homology. Many of the recombination events joined a site in one of the repeats with a site in another repeat. In two of the products, one of the recombining sites was at the end of one of the repeats. Removal of one of the EcoKI sites resulted in decreased recombination. We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination. The possible roles and consequences of this type of homologous interaction are discussed. PMID:9286991

  16. Mechanism of homologous recombination and implications for aging-related deletions in mitochondrial DNA.

    PubMed

    Chen, Xin Jie

    2013-09-01

    Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells.

  17. Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

    PubMed Central

    2013-01-01

    SUMMARY Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells. PMID:24006472

  18. Imatinib radiosensitises bladder cancer by targeting homologous recombination

    PubMed Central

    Qiao, Boling; Kerr, Martin; Groselj, Blaz; Teo, Mark TW; Knowles, Margaret A; Bristow, Robert G; Phillips, Roger M; Kiltie, Anne E

    2013-01-01

    Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumours are defective in non-homologous end-joining (NHEJ), and this phenotype might be exploited clinically by combining radiotherapy with a radiosensitising drug that targets homologous recombination (HR), thereby sparing normal tissues with intact NHEJ. The response of the HR protein RAD51 to radiation is inhibited by the small molecule tyrosine kinase inhibitor (TKI) imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using shRNA technology to mimic the invasive tumour phenotype, and also RAD51 knockdown (RAD51KD) cells to demonstrate imatinib’s pathway selectivity. Ku80KD, RAD51KD, non-silencing vector control and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ, and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (non-toxic) levels. Imatinib radiosensitised Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitised RAD51KD and RT112 cells, but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. PMID:23302228

  19. DEK is required for homologous recombination repair of DNA breaks.

    PubMed

    Smith, Eric A; Gole, Boris; Willis, Nicholas A; Soria, Rebeca; Starnes, Linda M; Krumpelbeck, Eric F; Jegga, Anil G; Ali, Abdullah M; Guo, Haihong; Meetei, Amom R; Andreassen, Paul R; Kappes, Ferdinand; Vinnedge, Lisa M Privette; Daniel, Jeremy A; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I

    2017-03-20

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

  20. DEK is required for homologous recombination repair of DNA breaks

    PubMed Central

    Smith, Eric A.; Gole, Boris; Willis, Nicholas A.; Soria, Rebeca; Starnes, Linda M.; Krumpelbeck, Eric F.; Jegga, Anil G.; Ali, Abdullah M.; Guo, Haihong; Meetei, Amom R.; Andreassen, Paul R.; Kappes, Ferdinand; Vinnedge, Lisa M. Privette; Daniel, Jeremy A.; Scully, Ralph; Wiesmüller, Lisa; Wells, Susanne I.

    2017-01-01

    DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition. PMID:28317934

  1. Minimum length of direct repeat sequences required for efficient homologous recombination induced by zinc finger nuclease in yeast.

    PubMed

    Ren, ChongHua; Yan, Qiang; Zhang, ZhiYing

    2014-10-01

    Zinc finger nuclease (ZFN) technology is a powerful molecular tool for targeted genome modifications and genetic engineering. However, screening for specific ZFs and validation of ZFN activity are labor intensive and time consuming. We previously designed a yeast-based ZFN screening and validation system by inserting a ZFN binding site flanked by a 164 bp direct repeat sequence into the middle of a Gal4 transcription factor, disrupting the open reading frame of the yeast Gal4 gene. Expression of the ZFN causes a double stranded break at its binding site, which promotes the cellular DNA repair system to restore expression of a functional Gal transcriptional factor via homologous recombination. Expression of Gal4 transcription factor leads to activation of three reporter genes in an AH109 yeast two-hybrid strain. However, the 164 bp direct repeat appears to generate spontaneous homologous recombination frequently, resulting in many false positive ZFNs. To overcome this, a series of DNA fragments of various lengths from 10 to 150 bp with 10 bp increase each and 164 bp direct repeats flanking the ZFN binding site were designed and constructed. The results demonstrated that the minimum length required for ZFN-induced homologous recombination was 30 bp, which almost eliminated spontaneous recombination. Using the 30 bp direct repeat sequence, ZFN could efficiently induce homologous recombination, while false positive ZFNs resulting from spontaneous homologous recombination were minimized. Thus, this study provided a simple, fast and sensitive ZFN screening and activity validation system in yeast.

  2. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. 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

  4. Homologous recombination as a replication fork escort: fork-protection and recovery.

    PubMed

    Costes, Audrey; Lambert, Sarah A E

    2012-12-27

    Homologous recombination is a universal mechanism that allows DNA repair and ensures the efficiency of DNA replication. The substrate initiating the process of homologous recombination is a single-stranded DNA that promotes a strand exchange reaction resulting in a genetic exchange that promotes genetic diversity and DNA repair. The molecular mechanisms by which homologous recombination repairs a double-strand break have been extensively studied and are now well characterized. However, the mechanisms by which homologous recombination contribute to DNA replication in eukaryotes remains poorly understood. Studies in bacteria have identified multiple roles for the machinery of homologous recombination at replication forks. Here, we review our understanding of the molecular pathways involving the homologous recombination machinery to support the robustness of DNA replication. In addition to its role in fork-recovery and in rebuilding a functional replication fork apparatus, homologous recombination may also act as a fork-protection mechanism. We discuss that some of the fork-escort functions of homologous recombination might be achieved by loading of the recombination machinery at inactivated forks without a need for a strand exchange step; as well as the consequence of such a model for the stability of eukaryotic genomes.

  5. Homologous Recombination as a Replication Fork Escort: Fork-Protection and Recovery

    PubMed Central

    Costes, Audrey; Lambert, Sarah A. E.

    2012-01-01

    Homologous recombination is a universal mechanism that allows DNA repair and ensures the efficiency of DNA replication. The substrate initiating the process of homologous recombination is a single-stranded DNA that promotes a strand exchange reaction resulting in a genetic exchange that promotes genetic diversity and DNA repair. The molecular mechanisms by which homologous recombination repairs a double-strand break have been extensively studied and are now well characterized. However, the mechanisms by which homologous recombination contribute to DNA replication in eukaryotes remains poorly understood. Studies in bacteria have identified multiple roles for the machinery of homologous recombination at replication forks. Here, we review our understanding of the molecular pathways involving the homologous recombination machinery to support the robustness of DNA replication. In addition to its role in fork-recovery and in rebuilding a functional replication fork apparatus, homologous recombination may also act as a fork-protection mechanism. We discuss that some of the fork-escort functions of homologous recombination might be achieved by loading of the recombination machinery at inactivated forks without a need for a strand exchange step; as well as the consequence of such a model for the stability of eukaryotic genomes. PMID:24970156

  6. Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes.

    PubMed

    Schwartz, Erin K; Heyer, Wolf-Dietrich

    2011-04-01

    Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81-Mms4/EME1, Slx1-Slx4/BTBD12/MUS312, XPF-ERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.

  7. Mismatch repair regulates homologous recombination, but has little influence on antigenic variation, in Trypanosoma brucei.

    PubMed

    Bell, Joanna S; McCulloch, Richard

    2003-11-14

    Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays.

  8. 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.

  9. PCR artifact in testing for homologous recombination in genomic editing in zebrafish

    PubMed Central

    Won, Minho

    2017-01-01

    We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5’ and 3’ ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish. PMID:28362803

  10. Genetic interactions among homologous recombination mutants in Candida albicans.

    PubMed

    Bellido, Alberto; Andaluz, Encarnación; Gómez-Raja, Jonathan; Álvarez-Barrientos, Alberto; Larriba, Germán

    2015-01-01

    rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Arabidopsis RAD51C gene is important for homologous recombination in meiosis and mitosis.

    PubMed

    Abe, Kiyomi; Osakabe, Keishi; Nakayama, Shigeki; Endo, Masaki; Tagiri, Akemi; Todoriki, Setsuko; Ichikawa, Hiroaki; Toki, Seiichi

    2005-10-01

    Rad51 is a homolog of the bacterial RecA recombinase, and a key factor in homologous recombination in eukaryotes. Rad51 paralogs have been identified from yeast to vertebrates. Rad51 paralogs are thought to play an important role in the assembly or stabilization of Rad51 that promotes homologous pairing and strand exchange reactions. We previously characterized two RAD51 paralogous genes in Arabidopsis (Arabidopsis thaliana) named AtRAD51C and AtXRCC3, which are homologs of human RAD51C and XRCC3, respectively, and described the interaction of their products in a yeast two-hybrid system. Recent studies showed the involvement of AtXrcc3 in DNA repair and functional role in meiosis. To determine the role of RAD51C in meiotic and mitotic recombination in higher plants, we characterized a T-DNA insertion mutant of AtRAD51C. Although the atrad51C mutant grew normally during vegetative developmental stage, the mutant produced aborted siliques, and their anthers did not contain mature pollen grains. Crossing of the mutant with wild-type plants showed defective male and female gametogeneses as evidenced by lack of seed production. Furthermore, meiosis was severely disturbed in the mutant. The atrad51C mutant also showed increased sensitivity to gamma-irradiation and cisplatin, which are known to induce double-strand DNA breaks. The efficiency of homologous recombination in somatic cells in the mutant was markedly reduced relative to that in wild-type plants.

  12. Fhit and CHK1 have opposing effects on homologous recombination repair.

    PubMed

    Hu, Baocheng; Wang, Hongyan; Wang, Xiang; Lu, Hua-Rui; Huang, Cuifen; Powell, Simon N; Huebner, Kay; Wang, Ya

    2005-10-01

    Fragile histidine triad (FHIT) gene deletion or promoter methylation and reduced Fhit protein expression occur in approximately 70% of human epithelial tumors and, in some cancers, are clearly associated with tumor progression. Specific Fhit signal pathways have not been identified. We previously reported that compared with Fhit+/+ cells, Fhit-/- cells with an overactivated ATR/CHK1 pathway show increased mutation frequency and resistance to DNA damage-induced killing, indicating that Fhit and the CHK1 pathway have opposing roles in cells responding to DNA damage. In this study, we show that cells, with or without Fhit expression, have similar DNA double-strand break induction levels and similar rejoining rates following ionizing radiation, indicating that the effect of Fhit on cell radiosensitivity is independent of nonhomologous end-joining. By combining I-SceI-induced-DNA double-strand break system and small interfering RNA approach, we also show that knocking down Fhit increases the efficiency of homologous recombination repair of cells, but knocking down Chk1 decreases the efficiency of homologous recombination repair, associated with the sensitivity to ionizing radiation-induced killing. Taken together, the results show that the role of Fhit in affecting the sensitivity of cells to ionizing radiation-induced killing is through the CHK1 pathway linked to homologous recombination repair. These results also illustrate the importance of balanced checkpoint activation in genomic stability and suggest a connection between the radioresistance and mutagenesis, carcinogenesis, as well as tumor progression in Fhit-deficient cells or tissue.

  13. Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination.

    PubMed

    Shao, Renfu; Barker, Stephen C

    2011-02-15

    The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse. Copyright © 2010 Elsevier B.V. All rights reserved.

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

    PubMed

    Andersen, Sabrina L; Zhang, Aimee; Dominska, Margaret; Moriel-Carretero, María; Herrera-Moyano, Emilia; Aguilera, Andrés; Petes, Thomas D

    2016-03-01

    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

  15. 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

  16. A BRCA1-interacting lncRNA regulates homologous recombination.

    PubMed

    Sharma, Vivek; Khurana, Simran; Kubben, Nard; Abdelmohsen, Kotb; Oberdoerffer, Philipp; Gorospe, Myriam; Misteli, Tom

    2015-11-01

    Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR.

  17. Cell cycle regulation of homologous recombination in Saccharomyces cerevisiae.

    PubMed

    Mathiasen, David P; Lisby, Michael

    2014-03-01

    Homologous recombination (HR) contributes to maintaining genome integrity by facilitating error-free repair of DNA double-strand breaks (DSBs) primarily during the S and G2 phases of the mitotic cell cycle, while nonhomologous end joining (NHEJ) is the preferred pathway for DSB repair in G1 phase. The decision to repair a DSB by NHEJ or HR is made primarily at the level of DSB end resection, which is inhibited by the Ku complex in G1 and promoted by the Sae2 and Mre11 nucleases in S/G2 . The cell cycle regulation of HR is accomplished both at the transcription level and at the protein level through post-translational modification, degradation and subcellular localization. Cyclin-dependent kinase Cdc28 plays an established key role in these events, while the role of transcriptional regulation and protein degradation are less well understood. Here, the cell cycle regulatory mechanisms for mitotic HR in Saccharomyces cerevisiae are reviewed, and evolutionarily conserved principles are highlighted. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  18. A BRCA1-interacting lncRNA regulates homologous recombination

    PubMed Central

    Sharma, Vivek; Khurana, Simran; Kubben, Nard; Abdelmohsen, Kotb; Oberdoerffer, Philipp; Gorospe, Myriam; Misteli, Tom

    2015-01-01

    Long non-coding RNAs (lncRNAs) are important players in diverse biological processes. Upon DNA damage, cells activate a complex signaling cascade referred to as the DNA damage response (DDR). Using a microarray screen, we identify here a novel lncRNA, DDSR1 (DNA damage-sensitive RNA1), which is induced upon DNA damage. DDSR1 induction is triggered in an ATM-NF-κB pathway-dependent manner by several DNA double-strand break (DSB) agents. Loss of DDSR1 impairs cell proliferation and DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect in the absence of DDSR1 is marked by aberrant accumulation of BRCA1 and RAP80 at DSB sites. In line with a role in regulating HR, DDSR1 interacts with BRCA1 and hnRNPUL1, an RNA-binding protein involved in DNA end resection. Our results suggest a role for the lncRNA DDSR1 in modulating DNA repair by HR. PMID:26412854

  19. Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model.

    PubMed Central

    Haber, James E; Ira, Gregorz; Malkova, Anna; Sugawara, Neal

    2004-01-01

    Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking. PMID:15065659

  20. Repairing a double-strand chromosome break by homologous recombination: revisiting Robin Holliday's model.

    PubMed

    Haber, James E; Ira, Gregorz; Malkova, Anna; Sugawara, Neal

    2004-01-29

    Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.

  1. Loss of Homologous Recombination or Non-homologous End-joining Leads to Radial Formation Following DNA Interstrand Crosslink Damage

    PubMed Central

    Hanlon Newell, Amy E.; Hemphill, Aaron; Akkari, Yassmine M.N.; Hejna, James; Moses, Robb E.; Olson, Susan B.

    2008-01-01

    High levels of interstrand cross-link damage in mammalian cells cause chromatid breaks and radial formations recognizable by cytogenetic examination. The mechanism of radial formation observed following DNA damage has yet to be determined. Due to recent findings linking homologous recombination and non-homologous end-joining to the action of the Fanconi anemia pathway, we speculated that radials might be the result of defects in either of the pathways of DNA repair. To test this hypothesis, we have investigated the role of homologous recombination proteins RAD51 and RAD52, non-homologous end-joining proteins Ku70 and LIG4, and protein MRE11 in radial formation and cell survival following interstrand crosslink damage with mitomycin C. For the studies we used small inhibitory RNA to deplete the proteins from cells, allowing for evaluation of radial formation and cell survival. In transformed normal human fibroblasts, depletion of these proteins increased interstrand crosslink sensitivity as manifest by decreased cell survival and increased radial formation. These results demonstrate that inactivation of proteins from either of the two separate DNA repair pathways increases cellular sensitivity to interstrand crosslinks, indicating each pathway plays a role in the normal response to interstrand crosslink damage. We can also conclude that homologous recombination or non-homologous end-joining are not required for radial formation, since radials occur with depletion of these pathways. PMID:18758156

  2. 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.

  3. Genetic battle between Helicobacter pylori and humans. The mechanism underlying homologous recombination in bacteria, which can infect human cells.

    PubMed

    Hanada, Katsuhiro; Yamaoka, Yoshio

    2014-10-01

    Helicobacter pylori is a gram-negative pathogenic bacterium that colonises the human stomach. The chronic infection it causes results in peptic ulcers and gastric cancers. H. pylori can easily establish a chronic infection even if the immune system attacks this pathogen with oxidative stress agents and immunoglobulins. This is attributed to bacterial defence mechanisms against these stresses. As a defence mechanism against oxidative stresses, in bacterial genomes, homologous recombination can act as a repair pathway of DNA's double-strand breaks (DSBs). Moreover, homologous recombination is also involved in the antigenic variation in H. pylori. Gene conversion alters genomic structures of babA and babB (encoding outer membrane proteins), resulting in escape from immunoglobulin attacks. Thus, homologous recombination in bacteria plays an important role in the maintenance of a chronic infection. In addition, H. pylori infection causes DSBs in human cells. Homologous recombination is also involved in the repair of DSBs in human cells. In this review, we describe the roles of homologous recombination with an emphasis on the maintenance of a chronic infection. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. 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

  5. Engineered Zinc Finger Nuclease–Mediated Homologous Recombination of the Human Rhodopsin Gene

    PubMed Central

    Greenwald, David L.; Cashman, Siobhan M.

    2010-01-01

    Purpose. Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Methods. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Results. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. Conclusions. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination. PMID:20671268

  6. Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases.

    PubMed Central

    Brenneman, M; Gimble, F S; Wilson, J H

    1996-01-01

    In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model. PMID:8622983

  7. Better understanding of homologous recombination through a 12-week laboratory course for undergraduates majoring in biotechnology.

    PubMed

    Li, Ming; Shen, Xiaodong; Zhao, Yan; Hu, Xiaomei; Hu, Fuquan; Rao, Xiancai

    2017-07-08

    Homologous recombination, a central concept in biology, is defined as the exchange of DNA strands between two similar or identical nucleotide sequences. Unfortunately, undergraduate students majoring in biotechnology often experience difficulties in understanding the molecular basis of homologous recombination. In this study, we developed and implemented a 12-week laboratory course for biotechnology undergraduates in which gene targeting in Streptococcus suis was used to facilitate their understanding of the basic concept and process of homologous recombination. Students worked in teams of two to select a gene of interest to create a knockout mutant using methods that relied on homologous recombination. By integrating abstract knowledge and practice in the process of scientific research, students gained hands-on experience in molecular biology techniques while learning about the principle and process of homologous recombination. The learning outcomes and survey-based assessment demonstrated that students substantially enhanced their understanding of how homologous recombination could be used to study gene function. Overall, the course was very effective for helping biotechnology undergraduates learn the theory and application of homologous recombination, while also yielding positive effects in developing confidence and scientific skills for future work in research. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):329-335, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

  8. Engineered zinc finger nuclease-mediated homologous recombination of the human rhodopsin gene.

    PubMed

    Greenwald, David L; Cashman, Siobhan M; Kumar-Singh, Rajendra

    2010-12-01

    Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination.

  9. Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

    PubMed Central

    Zelensky, Alex N.; Sanchez, Humberto; Ristic, Dejan; Vidic, Iztok; van Rossum-Fikkert, Sari E.; Essers, Jeroen; Wyman, Claire; Kanaar, Roland

    2013-01-01

    Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. PMID:23666627

  10. Selective isolation of cosmid clones by homologous recombination in Escherichia coli.

    PubMed Central

    Poustka, A; Rackwitz, H R; Frischauf, A M; Hohn, B; Lehrach, H

    1984-01-01

    A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid has been developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into lambda particles. Appropriate aliquots are then introduced into a rec+ host containing the sequence used for selection cloned into a plasmid vector without sequence homology to the cosmid vector. After a short time for recombination, the cosmids are packaged in vivo. Cosmids that have taken up the plasmid by homologous recombination are isolated by plating under conditions selecting for the antibiotic resistance markers carried by both vectors. The recombined cosmids can lose the inserted sequence by another homologous recombination event and, after packaging in vivo, these revertants can be identified on appropriate indicator plates. Images PMID:6330743

  11. 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

  12. 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

  13. Restriction-Stimulated Homologous Recombination of Plasmids by the Rece Pathway of Escherichia Coli

    PubMed Central

    Nussbaum, A.; Shalit, M.; Cohen, A.

    1992-01-01

    To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI(+) cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction. PMID:1732167

  14. High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.

    PubMed

    Mizutani, Kimihiko

    2015-01-01

    Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.

  15. Human MutL-complexes monitor homologous recombination independently of mismatch repair.

    PubMed

    Siehler, Simone Yasmin; Schrauder, Michael; Gerischer, Ulrike; Cantor, Sharon; Marra, Giancarlo; Wiesmüller, Lisa

    2009-02-01

    The role of mismatch repair proteins has been well studied in the context of DNA repair following DNA polymerase errors. Particularly in yeast, MSH2 and MSH6 have also been implicated in the regulation of genetic recombination, whereas MutL homologs appeared to be less important. So far, little is known about the role of the human MutL homolog hMLH1 in recombination, but recently described molecular interactions suggest an involvement. To identify activities of hMLH1 in this process, we applied an EGFP-based assay for the analysis of different mechanisms of DNA repair, initiated by a targeted double-stranded DNA break. We analysed 12 human cellular systems, differing in the hMLH1 and concomitantly in the hPMS1 and hPMS2 status via inducible protein expression, genetic reconstitution, or RNA interference. We demonstrate that hMLH1 and its complex partners hPMS1 and hPMS2 downregulate conservative homologous recombination (HR), particularly when involving DNA sequences with only short stretches of uninterrupted homology. Unexpectedly, hMSH2 is dispensable for this effect. Moreover, the damage-signaling kinase ATM and its substrates BLM and BACH1 are not strictly required, but the combined effect of ATM/ATR-signaling components may mediate the anti-recombinogenic effect. Our data indicate a protective role of hMutL-complexes in a process which may lead to detrimental genome rearrangements, in a manner which does not depend on mismatch repair.

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

    PubMed

    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.

  17. Allele-dependent recombination frequency: homology requirement in meiotic recombination at the hot spot in the mouse major histocompatibility complex.

    PubMed

    Yoshino, M; Sagai, T; Lindahl, K F; Toyoda, Y; Moriwaki, K; Shiroishi, T

    1995-05-20

    Meiotic recombination break joints in the mouse major histocompatibility complex (MHC) are clustered within short segments known as hot spots. We systematically investigated the requirement for sequence homology between two chromosomes for recombination activity at the hot spot next to the Lmp2 gene. The results indicated that a high rate of recombination required a high degree of similarity of overall genome structure at the hot spot. In particular, the same copy number of repetitive sequences within the hot spot was essential for a high frequency of recombination, suggesting that recombination in mouse meiosis is more sensitive to heterozygous deletion or insertion of DNA than to mismatches of single-base substitutions.

  18. 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.

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

    PubMed

    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.

  20. 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

  1. 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

  2. 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.

  3. Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila.

    PubMed

    David, Sophia; Sánchez-Busó, Leonor; Harris, Simon R; Marttinen, Pekka; Rusniok, Christophe; Buchrieser, Carmen; Harrison, Timothy G; Parkhill, Julian

    2017-06-01

    Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires' disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic "hotspots" of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila

  4. Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila

    PubMed Central

    Marttinen, Pekka; Rusniok, Christophe; Harrison, Timothy G.

    2017-01-01

    Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires’ disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic “hotspots” of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila

  5. Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange.

    PubMed

    Lambert, Sarah; Mizuno, Ken'ichi; Blaisonneau, Joël; Martineau, Sylvain; Chanet, Roland; Fréon, Karine; Murray, Johanne M; Carr, Antony M; Baldacci, Giuseppe

    2010-08-13

    Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

  6. Genome-Wide Survey of Mutual Homologous Recombination in a Highly Sexual Bacterial Species

    PubMed Central

    Yahara, Koji; Kawai, Mikihiko; Furuta, Yoshikazu; Takahashi, Noriko; Handa, Naofumi; Tsuru, Takeshi; Oshima, Kenshiro; Yoshida, Masaru; Azuma, Takeshi; Hattori, Masahira; Uchiyama, Ikuo; Kobayashi, Ichizo

    2012-01-01

    The nature of a species remains a fundamental and controversial question. The era of genome/metagenome sequencing has intensified the debate in prokaryotes because of extensive horizontal gene transfer. In this study, we conducted a genome-wide survey of outcrossing homologous recombination in the highly sexual bacterial species Helicobacter pylori. We conducted multiple genome alignment and analyzed the entire data set of one-to-one orthologous genes for its global strains. We detected mosaic structures due to repeated recombination events and discordant phylogenies throughout the genomes of this species. Most of these genes including the “core” set of genes and horizontally transferred genes showed at least one recombination event. Taking into account the relationship between the nucleotide diversity and the minimum number of recombination events per nucleotide, we evaluated the recombination rate in every gene. The rate appears constant across the genome, but genes with a particularly high or low recombination rate were detected. Interestingly, genes with high recombination included those for DNA transformation and for basic cellular functions, such as biosynthesis and metabolism. Several highly divergent genes with a high recombination rate included those for host interaction, such as outer membrane proteins and lipopolysaccharide synthesis. These results provide a global picture of genome-wide distribution of outcrossing homologous recombination in a bacterial species for the first time, to our knowledge, and illustrate how a species can be shaped by mutual homologous recombination. PMID:22534164

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

    PubMed

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

    1999-06-01

    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.

  8. Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells.

    PubMed

    Du, Li-Qing; Du, Xiao-Qing; Bai, Jian-Qiang; Wang, Yan; Yang, Qing-Shan; Wang, Xiao-Chun; Zhao, Peng; Wang, Hong; Liu, Qiang; Fan, Fei-Yue

    2012-05-01

    Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated. Induction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays. The result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells. These results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination-directed subnuclear foci.

  9. 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.

  10. Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae.

    PubMed Central

    Yoshida, Jumpei; Umezu, Keiko; Maki, Hisaji

    2003-01-01

    In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements. PMID:12750319

  11. PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus.

    PubMed

    Ronceret, Arnaud; Doutriaux, Marie-Pascale; Golubovskaya, Inna N; Pawlowski, Wojciech P

    2009-11-24

    Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.

  12. 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

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

    PubMed

    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.

  14. Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

    PubMed Central

    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

  15. Heterogeneity in the Frequency and Characteristics of Homologous Recombination in Pneumococcal Evolution

    PubMed Central

    Hanage, William P.; Harris, Simon R.; Bentley, Stephen; Fraser, Christophe

    2014-01-01

    The bacterium Streptococcus pneumoniae (pneumococcus) is one of the most important human bacterial pathogens, and a leading cause of morbidity and mortality worldwide. The pneumococcus is also known for undergoing extensive homologous recombination via transformation with exogenous DNA. It has been shown that recombination has a major impact on the evolution of the pathogen, including acquisition of antibiotic resistance and serotype-switching. Nevertheless, the mechanism and the rates of recombination in an epidemiological context remain poorly understood. Here, we proposed several mathematical models to describe the rate and size of recombination in the evolutionary history of two very distinct pneumococcal lineages, PMEN1 and CC180. We found that, in both lineages, the process of homologous recombination was best described by a heterogeneous model of recombination with single, short, frequent replacements, which we call micro-recombinations, and rarer, multi-fragment, saltational replacements, which we call macro-recombinations. Macro-recombination was associated with major phenotypic changes, including serotype-switching events, and thus was a major driver of the diversification of the pathogen. We critically evaluate biological and epidemiological processes that could give rise to the micro-recombination and macro-recombination processes. PMID:24786281

  16. Heterogeneity in the frequency and characteristics of homologous recombination in pneumococcal evolution.

    PubMed

    Mostowy, Rafal; Croucher, Nicholas J; Hanage, William P; Harris, Simon R; Bentley, Stephen; Fraser, Christophe

    2014-05-01

    The bacterium Streptococcus pneumoniae (pneumococcus) is one of the most important human bacterial pathogens, and a leading cause of morbidity and mortality worldwide. The pneumococcus is also known for undergoing extensive homologous recombination via transformation with exogenous DNA. It has been shown that recombination has a major impact on the evolution of the pathogen, including acquisition of antibiotic resistance and serotype-switching. Nevertheless, the mechanism and the rates of recombination in an epidemiological context remain poorly understood. Here, we proposed several mathematical models to describe the rate and size of recombination in the evolutionary history of two very distinct pneumococcal lineages, PMEN1 and CC180. We found that, in both lineages, the process of homologous recombination was best described by a heterogeneous model of recombination with single, short, frequent replacements, which we call micro-recombinations, and rarer, multi-fragment, saltational replacements, which we call macro-recombinations. Macro-recombination was associated with major phenotypic changes, including serotype-switching events, and thus was a major driver of the diversification of the pathogen. We critically evaluate biological and epidemiological processes that could give rise to the micro-recombination and macro-recombination processes.

  17. The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

    PubMed

    Knoll, Alexander; Higgins, James D; Seeliger, Katharina; Reha, Sarah J; Dangel, Natalie J; Bauknecht, Markus; Schröpfer, Susan; Franklin, F Christopher H; Puchta, Holger

    2012-04-01

    The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

  18. Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications.

    PubMed

    Rockwood, Jananie; Mao, Dominic; Grogan, Dennis W

    2013-09-01

    Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotide-mediated transformation of Sulfolobus and other micro-organisms.

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

    PubMed Central

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

    2016-01-01

    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

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

    PubMed Central

    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.

    2013-01-01

    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

  1. 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.

  2. High-level transgene expression by homologous recombination-mediated gene transfer

    PubMed Central

    Grandjean, Mélanie; Girod, Pierre-Alain; Calabrese, David; Kostyrko, Kaja; Wicht, Marianne; Yerly, Florence; Mazza, Christian; Beckmann, Jacques S.; Martinet, Danielle; Mermod, Nicolas

    2011-01-01

    Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expression. PMID:21652640

  3. A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair

    PubMed Central

    Jain, Suvi; Sugawara, Neal; Lydeard, John; Vaze, Moreshwar; Tanguy Le Gac, Nicolas; Haber, James E.

    2009-01-01

    A DNA double-strand break (DSB) is repaired by gene conversion (GC) if both ends of the DSB share homology with an intact DNA sequence. However, if homology is limited to only one of the DSB ends, repair occurs by break-induced replication (BIR). It is not known how the homology status of the DSB ends is first assessed and what other parameters govern the choice between these repair pathways. Our data suggest that a “recombination execution checkpoint” (REC) regulates the choice of the homologous recombination pathway employed to repair a given DSB. This choice is made prior to the initiation of DNA synthesis, and is dependent on the relative position and orientation of the homologous sequences used for repair. The RecQ family helicase Sgs1 plays a key role in regulating the choice of the recombination pathway. Surprisingly, break repair and gap repair are fundamentally different processes, both kinetically and genetically, as Pol32 is required only for gap repair. We propose that the REC may have evolved to preserve genome integrity by promoting conservative repair, especially when a DSB occurs within a repeated sequence. PMID:19204116

  4. Role of homologous recombination in adaptive diversification of extraintestinal Escherichia coli.

    PubMed

    Paul, Sandip; Linardopoulou, Elena V; Billig, Mariya; Tchesnokova, Veronika; Price, Lance B; Johnson, James R; Chattopadhyay, Sujay; Sokurenko, Evgeni V

    2013-01-01

    The contribution of homologous exchange (recombination) of core genes in the adaptive evolution of bacterial pathogens is not well understood. To investigate this, we analyzed fully assembled genomes of two Escherichia coli strains from sequence type 131 (ST131), a clonal group that is both the leading cause of extraintestinal E. coli infections and the main source of fluoroquinolone-resistant E. coli. Although the sequences of each of the seven multilocus sequence typing genes were identical in the two ST131 isolates, the strains diverged from one another by homologous recombination that affected at least 9% of core genes. This was on a par with the contribution to genomic diversity of horizontal gene transfer and point gene mutation. The genomic positions of recombinant and mobile genetic regions were partially linked, suggesting their concurrent occurrence. One of the genes affected by homologous recombination was fimH, which encodes mannose-specific type 1 fimbrial adhesin, resulting in functionally distinct copies of the gene in ST131 strains. One strain, a uropathogenic isolate, had a pathoadaptive variant of fimH that was acquired by homologous replacement into the commensal strain background. Close examination of FimH structure and function in additional ST131 isolates revealed that recombination led to acquisition of several functionally distinct variants that, upon homologous exchange, were targeted by a variety of pathoadaptive mutations under strong positive selection. Different recombinant fimH strains also showed a strong clonal association with ST131 isolates that had distinct fluoroquinolone resistance profiles. Thus, homologous recombination of core genes plays a significant role in adaptive diversification of bacterial pathogens, especially at the level of clonally related groups of isolates.

  5. Homologous recombination and double-strand break repair in the transformation of Rhizopus oryzae.

    PubMed

    Skory, C D

    2002-11-01

    Genetic transformation of the Mucorales fungi has been problematic, since DNA transformed into the host rarely integrates and usually is mitotically unstable in the absence of selective pressure. In this study, transformation of Rhizopus oryzae was investigated to determine if the fate of introduced DNA could be predicted based on double-strand break repair and recombination mechanisms found in other fungi. A transformation system was developed with uracil auxotrophs of Rhizopus oryzae that could be complemented with the pyrG gene isolated in this work. DNA transformed as circular plasmids was maintained extrachromosomally in high-molecular-weight (>23 kb) concatenated arrangement. Type-I crossover integration into the pyrG locus and type-III pyrG gene replacement events occurred in approximately 1-5% of transformants. Linearization of the plasmid pPyr225 with a single restriction enzyme that cleaves within the vector sequence almost always resulted in isolates with replicating concatenated plasmids that had been repaired by end-joining recombination that restored the restriction site. The addition of a 40-bp direct repeat on either side of this cleavage site led to repair by homologous recombination between the repeated sequences on the plasmid, resulting in loss of the restriction site. When plasmid pPyr225 was digested with two different enzymes that cleave within the vector sequence to release the pyrG containing fragment, only pyrG gene replacement recombination occurred in transformants. Linearization of plasmid pPyr225 within the pyrG gene itself gave the highest percentage (20%) of type-I integration at the pyrG locus. However, end-joining repair and gene replacement events were still the predominant types of recombination found in transformations with this plasmid topology.

  6. Distribution of Exchanges upon Homologous Recombination of Exogenous DNA in Xenopus Laevis Oocytes

    PubMed Central

    Carroll, D.; Lehman, C. W.; Jeong-Yu, S.; Dohrmann, P.; Dawson, R. J.; Trautman, J. K.

    1994-01-01

    Homologous recombination between DNA molecules injected into Xenopus oocyte nuclei was investigated by examining the recovery of information from differentially marked parental sequences. The injected recombination substrate was a linear DNA with terminal direct repeats of 1246 bp; one repeat differed from the other by eight single base-pair substitutions, distributed throughout the region of homology, each of which created or destroyed a restriction enzyme site. Recombination products were recovered and analyzed for their content of the diagnostic sites, either directly by Southern blot-hybridization or after cloning in bacteria. The majority (76%) of the cloned products appeared to be the result of simple exchanges-i.e., there was one sharp transition from sequences derived from one parent to sequences derived from the other. These simple exchanges were concentrated near the ends of the homologous interval and, thus, near the sites of the original molecular ends. Placing marked sites on only one side of the homologous overlap showed that marker recovery was governed largely by the positions of the molecular ends and not by the markers themselves. When a terminal nonhomology was present at one end of the substrate, the yield of recombinants was sharply decreased, but the pattern of exchanges was not affected, suggesting that products from end-blocked substrates arise by the same recombination pathway. Because of considerable evidence supporting a nonconservative, resection-annealing mechanism for recombination in oocytes, we interpret the distribution of exchanges as resulting from long-patch repair of extensive heteroduplex intermediates. PMID:7828826

  7. Implication of RuvABC and RecG in homologous recombination in Streptomyces ambofaciens.

    PubMed

    Hoff, Grégory; Bertrand, Claire; Piotrowski, Emilie; Thibessard, Annabelle; Leblond, Pierre

    2017-01-01

    Most bacterial organisms rely on homologous recombination to repair DNA double-strand breaks and for the post-replicative repair of DNA single-strand gaps. Homologous recombination can be divided into three steps: (i) a pre-synaptic step in which the DNA 3'-OH ends are processed, (ii) a recA-dependent synaptic step allowing the invasion of an intact copy and the formation of Holliday junctions, and (iii) a post-synaptic step consisting of migration and resolution of these junctions. Currently, little is known about factors involved in homologous recombination, especially for the post-synaptic step. In Escherichia coli, branch migration and resolution are performed by the RuvABC complex, but could also rely on the RecG helicase in a redundant manner. In this study, we show that recG and ruvABC are well-conserved among Streptomyces. ΔruvABC, ΔrecG and ΔruvABC ΔrecG mutant strains were constructed. ΔruvABC ΔrecG is only slightly affected by exposure to DNA damage (UV). We also show that conjugational recombination decreases in the absence of RuvABC and RecG, but that intra-chromosomal recombination is not affected. These data suggest that RuvABC and RecG are indeed involved in homologous recombination in Streptomyces ambofaciens and that alternative factors are able to take over Holliday junction in Streptomyces.

  8. [An homologous recombination strategy to directly clone mammalian telemeres

    SciTech Connect

    Not Available

    1992-01-01

    We have pursued three goals over the past year. The first involved determining whether the HARY vector could be used for homologous integration in the human genome. The second was to ascertain whether inserted sequences could be amplified in preference to the endogenous DHFR genes. The third was to determine if the HARY insertion could provide an anchor point for long range restriction mapping. The progress in each goal is described.

  9. Nascent DNA synthesis during homologous recombination is synergistically promoted by the rad51 recombinase and DNA homology.

    PubMed

    Mundia, Maureen M; Desai, Vatsal; Magwood, Alissa C; Baker, Mark D

    2014-05-01

    In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (3' extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 3' extension for homology lengths of 500 bp to 2 kb. At values below ∼500 bp, the frequency of 3' extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 3' extension. Overexpression of wild-type Rad51 stimulated the frequency of 3' extension by ∼3-fold for homology lengths <900 bp, but when homology was >2 kb, 3' extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 3' extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 3' extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 3' extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 3' extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 3' extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization.

  10. Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

    PubMed Central

    Mundia, Maureen M.; Desai, Vatsal; Magwood, Alissa C.; Baker, Mark D.

    2014-01-01

    In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (3′ extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 3′ extension for homology lengths of 500 bp to 2 kb. At values below ∼500 bp, the frequency of 3′ extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 3′ extension. Overexpression of wild-type Rad51 stimulated the frequency of 3′ extension by ∼3-fold for homology lengths <900 bp, but when homology was >2 kb, 3′ extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 3′ extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 3′ extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 3′ extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1–8, rescues the 3′ extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 3′ extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization. PMID:24583581

  11. A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile

    PubMed Central

    Joska, Tammy M.; Mashruwala, Ameya; Boyd, Jeffrey M.; Belden, William J.

    2014-01-01

    Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2 μm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits. PMID:24418681

  12. A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile.

    PubMed

    Joska, Tammy M; Mashruwala, Ameya; Boyd, Jeffrey M; Belden, William J

    2014-05-01

    Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2μm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits.

  13. Recombination sequences in plant mitochondrial genomes: diversity and homologies to known mitochondrial genes.

    PubMed Central

    Stern, D B; Palmer, J D

    1984-01-01

    Several plant mitochondrial genomes contain repeated sequences that are postulated to be sites of homologous intragenomic recombination (1-3). In this report, we have used filter hybridizations to investigate sequence relationships between the cloned mitochondrial DNA (mtDNA) recombination repeats from turnip, spinach and maize and total mtDNA isolated from thirteen species of angiosperms. We find that strong sequence homologies exist between the spinach and turnip recombination repeats and essentially all other mitochondrial genomes tested, whereas a major maize recombination repeat does not hybridize to any other mtDNA. The sequences homologous to the turnip repeat do not appear to function in recombination in any other genome, whereas the spinach repeat hybridizes to reiterated sequences within the mitochondrial genomes of wheat and two species of pokeweed that do appear to be sites of recombination. Thus, although intragenomic recombination is a widespread phenomenon in plant mitochondria, it appears that different sequences either serve as substrates for this function in different species, or else surround a relatively short common recombination site which does not cross-hybridize under our experimental conditions. Identified gene sequences from maize mtDNA were used in heterologous hybridizations to show that the repeated sequences implicated in recombination in turnip and spinach/pokeweed/wheat mitochondria include, or are closely linked to genes for subunit II of cytochrome c oxidase and 26S rRNA, respectively. Together with previous studies indicating that the 18S rRNA gene in wheat mtDNA is contained within a recombination repeat (3), these results imply an unexpectedly frequent association between recombination repeats and plant mitochondrial genes. Images PMID:6473104

  14. 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.

  15. V(D)J recombination coding junction formation without DNA homology: processing of coding termini.

    PubMed Central

    Boubnov, N V; Wills, Z P; Weaver, D T

    1993-01-01

    Coding junction formation in V(D)J recombination generates diversity in the antigen recognition structures of immunoglobulin and T-cell receptor molecules by combining processes of deletion of terminal coding sequences and addition of nucleotides prior to joining. We have examined the role of coding end DNA composition in junction formation with plasmid substrates containing defined homopolymers flanking the recombination signal sequence elements. We found that coding junctions formed efficiently with or without terminal DNA homology. The extent of junctional deletion was conserved independent of coding ends with increased, partial, or no DNA homology. Interestingly, G/C homopolymer coding ends showed reduced deletion regardless of DNA homology. Therefore, DNA homology cannot be the primary determinant that stabilizes coding end structures for processing and joining. PMID:8413286

  16. Effect of subinhibitory concentrations of antibiotics on intrachromosomal homologous recombination in Escherichia coli.

    PubMed

    López, Elena; Blázquez, Jesús

    2009-08-01

    Subinhibitory concentrations of some antibiotics, such as fluoroquinolones, have been reported to stimulate mutation and, consequently, bacterial adaptation to different stresses, including antibiotic pressure. In Escherichia coli, this stimulation is mediated by alternative DNA polymerases induced via the SOS response. Sublethal concentrations of the fluoroquinolone ciprofloxacin have been shown to stimulate recombination between divergent sequences in E. coli. However, the effect of ciprofloxacin on recombination between homologous sequences and its SOS dependence have not been studied. Moreover, the possible effects of other antibiotics on homologous recombination remain untested. The aim of this work was to study the effects of sublethal concentrations of ciprofloxacin and 10 additional antibiotics, including different molecular families with different molecular targets, on the rate of homologous recombination of DNA in E. coli. The antibiotics tested were ciprofloxacin, ampicillin, ceftazidime, imipenem, chloramphenicol, tetracycline, gentamicin, rifampin (rifampicin), trimethoprim, fosfomycin, and colistin. Our results indicate that only ciprofloxacin consistently stimulates the intrachromosomal recombinogenic capability of homologous sequences in E. coli. The ciprofloxacin-based stimulation occurs at concentrations and times that apparently do not dramatically compromise the viability of the whole population, and it is dependent on RecA and partially dependent on SOS induction. One of the main findings of this work is that, apart from quinolone antibiotics, none of the most used antibiotics, including trimethoprim (a known inducer of the SOS response), has a clear side effect on homologous recombination in E. coli. In addition to the already described effects of some antibiotics on mutagenicity, DNA transfer, and genetic transformability in naturally competent species, the effect of increasing intrachromosomal recombination of homologous DNA sequences can be

  17. 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.

  18. Recombination-independent mechanisms and pairing of homologous chromosomes during meiosis in plants.

    PubMed

    Da Ines, Olivier; Gallego, Maria E; White, Charles I

    2014-03-01

    Meiosis is the specialized eukaryotic cell division that permits the halving of ploidy necessary for gametogenesis in sexually reproducing organisms. This involves a single round of DNA replication followed by two successive divisions. To ensure balanced segregation, homologous chromosome pairs must migrate to opposite poles at the first meiotic division and this means that they must recognize and pair with each other beforehand. Although understanding of the mechanisms by which meiotic chromosomes find and pair with their homologs has greatly advanced, it remains far from being fully understood. With some notable exceptions such as male Drosophila, the recognition and physical linkage of homologs at the first meiotic division involves homologous recombination. However, in addition to this, it is clear that many organisms, including plants, have also evolved a series of recombination-independent mechanisms to facilitate homolog recognition and pairing. These implicate chromosome structure and dynamics, telomeres, centromeres, and, most recently, small RNAs. With a particular focus on plants, we present here an overview of understanding of these early, recombination-independent events that act in the pairing of homologous chromosomes during the first meiotic division.

  19. Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination

    PubMed Central

    Ceballos, Shannon J.; Heyer, Wolf-Dietrich

    2011-01-01

    Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. PMID:21704205

  20. [Construction of multifunctional genetically engineered pesticides-degrading bacteria by homologous recombination].

    PubMed

    Jiang, Jian-Dong; Gu, Li-Feng; Sun, Ji-Quan; Dai, Xian-Zhu; Wen, Yang; Li, Shun-Peng

    2005-11-01

    Construction of multifunctional pesticides-degrading genetically engineered microorganisms (GEMs) is increasing important in the bioremediation of various pesticides contaminants in environment. However, construction of genetically stable GEMs without any exogenous antibiotic resistance is thought to be one of the bottlenecks in GEMs construction. In this article, homologous recombination vectors with the recipient's 16S rDNA as homologous recombination directing sequence (HRDS) and sacB gene as double crossover recombinants positive selective marker were firstly constructed. The methyl parathion hydroalse gene (mpd) was inserted into the 16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7 x 10-(7) - 6.8 x 10(-7). Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed. The homologous recombination events were confirmed by PCR and southern blot methods. The obtained GEMs were genetically stable and could degrade methyl parathion and carbofuran simultaneously. The insertion of mpd gene into rrn site did not have any significant effect on recipient' s physiological and original degrading characteristics. The methyl parathion hydrolase (MPH) was expressed at a relatively high level in the recombinants and the recombinant MPH specific activity in cell lysate was higher than that of original bacterium (DLL-1) in every growth phase tested. The highest recombinant MPH specific activity was 6.22 mu/tg. In this article, we describe a first attempt to use rRNA-encoding regions of Sphingomonas strains as target site for expression of exogenous MPH, and constructed multifunctional pesticides degrading GEMs, which are genetically stable and promising for developing bioremediation strategies for the decontamination of pesticides polluted soils.

  1. Increase of homologous recombination frequency in vascular tissue of Arabidopsis plants exposed to salt stress.

    PubMed

    Boyko, Alex; Hudson, Darryl; Bhomkar, Prasanna; Kathiria, Palak; Kovalchuk, Igor

    2006-06-01

    Here we analyzed the influence of salt stress on plant genome stability. Homologous recombination events were detected in transgenic Arabidopsis plants that carried in their genome a beta-glucuronidase recombination marker. Recombination events were scored as blue sectors using a stereo microscope. Exposure to 50 mM salt resulted in a 3.0-fold increase in recombination frequency. To analyze the organ and tissue specificity of recombination events, we examined cross-sections of leaves, stems and roots. We found that nearly 30% of recombination events in plants grown under normal conditions and nearly 50% of events in plants grown on salt were undetected by the conventional method. Most of the recombination events represented a cluster/group of cells (12 on average), although events with single cells were also detected. Recombination events were very frequent in leaf mesophyll cells. On average, individual recombination events located on leaves contained more cells than events located on roots or stems. Analysis of recombination events in cross-sectioned tissue of salt-treated plants revealed a shift in the distribution of recombination events towards the vascular tissue. We discuss the significance of the finding for plant stress physiology.

  2. Recombinational DNA repair in a cellular context: a search for the homology search.

    PubMed

    Weiner, Allon; Zauberman, Nathan; Minsky, Abraham

    2009-10-01

    Double-strand DNA breaks (DSBs) are the most detrimental lesion that can be sustained by the genetic complement, and their inaccurate mending can be just as damaging. According to the consensual view, precise DSB repair relies on homologous recombination. Here, we review studies on DNA repair, chromatin diffusion and chromosome confinement, which collectively imply that a genome-wide search for a homologous template, generally thought to be a pivotal stage in all homologous DSB repair pathways, is improbable. The implications of this assertion for the scope and constraints of DSB repair pathways and for the ability of diverse organisms to cope with DNA damage are discussed.

  3. 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.

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

    PubMed

    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.

  5. 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

  6. Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution

    PubMed Central

    Lee, Christopher; Kim, Namshin; Roy, Meenakshi; Graveley, Brenton R.

    2010-01-01

    The arthropod Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of protein isoforms via combinatorial splicing of numerous alternative exons encoding immunoglobulin variable domains organized into three clusters referred to as the exon 4, 6, and 9 clusters. Dscam protein diversity is important for nervous system development and immune functions. We have performed extensive phylogenetic analyses of Dscam from 20 arthropods (each containing between 46 and 96 alternative exons) to reconstruct the detailed history of exon duplication and loss events that built this remarkable system over 450 million years of evolution. Whereas the structure of the exon 4 cluster is ancient, the exon 6 and 9 clusters have undergone massive, independent expansions in each insect lineage. An analysis of nearly 2000 duplicated exons enabled detailed reconstruction of the timing, location, and boundaries of these duplication events. These data clearly show that new Dscam exons have arisen continuously throughout arthropod evolution and that this process is still occurring in the exon 6 and 9 clusters. Recently duplicated regions display boundaries corresponding to a single exon and the adjacent intron. The boundaries, homology, location, clustering, and relative frequencies of these duplication events strongly suggest that staggered homologous recombination is the major mechanism by which new Dscam exons evolve. These data provide a remarkably detailed picture of how complex gene structure evolves and reveal the molecular mechanism behind this process. PMID:19934230

  7. Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution.

    PubMed

    Lee, Christopher; Kim, Namshin; Roy, Meenakshi; Graveley, Brenton R

    2010-01-01

    The arthropod Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of protein isoforms via combinatorial splicing of numerous alternative exons encoding immunoglobulin variable domains organized into three clusters referred to as the exon 4, 6, and 9 clusters. Dscam protein diversity is important for nervous system development and immune functions. We have performed extensive phylogenetic analyses of Dscam from 20 arthropods (each containing between 46 and 96 alternative exons) to reconstruct the detailed history of exon duplication and loss events that built this remarkable system over 450 million years of evolution. Whereas the structure of the exon 4 cluster is ancient, the exon 6 and 9 clusters have undergone massive, independent expansions in each insect lineage. An analysis of nearly 2000 duplicated exons enabled detailed reconstruction of the timing, location, and boundaries of these duplication events. These data clearly show that new Dscam exons have arisen continuously throughout arthropod evolution and that this process is still occurring in the exon 6 and 9 clusters. Recently duplicated regions display boundaries corresponding to a single exon and the adjacent intron. The boundaries, homology, location, clustering, and relative frequencies of these duplication events strongly suggest that staggered homologous recombination is the major mechanism by which new Dscam exons evolve. These data provide a remarkably detailed picture of how complex gene structure evolves and reveal the molecular mechanism behind this process.

  8. Change of gene structure and function by non-homologous end-joining, homologous recombination, and transposition of DNA.

    PubMed

    Goettel, Wolfgang; Messing, Joachim

    2009-06-01

    An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization

  9. Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas.

    PubMed

    Pennington, Kathryn P; Walsh, Tom; Harrell, Maria I; Lee, Ming K; Pennil, Christopher C; Rendi, Mara H; Thornton, Anne; Norquist, Barbara M; Casadei, Silvia; Nord, Alexander S; Agnew, Kathy J; Pritchard, Colin C; Scroggins, Sheena; Garcia, Rochelle L; King, Mary-Claire; Swisher, Elizabeth M

    2014-02-01

    Hallmarks of germline BRCA1/2-associated ovarian carcinomas include chemosensitivity and improved survival. The therapeutic impact of somatic BRCA1/2 mutations and mutations in other homologous recombination DNA repair genes is uncertain. Using targeted capture and massively parallel genomic sequencing, we assessed 390 ovarian carcinomas for germline and somatic loss-of-function mutations in 30 genes, including BRCA1, BRCA2, and 11 other genes in the homologous recombination pathway. Thirty-one percent of ovarian carcinomas had a deleterious germline (24%) and/or somatic (9%) mutation in one or more of the 13 homologous recombination genes: BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D. Nonserous ovarian carcinomas had similar rates of homologous recombination mutations to serous carcinomas (28% vs. 31%, P = 0.6), including clear cell, endometrioid, and carcinosarcoma. The presence of germline and somatic homologous recombination mutations was highly predictive of primary platinum sensitivity (P = 0.0002) and improved overall survival (P = 0.0006), with a median overall survival of 66 months in germline homologous recombination mutation carriers, 59 months in cases with a somatic homologous recombination mutation, and 41 months for cases without a homologous recombination mutation. Germline or somatic mutations in homologous recombination genes are present in almost one third of ovarian carcinomas, including both serous and nonserous histologies. Somatic BRCA1/2 mutations and mutations in other homologous recombination genes have a similar positive impact on overall survival and platinum responsiveness as germline BRCA1/2 mutations. The similar rate of homologous recombination mutations in nonserous carcinomas supports their inclusion in PARP inhibitor clinical trials. ©2013 AACR.

  10. Unequal sister chromatid and homolog recombination at a tandem duplication of the A1 locus in maize.

    PubMed

    Yandeau-Nelson, Marna D; Xia, Yiji; Li, Jin; Neuffer, M Gerald; Schnable, Patrick S

    2006-08-01

    Tandemly arrayed duplicate genes are prevalent. The maize A1-b haplotype is a tandem duplication that consists of the components, alpha and beta. The rate of meiotic unequal recombination at A1-b is ninefold higher when a homolog is present than when it is absent (i.e., hemizygote). When a sequence heterologous homolog is available, 94% of recombinants (264/281) are generated via recombination with the homolog rather than with the sister chromatid. In addition, 83% (220/264) of homolog recombination events involved alpha rather than beta. These results indicate that: (1) the homolog is the preferred template for unequal recombination and (2) pairing of the duplicated segments with the homolog does not occur randomly but instead favors a particular configuration. The choice of recombination template (i.e., homolog vs. sister chromatid) affects the distribution of recombination breakpoints within a1. Rates of unequal recombination at A1-b are similar to the rate of recombination between nonduplicated a1 alleles. Unequal recombination is therefore common and is likely to be responsible for the generation of genetic variability, even within inbred lines.

  11. Unequal Sister Chromatid and Homolog Recombination at a Tandem Duplication of the a1 Locus in Maize

    PubMed Central

    Yandeau-Nelson, Marna D.; Xia, Yiji; Li, Jin; Neuffer, M. Gerald; Schnable, Patrick S.

    2006-01-01

    Tandemly arrayed duplicate genes are prevalent. The maize A1-b haplotype is a tandem duplication that consists of the components, α and β. The rate of meiotic unequal recombination at A1-b is ninefold higher when a homolog is present than when it is absent (i.e., hemizygote). When a sequence heterologous homolog is available, 94% of recombinants (264/281) are generated via recombination with the homolog rather than with the sister chromatid. In addition, 83% (220/264) of homolog recombination events involved α rather than β. These results indicate that: (1) the homolog is the preferred template for unequal recombination and (2) pairing of the duplicated segments with the homolog does not occur randomly but instead favors a particular configuration. The choice of recombination template (i.e., homolog vs. sister chromatid) affects the distribution of recombination breakpoints within a1. Rates of unequal recombination at A1-b are similar to the rate of recombination between nonduplicated a1 alleles. Unequal recombination is therefore common and is likely to be responsible for the generation of genetic variability, even within inbred lines. PMID:16751673

  12. The effect of thermal dose on hyperthermia-mediated inhibition of DNA repair through homologous recombination.

    PubMed

    van den Tempel, Nathalie; Laffeber, Charlie; Odijk, Hanny; van Cappellen, Wiggert A; van Rhoon, Gerard C; Franckena, Martine; Kanaar, Roland

    2017-07-04

    Hyperthermia has a number of biological effects that sensitize tumors to radiotherapy in the range between 40-44 °C. One of these effects is heat-induced degradation of BRCA2 that in turn causes reduced RAD51 focus formation, which results in an attenuation of DNA repair through homologous recombination. Prompted by this molecular insight into how hyperthermia attenuates homologous recombination, we now quantitatively explore time and temperature dynamics of hyperthermia on BRCA2 levels and RAD51 focus formation in cell culture models, and link this to their clonogenic survival capacity after irradiation (0-6 Gy). For treatment temperatures above 41 °C, we found a decrease in cell survival, an increase in sensitization towards irradiation, a decrease of BRCA2 protein levels, and altered RAD51 focus formation. When the temperatures exceeded 43 °C, we found that hyperthermia alone killed more cells directly, and that processes other than homologous recombination were affected by the heat. This study demonstrates that optimal inhibition of HR is achieved by subjecting cells to hyperthermia at 41-43 °C for 30 to 60 minutes. Our data provides a guideline for the clinical application of novel combination treatments that could exploit hyperthermia's attenuation of homologous recombination, such as the combination of hyperthermia with PARP-inhibitors for non-BRCA mutations carriers.

  13. A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity.

    PubMed

    Uringa, Evert-Jan; Baldeyron, Céline; Odijk, Hanny; Wassenaar, Evelyne; van Cappellen, Wiggert A; Maas, Alex; Hoeijmakers, Jan H J; Baarends, Willy M; Kanaar, Roland; Essers, Jeroen

    2015-01-01

    Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51(+/wt-GFP) embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51(+/wt-GFP) embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  15. 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.

  16. A defect in homologous recombination leads to increased translesion synthesis in E. coli.

    PubMed

    Naiman, Karel; Pagès, Vincent; Fuchs, Robert P

    2016-09-19

    DNA damage tolerance pathways allow cells to duplicate their genomes despite the presence of replication blocking lesions. Cells possess two major tolerance strategies, namely translesion synthesis (TLS) and homology directed gap repair (HDGR). TLS pathways involve specialized DNA polymerases that are able to synthesize past DNA lesions with an intrinsic risk of causing point mutations. In contrast, HDGR pathways are essentially error-free as they rely on the recovery of missing information from the sister chromatid by RecA-mediated homologous recombination. We have investigated the genetic control of pathway choice between TLS and HDGR in vivo in Escherichia coli In a strain with wild type RecA activity, the extent of TLS across replication blocking lesions is generally low while HDGR is used extensively. Interestingly, recA alleles that are partially impaired in D-loop formation confer a decrease in HDGR and a concomitant increase in TLS. Thus, partial defect of RecA's capacity to invade the homologous sister chromatid increases the lifetime of the ssDNA.RecA filament, i.e. the 'SOS signal'. This increase favors TLS by increasing both the TLS polymerase concentration and the lifetime of the TLS substrate, before it becomes sequestered by homologous recombination. In conclusion, the pathway choice between error-prone TLS and error-free HDGR is controlled by the efficiency of homologous recombination. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones.

    PubMed

    Arenhart, Sandra; Silva, José Valter Joaquim; Flores, Eduardo Furtado; Weiblen, Rudi; Gil, Laura Helena Vega Gonzales

    The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  18. Homologous recombination in pestiviruses: identification of three putative novel events between different subtypes/genogroups.

    PubMed

    Weber, Matheus Nunes; Streck, André Felipe; Silveira, Simone; Mósena, Ana Cristina Sbaraini; Silva, Mariana Soares da; Canal, Cláudio Wageck

    2015-03-01

    Viruses from the genus Pestivirus of the family Flaviviridae have a non-segmented, single-stranded RNA genome and can cause diseases in animals from the order Artiodactyla. Homologous recombination is rarely reported in this virus family. To detect possible recombination events, all complete pestivirus genomes that are available in GenBank were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Three putative recombinant viruses derived from recombination from different pestivirus subtypes/genogroups were detected: Bovine viral diarrhea virus 1 (BVDV-1) strain 3156, BVDV-2 strain JZ05-1 and Classical swine fever virus (CSFV) strain IND/UK/LAL-290. The present study demonstrated that the pestivirus classification cannot be based only on the analysis of one fragment of the genome because genetic conversions can lead to errors. The designation of the recombinant forms (RF) provides a more informative structure for the nomenclature of the genetic variant. The present work reinforces that homologous recombination occurs in pestivirus populations under natural replication and describes the first evidence of recombination in BVDV-2. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Engineering the Caenorhabditis elegans Genome Using Cas9-Triggered Homologous Recombination

    PubMed Central

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

    2013-01-01

    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 followed by 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 efficiently repaired 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

  20. Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA.

    PubMed Central

    Kucherlapati, R S; Eves, E M; Song, K Y; Morse, B S; Smithies, O

    1984-01-01

    We have used the eukaryotic-prokaryotic shuttle vector pSV2Neo to demonstrate that cultured mammalian somatic cells have the enzymatic machinery to mediate homologous recombination and that the frequency of this recombination can be enhanced by pretreatment of the input DNA. Two nonoverlapping deletion mutants of pSV2Neo were constructed, each affecting the bacterial aminoglycoside 3'-phosphorylase gene (the neo gene), which confers resistance to aminoglycoside antibiotics on bacteria and resistance to the antibiotic G418 on mammalian cells. Mammalian cells transfected with either deletion plasmid alone yield no G418 -resistant colonies. Cells cotransfected with both deletion plasmids yield G418 -resistant colonies with high frequency. We show that these resistant colonies result from recombination involving homologous crossing-over or gene conversion between the deletion plasmids by rescuing from the resistant cells both types of reciprocal recombinant, full-length plasmids, and doubly deleted plasmids. Cutting one of the input plasmids to generate a double-stranded gap in the neo gene considerably enhances the frequency of homologous recombination within the gene. This suggests that targeting exogenous DNA to specific sites in mammalian chromosomes could be facilitated by suitable pretreatment of the DNA. Images PMID:6328502

  1. Ecological genomics in Xanthomonas: the nature of genetic adaptation with homologous recombination and host shifts.

    PubMed

    Huang, Chao-Li; Pu, Pei-Hua; Huang, Hao-Jen; Sung, Huang-Mo; Liaw, Hung-Jiun; Chen, Yi-Min; Chen, Chien-Ming; Huang, Ming-Ban; Osada, Naoki; Gojobori, Takashi; Pai, Tun-Wen; Chen, Yu-Tin; Hwang, Chi-Chuan; Chiang, Tzen-Yuh

    2015-03-15

    Comparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants. Using whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting. Altogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification.

  2. UV irradiation induces homologous recombination genes in the model archaeon, Halobacterium sp. NRC-1

    PubMed Central

    McCready, Shirley; Müller, Jochen A; Boubriak, Ivan; Berquist, Brian R; Ng, Wooi Loon; DasSarma, Shiladitya

    2005-01-01

    Background A variety of strategies for survival of UV irradiation are used by cells, ranging from repair of UV-damaged DNA, cell cycle arrest, tolerance of unrepaired UV photoproducts, and shielding from UV light. Some of these responses involve UV-inducible genes, including the SOS response in bacteria and an array of genes in eukaryotes. To address the mechanisms used in the third branch of life, we have studied the model archaeon, Halobacterium sp. strain NRC-1, which tolerates high levels of solar radiation in its natural hypersaline environment. Results Cells were irradiated with 30–70 J/m2 UV-C and an immunoassay showed that the resulting DNA damage was largely repaired within 3 hours in the dark. Under such conditions, transcriptional profiling showed the most strongly up-regulated gene was radA1, the archaeal homolog of rad51/recA, which was induced 7-fold. Additional genes involved in homologous recombination, such as arj1 (recJ-like exonuclease), dbp (eukaryote-like DNA binding protein of the superfamily I DNA and RNA helicases), and rfa3 (replication protein A complex), as well as nrdJ, encoding for cobalamin-dependent ribonucleotide reductase involved in DNA metabolism, were also significantly induced in one or more of our experimental conditions. Neither prokaryotic nor eukaryotic excision repair gene homologs were induced and there was no evidence of an SOS-like response. Conclusion These results show that homologous recombination plays an important role in the cellular response of Halobacterium sp. NRC-1 to UV damage. Homologous recombination may permit rescue of stalled replication forks, and/or facilitate recombinational repair. In either case, this provides a mechanism for the observed high-frequency recombination among natural populations of halophilic archaea. PMID:16176594

  3. E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis.

    PubMed

    Ishag, Hassan Z A; Xiong, Qiyan; Liu, Maojun; Feng, Zhixin; Shao, Guoqing

    2017-05-01

    Mycoplasma hyorhinis is an opportunistic pathogen of pigs. Recently, it has been shown to transform cell cultures, increasing the attention of the researchers. Studies on the pathogenesis require specific genetic tool that is not yet available for the pathogen. To address this limitation, we constructed two suicide plasmids pGEMT-tetM/LR and pGEMT-recA-tetM/LR having a tetracycline resistance marker flanked by two hemolysin gene arms. The latter plasmid encodes an E. coli recA, a gene involved in DNA recombination, repair and maintenance of DNA. Using inactivation of the hemolysin gene, which results in a detectable and measurable phenotype, we found that each plasmid can disrupt the hemolysin gene of M. hyorhinis through a double cross-over homologous recombination. However, inclusion of the E. coli recA gene in the construct resulted in 9-fold increase in the frequency of hemolysin gene mutants among the screened tetracycline resistance colonies. The resultant hemolysin mutant strain lacks the ability to lyse mouse bed blood cells (RBC) when tested in vitro (p<0.001). The host-plasmid system described in this study, has applications for the genetic manipulation of this pathogen and potentially other mycoplasmas.

  4. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair.

    PubMed

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw; Syljuåsen, Randi G; Lundin, Cecilia; Bartek, Jiri; Helleday, Thomas

    2005-02-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

  5. Recombination-independent recognition of DNA homology for repeat-induced point mutation.

    PubMed

    Gladyshev, Eugene; Kleckner, Nancy

    2017-06-01

    Numerous cytogenetic observations have shown that homologous chromosomes (or individual chromosomal loci) can engage in specific pairing interactions in the apparent absence of DNA breakage and recombination, suggesting that canonical recombination-mediated mechanisms may not be the only option for sensing DNA/DNA homology. One proposed mechanism for such recombination-independent homology recognition involves direct contacts between intact double-stranded DNA molecules. The strongest in vivo evidence for the existence of such a mechanism is provided by the phenomena of homology-directed DNA modifications in fungi, known as repeat-induced point mutation (RIP, discovered in Neurospora crassa) and methylation-induced premeiotically (MIP, discovered in Ascobolus immersus). In principle, Neurospora RIP can detect the presence of gene-sized DNA duplications irrespectively of their origin, underlying nucleotide sequence, coding capacity or relative, as well as absolute positions in the genome. Once detected, both sequence copies are altered by numerous cytosine-to-thymine (C-to-T) mutations that extend specifically over the duplicated region. We have recently shown that Neurospora RIP does not require MEI-3, the only RecA/Rad51 protein in this organism, consistent with a recombination-independent mechanism. Using an ultra-sensitive assay for RIP mutation, we have defined additional features of this process. We have shown that RIP can detect short islands of homology of only three base-pairs as long as many such islands are arrayed with a periodicity of 11 or 12 base-pairs along a pair of DNA molecules. While the presence of perfect homology is advantageous, it is not required: chromosomal segments with overall sequence identity of only 35-36 % can still be recognized by RIP. Importantly, in order for this process to work efficiently, participating DNA molecules must be able to co-align along their lengths. Based on these findings, we have proposed a model, in which

  6. RadB acts in homologous recombination in the archaeon Haloferax volcanii, consistent with a role as recombination mediator.

    PubMed

    Wardell, Kayleigh; Haldenby, Sam; Jones, Nathan; Liddell, Susan; Ngo, Greg H P; Allers, Thorsten

    2017-07-01

    Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination - it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. 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

  8. Somatic homologous recombination in planta: the recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects.

    PubMed

    Swoboda, P; Hohn, B; Gal, S

    1993-02-01

    We have previously described a non-selective method for scoring somatic recombination in the genome of whole plants. The recombination substrate consists of a defective partial dimer of Cauliflower Mosaic Virus (CaMV) sequences, which can code for production of viable virus only upon homologous recombination; this leads to disease symptoms on leaves. Brassica napus plants (rapeseed) harbouring the recombination substrate as a transgene were used to examine the time in plant development at which recombination takes place. The analysis of three transgene loci revealed recombination frequencies specific for each locus. Recombination frequencies were increased if more than one transgene locus was present per genome, either in allelic (homozygosity of the transgene locus) or in non-allelic positions. In both cases, the overall recombination frequency was found to be elevated to approximately the sum of the frequencies for the individual transgene loci or slightly higher, suggesting that the respective transgene loci behave largely independently of each other. For all plants tested (single locus, two or multiple loci) maximal recombination frequencies were of the order of 10(-6) events per cell division.

  9. Type III restriction is alleviated by bacteriophage (RecE) homologous recombination function but enhanced by bacterial (RecBCD) function.

    PubMed

    Handa, Naofumi; Kobayashi, Ichizo

    2005-11-01

    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.

  10. Change of Gene Structure and Function by Non-Homologous End-Joining, Homologous Recombination, and Transposition of DNA

    PubMed Central

    Goettel, Wolfgang; Messing, Joachim

    2009-01-01

    An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization

  11. The Functions of BRCA2 in Homologous Recombinational Repair

    DTIC Science & Technology

    2006-07-01

    frequency Results on protein purification: We have expressed the human Rad51 protein in insect cells Sf9 using the baculovirus system. The Rad51 protein...fragments of BRCA2 in Sf9 cells. The BRC1-4, BRC5-8, and BRC1-8 domains encode the proteins of 80-kDa, 65- kDa, and 138-kDa, respectively. The

  12. Heteroduplex formation, mismatch resolution, and genetic sectoring during homologous recombination in the hyperthermophilic archaeon sulfolobus acidocaldarius.

    PubMed

    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 acidocaldariuspyrE 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.

  13. The Functions of BRCA2 in Homologous Recombinational Repair

    DTIC Science & Technology

    2005-07-01

    expressed the human Rad51 protein in insect cells Sf9 using the baculovirus system. The Rad5I protein was subsequently purified to Fig. 4. Purified...fragment and untagged Rad5 1 B (or Rad5lC) proteins in Sf9 insect cells. We tested BRCI-4/Rad51B, BRC1-8/Rad51B, BRCI-4/Rad51C and BRC 1 -8/Rad5 IC...repeats of BRCA2 and Rad5 I B (or Rad5 1 C) was found. 9 3. The human Rad51 protein was expressed in insect cells and purified to homogeneity using

  14. 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

  15. Homologous recombination in Agrobacterium: potential implications for the genomic species concept in bacteria.

    PubMed

    Costechareyre, Denis; Bertolla, Franck; Nesme, Xavier

    2009-01-01

    According to current taxonomical rules, a bona fide bacterial species is a genomic species characterized by the genomic similarity of its members. It has been proposed that the genomic cohesion of such clusters may be related to sexual isolation, which limits gene flow between too divergent bacteria. Homologous recombination is one of the most studied mechanisms responsible for this genetic isolation. Previous studies on several bacterial models showed that recombination frequencies decreased exponentially with increasing DNA sequence divergence. In the present study, we investigated this relationship in the Agrobacterium tumefaciens species complex, which allowed us to focus on sequence divergence in the vicinity of the genetic boundaries of genomic species. We observed that the sensitivity of the recombination frequency to DNA divergence fitted a log-linear function until approximately 10% sequence divergence. The results clearly revealed that there was no sharp drop in recombination frequencies at the point where the sequence divergence distribution showed a "gap" delineating genomic species. The ratio of the recombination frequency in homogamic conditions relative to this frequency in heterogamic conditions, that is, sexual isolation, was found to decrease from 8 between the most distant strains within a species to 9 between the most closely related species, for respective increases from 4.3% to 6.4% mismatches in the marker gene chvA. This means that there was only a 1.13-fold decrease in recombination frequencies for recombination events at both edges of the species border. Hence, from the findings of this investigation, we conclude that--at least in this taxon--sexual isolation based on homologous recombination is likely not high enough to strongly hamper gene flow between species as compared with gene flow between distantly related members of the same species. The 70% relative binding ratio cutoff used to define bacterial species is likely correlated to

  16. Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

    PubMed Central

    Nefedov, Mikhail; Carbone, Lucia; Field, Matthew; Schein, Jacquie; de Jong, Pieter J.

    2011-01-01

    We have developed a new approach to screen bacterial artificial chromosome (BAC) libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380) with temperature inducible homologous recombination (HR) capability. We amplified one library segment, induced HR at 42°C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies. PMID:20981149

  17. 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

  18. Structural Studies of DNA End Detection and Resection in Homologous Recombination

    PubMed Central

    Schiller, Christian Bernd; Seifert, Florian Ulrich; Linke-Winnebeck, Christian; Hopfner, Karl-Peter

    2014-01-01

    DNA double-strand breaks are repaired by two major pathways, homologous recombination or nonhomologous end joining. The commitment to one or the other pathway proceeds via different steps of resection of the DNA ends, which is controlled and executed by a set of DNA double-strand break sensors, endo- and exonucleases, helicases, and DNA damage response factors. The molecular choreography of the underlying protein machinery is beginning to emerge. In this review, we discuss the early steps of genetic recombination and double-strand break sensing with an emphasis on structural and molecular studies. PMID:25081516

  19. A Conditional Mouse Model for Measuring the Frequency of Homologous Recombination Events In Vivo in the Absence of Essential Genes▿‡

    PubMed Central

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

    2011-01-01

    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

  20. 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.

  1. Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination

    PubMed Central

    Chemler, Joseph A.; Tripathi, Ashootosh; Hansen, Douglas A.; O'Neil-Johnson, Mark; Williams, Russell B.; Starks, Courtney; Park, Sung Ryeol; Sherman, David H.

    2015-01-01

    The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as bio-synthetic assembly lines. The modular nature of these megaenzymes 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

  2. From meiosis to postmeiotic events: homologous recombination is obligatory but flexible.

    PubMed

    Székvölgyi, Lóránt; Nicolas, Alain

    2010-02-01

    Sexual reproduction depends on the success of faithful chromosome transmission during meiosis to yield viable gametes. Central to meiosis is the process of recombination between paternal and maternal chromosomes, which boosts the genetic diversity of progeny and ensures normal homologous chromosome segregation. Imperfections in meiotic recombination are the source of de novo germline mutations, abnormal gametes, and infertility. Thus, not surprisingly, cells have developed a variety of mechanisms and tight controls to ensure sufficient and well-distributed recombination events within their genomes, the details of which remain to be fully elucidated. Local and genome-wide studies of normal and genetically engineered cells have uncovered a remarkable stochasticity in the number and positioning of recombination events per chromosome and per cell, which reveals an impressive level of flexibility. In this minireview, we summarize our contemporary understanding of meiotic recombination and its control mechanisms, and address the seemingly paradoxical and poorly understood diversity of recombination sites. Flexibility in the distribution of meiotic recombination events within genomes may reside in regulation at the chromatin level, with histone modifications playing a recently recognized role.

  3. A protocol for construction of gene targeting vectors and generation of homologous recombinant embryonic stem cells.

    PubMed

    Bouabe, Hicham; Okkenhaug, Klaus

    2013-01-01

    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 cells (ESCs) 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, and short- and long-range deletions, inversions. Site-specific modification into the genome of ESCs can be reached by homologous recombination using targeting vectors. Here, we describe a protocol to generate targeting constructs and homologous recombinant ESCs.

  4. 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

  5. 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.

  6. 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.

  7. Homologous genetic recombination in the yellow head complex of nidoviruses infecting Penaeus monodon shrimp.

    PubMed

    Wijegoonawardane, Priyanjalie K M; Sittidilokratna, Nusra; Petchampai, Natthida; Cowley, Jeff A; Gudkovs, Nicholas; Walker, Peter J

    2009-07-20

    Yellow head virus (YHV) is a highly virulent pathogen of Penaeus monodon shrimp. It is one of six known genotypes in the yellow head complex of nidoviruses which also includes mildly pathogenic gill-associated virus (GAV, genotype 2) and four other genotypes (genotypes 3-6) that have been detected only in healthy shrimp. In this study, comparative phylogenetic analyses conducted on replicase- (ORF1b) and glycoprotein- (ORF3) gene amplicons identified 10 putative natural recombinants amongst 28 viruses representing all six genotypes from across the Indo-Pacific region. The approximately 4.6 kb genomic region spanning the two amplicons was sequenced for three putative recombinant viruses from Vietnam (genotype 3/5), the Philippines (genotype 5/2) and Indonesia (genotype 3/2). SimPlot analysis using these and representative parental virus sequences confirmed that each was a recombinant genotype and identified a recombination hotspot in a region just upstream of the ORF1b C-terminus. Maximum-likelihood breakpoint analysis predicted identical crossover positions in the Vietnamese and Indonesian recombinants, and a crossover position 12 nt upstream in the Philippine recombinant. Homologous genetic recombination in the same genome region was also demonstrated in recombinants generated experimentally in shrimp co-infected with YHV and GAV. The high frequency with which natural recombinants were identified indicates that genetic exchange amongst genotypes is occurring commonly in Asia and playing a significant role in expanding the genetic diversity in the yellow head complex. This is the first evidence of genetic recombination in viruses infecting crustaceans and has significant implications for the pathogenesis of infection and diagnosis of these newly emerging invertebrate pathogens.

  8. (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 below.

  9. Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome.

    PubMed

    Yu, Zhongsheng; Chen, Hanqing; Liu, Jiyong; Zhang, Hongtao; Yan, Yan; Zhu, Nannan; Guo, Yawen; Yang, Bo; Chang, Yan; Dai, Fei; Liang, Xuehong; Chen, Yixu; Shen, Yan; Deng, Wu-Min; Chen, Jianming; Zhang, Bo; Li, Changqing; Jiao, Renjie

    2014-04-15

    Modifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1) to precisely generate genomic deletions; (2) to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3) to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the "ends-in" recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair); either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5 kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome.

  10. 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

  11. FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells.

    PubMed

    Simandlova, Jitka; Zagelbaum, Jennifer; Payne, Miranda J; Chu, Wai Kit; Shevelev, Igor; Hanada, Katsuhiro; Chatterjee, Sujoy; Reid, Dylan A; Liu, Ying; Janscak, Pavel; Rothenberg, Eli; Hickson, Ian D

    2013-11-22

    Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.

  12. Mos1 transposition as a tool to engineer the Caenorhabditis elegans genome by homologous recombination.

    PubMed

    Robert, Valérie J P; Katic, Iskra; Bessereau, Jean-Louis

    2009-11-01

    Gene knockouts and knock-ins have emerged as powerful tools to study gene function in model organisms. The construction of such engineered alleles requires that homologous recombination between a transgenic fragment carrying the modifications desired in the genome and the locus to engineer occurs at high frequencies. Homologous recombination frequency is significantly increased in the vicinity of a DNA double-strand break. Based on this observation, a new generation of transgene-instructed genome engineering protocols was developed. Here, we present MosTIC (for "Mos1 excision-induced transgene-instructed gene conversion"), a new technique that provides a means to engineer the Caenorhabditis elegans genome. MosTIC is initiated by the mobilization of Mos1, a Drosophila transposon experimentally introduced in C. elegans. During MosTIC, a Mos1 insertion localized in the genomic region to engineer is mobilized after germline expression of the Mos transposase. Mos1 excision generates a DNA double-strand break, which is repaired by homologous recombination using a transgenic repair template. This results in the transfer of information from the transgene into the genome. Depending on the method used to trigger Mos1 excision, two alternative MosTIC protocols are available, which are presented here in detail. This technique can be used for a wide range of applications, such as structure-function analysis, protein localization and purification, genetic screens or generation of single copy transgenes at a defined locus in the genome.

  13. Resolving RAD51C function in late stages of homologous recombination

    PubMed Central

    Sharan, Shyam K; Kuznetsov, Sergey G

    2007-01-01

    DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction. PMID:17547768

  14. High Rates of Homologous Recombination in the Mite Endosymbiont and Opportunistic Human Pathogen Orientia tsutsugamushi

    PubMed Central

    Sonthayanon, Piengchan; Peacock, Sharon J.; Chierakul, Wirongrong; Wuthiekanun, Vanaporn; Blacksell, Stuart D.; Holden, Mathew T. G.; Bentley, Stephen D.; Feil, Edward J.; Day, Nicholas P. J.

    2010-01-01

    Orientia tsutsugamushi is an intracellular α-proteobacterium which resides in trombiculid mites, and is the causative agent of scrub typhus in East Asia. The genome sequence of this species has revealed an unprecedented number of repeat sequences, most notably of the genes encoding the conjugative properties of a type IV secretion system (T4SS). Although this observation is consistent with frequent intragenomic recombination, the extent of homologous recombination (gene conversion) in this species is unknown. To address this question, and to provide a protocol for the epidemiological surveillance of this important pathogen, we have developed a multilocus sequence typing (MLST) scheme based on 7 housekeeping genes (gpsA, mdh, nrdB, nuoF, ppdK, sucD, sucB). We applied this scheme to the two published genomes, and to DNA extracted from blood taken from 84 Thai scrub typhus patients, from 20 cultured Thai patient isolates, 1 Australian patient sample, and from 3 cultured type strains. These data demonstrated that the O. tsutsugamushi population was both highly diverse [Simpson's index (95% CI) = 0.95 (0.92–0.98)], and highly recombinogenic. These results are surprising given the intracellular life-style of this species, but are broadly consistent with results obtained for Wolbachia, which is an α-proteobacterial reproductive parasite of arthropods. We also compared the MLST data with ompA sequence data and noted low levels of consistency and much higher discrimination by MLST. Finally, twenty-five percent of patients in this study were simultaneously infected with multiple sequence types, suggesting multiple infection caused by either multiple mite bites, or multiple strains co-existing within individual mites. PMID:20651929

  15. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) for directed enzyme evolution.

    PubMed

    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.

  16. 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

  17. 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

  18. Homologous recombination is a force in the evolution of canine distemper virus.

    PubMed

    Yuan, Chaowen; Liu, Wenxin; Wang, Yingbo; Hou, Jinlong; Zhang, Liguo; Wang, Guoqing

    2017-01-01

    Canine distemper virus (CDV) is the causative agent of canine distemper (CD) that is a highly contagious, lethal, multisystemic viral disease of receptive carnivores. The prevalence of CDV is a major concern in susceptible animals. Presently, it is unclear whether intragenic recombination can contribute to gene mutations and segment reassortment in the virus. In this study, 25 full-length CDV genome sequences were subjected to phylogenetic and recombinational analyses. The results of phylogenetic analysis, intragenic recombination, and nucleotide selection pressure indicated that mutation and recombination occurred in the six individual genes segment (H, F, P, N, L, M) of the CDV genome. The analysis also revealed pronounced genetic diversity in the CDV genome according to the geographically distinct lineages (genotypes), namely Asia-1, Asia-2, Asia-3, Europe, America-1, and America-2. The six recombination events were detected using SimPlot and RDP programs. The analysis of selection pressure demonstrated that a majority of the nucleotides in the CDV individual gene were under negative selection. Collectively, these data suggested that homologous recombination acts as a key force driving the genetic diversity and evolution of canine distemper virus.

  19. Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

    PubMed Central

    Simon, J R; Moore, P D

    1987-01-01

    Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA. Images PMID:3302673

  20. Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion

    PubMed Central

    Singh, Nishant; Bhattacharya, Alok; Bhattacharya, Sudha

    2013-01-01

    Homologous recombination (HR) has not been demonstrated in the parasitic protists Entamoeba histolytica or Entamoeba invadens, as no convenient method is available to measure it. However, HR must exist to ensure genome integrity, and possible genetic exchange, especially during stage conversion from trophozoite to cyst. Here we show the up regulation of mitotic and meiotic HR genes in Entamoeba during serum starvation, and encystation. To directly demonstrate HR we use a simple PCR-based method involving inverted repeats, which gives a reliable read out, as the recombination junctions can be determined by sequencing the amplicons. Using this read out, we demonstrate enhanced HR under growth stress in E. histolytica, and during encystation in E. invadens. We also demonstrate recombination between chromosomal inverted repeats. This is the first experimental demonstration of HR in Entamoeba and will help future investigations into this process, and to explore the possibility of meiosis in Entamoeba. PMID:24098652

  1. Homologous recombination occurs in Entamoeba and is enhanced during growth stress and stage conversion.

    PubMed

    Singh, Nishant; Bhattacharya, Alok; Bhattacharya, Sudha

    2013-01-01

    Homologous recombination (HR) has not been demonstrated in the parasitic protists Entamoeba histolytica or Entamoeba invadens, as no convenient method is available to measure it. However, HR must exist to ensure genome integrity, and possible genetic exchange, especially during stage conversion from trophozoite to cyst. Here we show the up regulation of mitotic and meiotic HR genes in Entamoeba during serum starvation, and encystation. To directly demonstrate HR we use a simple PCR-based method involving inverted repeats, which gives a reliable read out, as the recombination junctions can be determined by sequencing the amplicons. Using this read out, we demonstrate enhanced HR under growth stress in E. histolytica, and during encystation in E. invadens. We also demonstrate recombination between chromosomal inverted repeats. This is the first experimental demonstration of HR in Entamoeba and will help future investigations into this process, and to explore the possibility of meiosis in Entamoeba.

  2. The role of Deinococcus radiodurans RecFOR proteins in homologous recombination.

    PubMed

    Satoh, Katsuya; Kikuchi, Masahiro; Ishaque, Abu M; Ohba, Hirofumi; Yamada, Mitsugu; Tejima, Kouhei; Onodera, Takefumi; Narumi, Issay

    2012-04-01

    Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. An optimized RAD51 inhibitor that disrupts homologous recombination without requiring Michael acceptor reactivity

    PubMed Central

    Budke, Brian; Kalin, Jay H.; Pawlowski, Michal; Zelivianskaia, Anna S.; Wu, Megan; Kozikowski, Alan P.; Connell, Philip P.

    2013-01-01

    Homologous recombination (HR) is an essential process in cells that provides repair of DNA double-strand breaks and lesions that block DNA replication. RAD51 is an evolutionarily conserved protein that is central to HR. Overexpression of RAD51 protein is common in cancer cells and represents a potential therapeutic target in oncology. We previously described a chemical inhibitor of RAD51, called RI-1 (referred to as compound 1 in this report). The chloromaleimide group of this compound is thought to act as a Michael acceptor and react with the thiol group on C319 of RAD51, using a conjugate addition-elimination mechanism. In order to reduce the likelihood of off-target effects and to improve compound stability in biological systems, we developed an analog of compound 1 that lacks maleimide-based reactivity but retains RAD51 inhibitory activity. This compound, 1-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)-4-morpholino-1H-pyrrole-2,5-dione, named RI-2 (referred to as compound 7a in this report), appears to bind reversibly to the same site on the RAD51 protein as does compound 1. Like compound 1, compound 7a specifically inhibits HR repair in human cells. PMID:23231413

  4. On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage.

    PubMed

    Shcherbakov, Victor P; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Kudryashova, Elena

    2011-01-02

    The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase.

  5. Suppression of Meiotic Recombination by CENP-B Homologs in Schizosaccharomyces pombe

    PubMed Central

    Johansen, Peter; Cam, Hugh P.

    2015-01-01

    Meiotic homologous recombination (HR) is not uniform across eukaryotic genomes, creating regions of HR hot- and coldspots. Previous study reveals that the Spo11 homolog Rec12 responsible for initiation of meiotic double-strand breaks in the fission yeast Schizosaccharomyces pombe is not targeted to Tf2 retrotransposons. However, whether Tf2s are HR coldspots is not known. Here, we show that the rates of HR across Tf2s are similar to a genome average but substantially increase in mutants deficient for the CENP-B homologs. Abp1, which is the most prominent of the CENP-B family members and acts as the primary determinant of HR suppression at Tf2s, is required to prevent gene conversion and maintain proper recombination exchange of homologous alleles flanking Tf2s. In addition, Abp1-mediated suppression of HR at Tf2s requires all three of its domains with distinct functions in transcriptional repression and higher-order genome organization. We demonstrate that HR suppression of Tf2s can be robustly maintained despite disruption to chromatin factors essential for transcriptional repression and nuclear organization of Tf2s. Intriguingly, we uncover a surprising cooperation between the histone methyltransferase Set1 responsible for histone H3 lysine 4 methylation and the nonhomologous end joining pathway in ensuring the suppression of HR at Tf2s. Our study identifies a molecular pathway involving functional cooperation between a transcription factor with epigenetic regulators and a DNA repair pathway to regulate meiotic recombination at interspersed repeats. PMID:26354768

  6. Inactivation of ku80 in the mushroom-forming fungus Schizophyllum commune increases the relative incidence of homologous recombination.

    PubMed

    de Jong, Jan F; Ohm, Robin A; de Bekker, Charissa; Wösten, Han A B; Lugones, Luis G

    2010-09-01

    Schizophyllum commune is the only mushroom-forming fungus in which targeted gene deletions by homologous recombination have been reported. However, these deletions occur with a low frequency. To overcome this, the ku80 gene of S. commune was deleted. This gene is involved in the nonhomologous end-joining system for DNA repair. The Deltaku80 strain was not affected in growth and development. However, the transformation efficiency was reduced up to 100-fold. This was accompanied by a strong increase in the relative number of transformants with a homologous integration of a knockout construct. Genes sc15, jmj3 and pri2 were deleted in the Deltaku80 strain. In total, seven out of 10 transformants showed a gene deletion. This frequency will facilitate a systematic analysis of gene function in S. commune.

  7. The detection of inherent homologous recombination between repeat sequences in H. pylori 26695 by the PCR-based method.

    PubMed

    Fu, Yajuan; Zepeda-Gurrola, Reyna Cristina; Aguilar-Gutiérrez, Germán Rubén; Lara-Ramírez, Edgar E; De Luna-Santillana, Erick J; Rodríguez-Luna, Isabel Cristina; Sánchez-Varela, Alejandro; Carreño-López, Ricardo; Moreno-Medina, Víctor Ricardo; Rodríguez-Pérez, Mario A; López-Vidal, Yolanda; Guo, Xianwu

    2014-02-01

    Helicobacter pylori infects more than half of the world's population, making it the most widespread infection of bacteria. It has high genetic diversity and has been considered as one of the most variable bacterial species. In the present study, a PCR-based method was used to detect the presence and the relative frequency of homologous recombination between repeat sequences (>500 bp) in H. pylori 26695. All the recombinant structures have been confirmed by sequencing. The inversion generated between inverted repeats showed distinct features from the recombination for duplication or deletion between direct repeats. Meanwhile, we gave the mathematic reasoning of a general formula for the calculation of relative recombination frequency and indicated the conditions for its application. This formula could be extensively applied to detect the frequency of homologous recombination, site-specific recombination, and other types of predictable recombination. Our results should be helpful for better understanding the genome evolution and adaptation of bacteria.

  8. Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

    PubMed Central

    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. Histone H2AX and the small RNA pathway modulate both non-homologous end-joining and homologous recombination in plants.

    PubMed

    Qi, Yiping; Zhang, Yong; Baller, Joshua A; Voytas, Daniel F

    2016-01-01

    Using a zinc finger nuclease (ZFN) that creates a site-specific DNA double-strand break (DSB) at the endogenous ADH1 locus, we provide genetic evidence that histone H2AX and DSB-induced small RNAs modulate DNA repair by both non-homologous end-joining (NHEJ) and homologous recombination (HR) in the model plant Arabidopsis thaliana. Published by Elsevier B.V.

  10. Is it time to split strategies to treat homologous recombinant deficiency in pancreas cancer?

    PubMed

    Teo, Min Yuen; O'Reilly, Eileen M

    2016-10-01

    Pancreatic cancer is a highly lethal malignancy which tends to present with late stage disease. To date, identification of oncogenic drivers and aberrations has not led to effective targeted therapy. Approximately 5-15% of pancreatic cancer has an inheritable component. In fact, pancreatic adenocarcinoma is now recognized as a BRCA1/2-related cancer. Germline BRCA1/2 mutations can be found in up to 3.6-7% of unselected pancreatic cancer patients although the rates are significantly higher amongst patients with Ashkenazi Jewish ancestry. Germline mutations of other components of DNA repair and homologous recombination have also been identified although at much lower frequency. Large sequencing efforts have further identified somatic mutations in these genes in a small subset of pancreatic cancers. Small series and case reports have suggested that pancreatic cancers harboring BRCA1/2 or other homologous repair gene mutations demonstrate enhanced response to platinum-based chemotherapy although this has not been prospectively validated. Clinical trials with poly (ADP-ribose) polymerase (PARP) inhibitors as monotherapy or in combination with chemotherapy in different clinical settings are currently on-going. A subtype of pancreatic adenocarcinoma as characterized by deficiency in homologous recombination exists although the optimal management strategy remains to be fully elucidated.

  11. New Targeted Agents in Gynecologic Cancers: Synthetic Lethality, Homologous Recombination Deficiency, and PARP Inhibitors.

    PubMed

    Liu, Fong W; Tewari, Krishnansu S

    2016-03-01

    Inhibitors of poly (ADP-ribose) polymerase (PARP) have emerged as a new class of anti-cancer drugs, specifically for malignancies bearing aberrations of the homologous recombination pathway, like those with mutations in the BRCA 1 and BRCA 2 genes. Olaparib, a potent PARP1 and PARP2 inhibitor, has been shown to significantly increase progression-free survival (PFS) in women with recurrent ovarian cancer related to a germline BRCA mutation and is currently approved fourth-line treatment in these patients. PARP inhibitors (PARPi) target the genetic phenomenon known as synthetic lethality to exploit faulty DNA repair mechanisms. While ovarian cancer is enriched with a population of tumors with known homologous recombination defects, investigations are underway to help identify pathways in other gynecologic cancers that may demonstrate susceptibility to PARPi through synthetically lethal mechanisms. The ARIEL2 trial prospectively determined a predictive assay to identify patients with HRD. The future of cancer therapeutics will likely incorporate these HRD assays to determine the best treatment plan for patients. While the role of PARPi is less clear in non-ovarian gynecologic cancers, the discovery of a predictive assay for HRD may open the door for clinical trials in these other gynecologic cancers enriched with patients with HRD. Identification of patients with tumors deficient in homologous repair or have HRD-like behavior moves cancer treatment towards individualized therapies in order to maximize treatment effect and quality of life for women living with gynecologic cancers.

  12. The dual role of HOP2 in mammalian meiotic homologous recombination

    PubMed Central

    Pezza, Roberto J.; Voloshin, Oleg N.; Volodin, Alexander A.; Boateng, Kingsley A.; Bellani, Marina A.; Mazin, Alexander V.; Camerini-Otero, R. Daniel

    2014-01-01

    Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis. PMID:24304900

  13. Inference of homologous recombination in bacteria using whole-genome sequences.

    PubMed

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

    2010-12-01

    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/.

  14. Homologous recombination intermediates between two duplex DNA catalysed by human cell extracts.

    PubMed Central

    Lopez, B; Rousset, S; Coppey, J

    1987-01-01

    Using as substrates, 1: the replicative form (RF) of phage M13 mp8 in which the reading frame of the lac Z' gene was disrupted by insertion of an octonucleotide, and 2: a restriction fragment one kb long, containing the functional lac Z' gene (isolated from wild type M13 mp8), we show that nuclear extracts from human cells (3 lines tested) promote the targeted replacement of the altered sequence by the functional one. Following incubation with the extracts, the DNA's were introduced in JM 109 bacteria (rec A- and lac Z'-) which were grown in presence of a colorimetric indicator of beta-galactosidase activity. Homologous recombination gives rise to the genotypical modification: lac Z'+ instead of lac Z'- in the bacteriophage DNA. This is revealed by phenotypical expression of the lac Z' gene product in replicating bacteriophage, i.e. the formation of blue instead of white plaques. The frequency of recombination (blue/total plaques) is increased by a factor of 50-80 as a function of protein concentration and of incubation time. The maximal frequency observed is 5 X 10(-5). There is no increase over the background when extracts are boiled. Electrophoresis and electron microscopy of DNA's incubated with the extracts show the formation of recombination intermediates with single strand exchange. Restriction analysis of recombined DNA confirms that the process corresponds to targeted sequence exchange. These data allow to propose three steps for homologous recombination between two duplex DNA's: i) unpairing of the two duplexes; ii) single-strand exchange and synaptic pairing; iii) resolution of the cross-junctions. The three steps correspond to those predicted by the gene conversion model of Holliday. Images PMID:3302944

  15. Telomerase Deficiency Affects the Formation of Chromosomal Translocations by Homologous Recombination in Saccharomyces cerevisiae

    PubMed Central

    Meyer, Damon H.; Bailis, Adam M.

    2008-01-01

    Telomerase is a ribonucleoprotein complex required for the replication and protection of telomeric DNA in eukaryotes. Cells lacking telomerase undergo a progressive loss of telomeric DNA that results in loss of viability and a concomitant increase in genome instability. We have used budding yeast to investigate the relationship between telomerase deficiency and the generation of chromosomal translocations, a common characteristic of cancer cells. Telomerase deficiency increased the rate of formation of spontaneous translocations by homologous recombination involving telomere proximal sequences during crisis. However, telomerase deficiency also decreased the frequency of translocation formation following multiple HO-endonuclease catalyzed DNA double-strand breaks at telomere proximal or distal sequences before, during and after crisis. This decrease correlated with a sequestration of the central homologous recombination factor, Rad52, to telomeres determined by chromatin immuno-precipitation. This suggests that telomerase deficiency results in the sequestration of Rad52 to telomeres, limiting the capacity of the cell to repair double-strand breaks throughout the genome. Increased spontaneous translocation formation in telomerase-deficient yeast cells undergoing crisis is consistent with the increased incidence of cancer in elderly humans, as the majority of our cells lack telomerase. Decreased translocation formation by recombinational repair of double-strand breaks in telomerase-deficient yeast suggests that the reemergence of telomerase expression observed in many human tumors may further stimulate genome rearrangement. Thus, telomerase may exert a substantial effect on global genome stability, which may bear significantly on the appearance and progression of cancer in humans. PMID:18830407

  16. 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.

  17. Role of the ruvB gene in homologous and homeologous recombination in Rhizobium etli.

    PubMed

    Martinez-Salazar, J M; Romero, D

    2000-02-08

    The Rhizobium etli ruvA and ruvB genes were cloned through a PCR-based approach, using degenerate primers matching conserved sectors in the amino acid sequences of RuvB from eight bacterial species. Comparative analysis of the predicted polypeptides for RuvA and RuvB of R. etli showed highly conserved blocks with the corresponding homologs in other bacteria; RuvB depicts characteristic motifs for DNA helicases (ATP-binding and DEXH-box motifs). An R. etli ruvB::loxP Sp mutant was constructed by interposon mutagenesis. This mutant was highly sensitive to DNA-damaging agents, such as methyl methanesulfonate and nitrofurantoin, implying a deficiency in DNA repair. Homologous and homeologous conjugational recombination was reduced almost tenfold in the ruvB::loxP Sp mutant; a recombination defect was also observed in assays employing recombination between small plasmids, albeit at a smaller magnitude. Although the ruvA and ruvB genes are contiguous in R. etli, complementation studies suggest that they are expressed independently.

  18. Compilation of DNA strand exchange sites for non-homologous recombination in somatic cells.

    PubMed Central

    Konopka, A K

    1988-01-01

    DNA sequences of 496 somatic cell illegitimate crossing over regions were compiled and analyzed. Sites for non-homologous recombination on linear DNAs transfected into mammalian cells (Transfected Linear DNAs; TLD) were analyzed separately from the remaining illegitimate recombination regions (IRR). Trinucleotides that are preferentially cleaved by rat liver topoisomerase I in vitro (CAT, CTY, GTY, RAT where R = purine, Y = pyrimidine) were present in the 10 base pair (bp) vicinity of the cross-over sites in 92% of IRR and 93% of TLD. Multiple repeats of these trinucleotides have been observed in 39% of IRR and 38% of TLD. Runs of five or more contiguous purines (or pyrimidines on the complementary strand) were found in 26% of IRR and 14% of TLD. Adenine-Thymine rich regions of five or more bases were found in 14% of IRR and 21% of TLD. Alternating purine-pyrimidine tracks longer than four nucleotides in length were found in 11% of IRR, though only in 4% of TLD. I discuss the possible biological significance of these findings and present an appendix containing the sequences in the 10 bp vicinity of the non-homologous recombination sites analyzed. PMID:2832826

  19. Chronic hypoxia decreases synthesis of homologous recombination proteins to offset chemoresistance and radioresistance.

    PubMed

    Chan, Norman; Koritzinsky, Marianne; Zhao, Helen; Bindra, Ranjit; Glazer, Peter M; Powell, Simon; Belmaaza, Abdellah; Wouters, Brad; Bristow, Robert G

    2008-01-15

    Hypoxic and/or anoxic tumor cells can have increased rates of mutagenesis and altered DNA repair protein expression. Yet very little is known regarding the functional consequences of any hypoxia-induced changes in the expression of proteins involved in DNA double-strand break repair. We have developed a unique hypoxic model system using H1299 cells expressing an integrated direct repeat green fluorescent protein (DR-GFP) homologous recombination (HR) reporter system to study HR under prolonged chronic hypoxia (up to 72 h under 0.2% O(2)) without bias from altered proliferation, cell cycle checkpoint activation, or severe cell toxicity. We observed decreased expression of HR proteins due to a novel mechanism involving decreased HR protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O(2) as measured by the DR-GFP reporter system. This decrease in functional HR resulted in increased sensitivity to the DNA cross-linking agents mitomycin C and cisplatin but not to the microtubule-interfering agent, paclitaxel. Chronically hypoxic H1299 cells that had decreased functional HR were relatively radiosensitive [oxygen enhancement ratio (OER), 1.37] when compared with acutely hypoxic or anoxic cells (OER, 1.96-2.61). Using CAPAN1 cells isogenic for BRCA2 and siRNA to RAD51, we confirmed that the hypoxia-induced radiosensitivity was due to decreased HR capacity. Persistent down-regulation of HR function by the tumor microenvironment could result in low-fidelity DNA repair and have significant implications for response to therapy and genetic instability in human cancers.

  20. Frequency of nonallelic homologous recombination is correlated with length of homology: evidence that ectopic synapsis precedes ectopic crossing-over.

    PubMed

    Liu, Pengfei; Lacaria, Melanie; Zhang, Feng; Withers, Marjorie; Hastings, P J; Lupski, James R

    2011-10-07

    Genomic disorders constitute a class of diseases that are associated with DNA rearrangements resulting from region-specific genome instability, that is, genome architecture incites genome instability. Nonallelic homologous recombination (NAHR) or crossing-over in meiosis between sequences that are not in allelic positions (i.e., paralogous sequences) can result in recurrent deletions or duplications causing genomic disorders. Previous studies of NAHR have focused on description of the phenomenon, but it remains unclear how NAHR occurs during meiosis and what factors determine its frequency. Here we assembled two patient cohorts with reciprocal genomic disorders; deletion associated Smith-Magenis syndrome and duplication associated Potocki-Lupski syndrome. By assessing the full spectrum of rearrangement types from the two cohorts, we find that complex rearrangements (those with more than one breakpoint) are more prevalent in copy-number gains (17.7%) than in copy-number losses (2.3%); an observation that supports a role for replicative mechanisms in complex rearrangement formation. Interestingly, for NAHR-mediated recurrent rearrangements, we show that crossover frequency is positively associated with the flanking low-copy repeat (LCR) length and inversely influenced by the inter-LCR distance. To explain this, we propose that the probability of ectopic chromosome synapsis increases with increased LCR length, and that ectopic synapsis is a necessary precursor to ectopic crossing-over. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  1. Frequency of Nonallelic Homologous Recombination Is Correlated with Length of Homology: Evidence that Ectopic Synapsis Precedes Ectopic Crossing-Over

    PubMed Central

    Liu, Pengfei; Lacaria, Melanie; Zhang, Feng; Withers, Marjorie; Hastings, P.J.; Lupski, James R.

    2011-01-01

    Genomic disorders constitute a class of diseases that are associated with DNA rearrangements resulting from region-specific genome instability, that is, genome architecture incites genome instability. Nonallelic homologous recombination (NAHR) or crossing-over in meiosis between sequences that are not in allelic positions (i.e., paralogous sequences) can result in recurrent deletions or duplications causing genomic disorders. Previous studies of NAHR have focused on description of the phenomenon, but it remains unclear how NAHR occurs during meiosis and what factors determine its frequency. Here we assembled two patient cohorts with reciprocal genomic disorders; deletion associated Smith-Magenis syndrome and duplication associated Potocki-Lupski syndrome. By assessing the full spectrum of rearrangement types from the two cohorts, we find that complex rearrangements (those with more than one breakpoint) are more prevalent in copy-number gains (17.7%) than in copy-number losses (2.3%); an observation that supports a role for replicative mechanisms in complex rearrangement formation. Interestingly, for NAHR-mediated recurrent rearrangements, we show that crossover frequency is positively associated with the flanking low-copy repeat (LCR) length and inversely influenced by the inter-LCR distance. To explain this, we propose that the probability of ectopic chromosome synapsis increases with increased LCR length, and that ectopic synapsis is a necessary precursor to ectopic crossing-over. PMID:21981782

  2. Either non-homologous ends joining or homologous recombination is required to repair double-strand breaks in the genome of macrophage-internalized Mycobacterium tuberculosis.

    PubMed

    Brzostek, Anna; Szulc, Izabela; 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.

  3. Structure of REC2, a recombinational repair gene of Ustilago maydis, and its function in homologous recombination between plasmid and chromosomal sequences.

    PubMed Central

    Rubin, B P; Ferguson, D O; Holloman, W K

    1994-01-01

    Mutation in the REC2 gene of Ustilago maydis leads to defects in DNA repair, recombination, and meiosis. Analysis of the primary sequence of the Rec2 protein reveals a region with significant homology to bacterial RecA protein and to the yeast recombination proteins Dmc1, Rad51, and Rad57. This homologous region in the U. maydis Rec2 protein was found to be functionally sensitive to mutation, lending support to the hypothesis that Rec2 has a functional RecA-like domain essential for activity in recombination and repair. Homologous recombination between plasmid and chromosomal DNA sequences is reduced substantially in the rec2 mutant following transformation. The frequency can be restored to a level approaching, but not exceeding, that observed in the wild-type strain if transformation is performed with cells containing multiple copies of REC2. Images PMID:8065360

  4. 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.

  5. DNA End Resection: Nucleases Team Up with the Right Partners to Initiate Homologous Recombination.

    PubMed

    Cejka, Petr

    2015-09-18

    The repair of DNA double-strand breaks by homologous recombination commences by nucleolytic degradation of the 5'-terminated strand of the DNA break. This leads to the formation of 3'-tailed DNA, which serves as a substrate for the strand exchange protein Rad51. The nucleoprotein filament then invades homologous DNA to drive template-directed repair. In this review, I discuss mainly the mechanisms of DNA end resection in Saccharomyces cerevisiae, which includes short-range resection by Mre11-Rad50-Xrs2 and Sae2, as well as processive long-range resection by Sgs1-Dna2 or Exo1 pathways. Resection mechanisms are highly conserved between yeast and humans, and analogous machineries are found in prokaryotes as well.

  6. Paths from DNA damage and signaling to genome rearrangements via homologous recombination.

    PubMed

    Nickoloff, Jac A

    2017-07-24

    DNA damage is a constant threat to genome integrity. DNA repair and damage signaling networks play a central role maintaining genome stability, suppressing tumorigenesis, and determining tumor response to common cancer chemotherapeutic agents and radiotherapy. DNA double-strand breaks (DSBs) are critical lesions induced by ionizing radiation and when replication forks encounter damage. DSBs can result in mutations and large-scale genome rearrangements reflecting mis-repair by non-homologous end joining or homologous recombination. Ionizing radiation induces genetic change immediately, and it also triggers delayed events weeks or even years after exposure, long after the initial damage has been repaired or diluted through cell division. This review covers DNA damage signaling and repair pathways and cell fate following genotoxic insult, including immediate and delayed genome instability and cell survival/cell death pathways. Copyright © 2017. Published by Elsevier B.V.

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

    USDA-ARS?s Scientific Manuscript database

    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, ...

  8. Homologous Recombination and Xylella fastidiosa Host-Pathogen Associations in South America.

    PubMed

    Coletta-Filho, Helvécio D; Francisco, Carolina S; Lopes, João R S; Muller, Christiane; Almeida, Rodrigo P P

    2017-03-01

    Homologous recombination affects the evolution of bacteria such as Xylella fastidiosa, a naturally competent plant pathogen that requires insect vectors for dispersal. This bacterial species is taxonomically divided into subspecies, with phylogenetic clusters within subspecies that are host specific. One subspecies, pauca, is primarily limited to South America, with the exception of recently reported strains in Europe and Costa Rica. Despite the economic importance of X. fastidiosa subsp. pauca in South America, little is known about its genetic diversity. Multilocus sequence typing (MLST) has previously identified six sequence types (ST) among plant samples collected in Brazil (both subsp. pauca and multiplex). Here, we report on a survey of X. fastidiosa genetic diversity (MLST based) performed in six regions in Brazil and two in Argentina, by sampling five different plant species. In addition to the six previously reported ST, seven new subsp. pauca and two new subsp. multiplex ST were identified. The presence of subsp. multiplex in South America is considered to be the consequence of a single introduction from its native range in North America more than 80 years ago. Different phylogenetic approaches clustered the South American ST into four groups, with strains infecting citrus (subsp. pauca); coffee and olive (subsp. pauca); coffee, hibiscus, and plum (subsp. pauca); and plum (subsp. multiplex). In areas where these different genetic clusters occurred sympatrically, we found evidence of homologous recombination in the form of bidirectional allelic exchange between subspp. pauca and multiplex. In fact, the only strain of subsp. pauca isolated from a plum host had an allele that originated from subsp. multiplex. These signatures of bidirectional homologous recombination between endemic and introduced ST indicate that gene flow occurs in short evolutionary time frames in X. fastidiosa, despite the ecological isolation (i.e., host plant species) of genotypes.

  9. Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination*

    PubMed Central

    Zhang, Qiang; Chen, Qi-he; Fu, Ming-liang; Wang, Jin-ling; Zhang, Hong-bo; He, Guo-qing

    2008-01-01

    The bglS gene encoding endo-l,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFα1S), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-l,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h·ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer. PMID:18600782

  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 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.

  11. Early days of DNA repair: discovery of nucleotide excision repair and homology-dependent recombinational repair.

    PubMed

    Rupp, W Dean

    2013-12-13

    The discovery of nucleotide excision repair in 1964 showed that DNA could be repaired by a mechanism that removed the damaged section of a strand and replaced it accurately by using the remaining intact strand as the template. This result showed that DNA could be actively metabolized in a process that had no precedent. In 1968, experiments describing postreplication repair, a process dependent on homologous recombination, were reported. The authors of these papers were either at Yale University or had prior Yale connections. Here we recount some of the events leading to these discoveries and consider the impact on further research at Yale and elsewhere.

  12. DNA Replication Triggered by Double-Stranded Breaks in E. coli: Dependence on Homologous Recombination Functions

    PubMed Central

    Asai, Tsuneaki; Bates, David B.; Kogoma, Tokio

    2010-01-01

    Summary Homologous recombination-dependent DNA replication (RDR) of a λ cos site-carrying plasmid is demonstrated in E. coli cells when the cells express λ terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot χ. χ is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the θ mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (or/Ms) on the chromosome. PMID:7923355

  13. RI-1: a chemical inhibitor of RAD51 that disrupts homologous recombination in human cells

    PubMed Central

    Budke, Brian; Logan, Hillary L.; Kalin, Jay H.; Zelivianskaia, Anna S.; Cameron McGuire, William; Miller, Luke L.; Stark, Jeremy M.; Kozikowski, Alan P.; Bishop, Douglas K.; Connell, Philip P.

    2012-01-01

    Homologous recombination serves multiple roles in DNA repair that are essential for maintaining genomic stability. We here describe RI-1, a small molecule that inhibits the central recombination protein RAD51. RI-1 specifically reduces gene conversion in human cells while stimulating single strand annealing. RI-1 binds covalently to the surface of RAD51 protein at cysteine 319 that likely destabilizes an interface used by RAD51 monomers to oligomerize into filaments on DNA. Correspondingly, the molecule inhibits the formation of subnuclear RAD51 foci in cells following DNA damage, while leaving replication protein A focus formation unaffected. Finally, it potentiates the lethal effects of a DNA cross-linking drug in human cells. Given that this inhibitory activity is seen in multiple human tumor cell lines, RI-1 holds promise as an oncologic drug. Furthermore, RI-1 represents a unique tool to dissect the network of reaction pathways that contribute to DNA repair in cells. PMID:22573178

  14. Homologous recombination-mediated double-strand break repair in mouse testicular extracts and comparison with different germ cell stages.

    PubMed

    Srivastava, Niloo; Raman, Mercy J

    2007-01-01

    Homologous recombination (HR) is established as a significant contributor to double-strand break (DSB) repair in mammalian somatic cells; however, its role in mammalian germ cells has not been characterized, although being conservative in nature it is anticipated to be the major pathway in germ cells. The germ cell system has inherent limitations by which intact cell approaches are not feasible. The present study, therefore, investigates HR-mediated DSB repair in mouse germ cell extracts by using an in vitro plasmid recombination assay based on functional rescue of a neomycin (neo) gene. A significantly high-fold increase in neo+ (Kan(R)) colonies following incubation of two plasmid substrates (neo delta1 and neo delta2) with testicular extracts demonstrated the extracts' ability to catalyze intermolecular recombination. A significant enhancement in recombinants upon linearization of one of the plasmids suggested the existence of an HR-mediated DSB repair activity. Comparison of the activity at sequential developmental stages, spermatogonia, spermatocytes and spermatids revealed its presence at all the stages; spermatocyte being the most proficient stage. Further, restriction analysis of recombinant plasmids indicated the predominance of gene conversion in enriched spermatocytes (mostly pachytenes), in contrast to gonial and spermatid extracts that showed higher reciprocal exchange. In conclusion, this study demonstrates HR repair activity at all stages of male germ cells, suggesting an important role of HR-mediated DSB repair during mammalian spermatogenesis. Further, the observed preference of gene conversion over reciprocal exchange at spermatocyte stage correlates with the close association of gene conversion with the meiotic recombination program.

  15. Alleles of the homologous recombination gene, RAD59, identify multiple responses to disrupted DNA replication in Saccharomyces cerevisiae.

    PubMed

    Liddell, Lauren C; Manthey, Glenn M; Owens, Shannon N; Fu, Becky X H; Bailis, Adam M

    2013-10-14

    In Saccharomyces cerevisiae, Rad59 is required for multiple homologous recombination mechanisms and viability in DNA replication-defective rad27 mutant cells. Recently, four rad59 missense alleles were found to have distinct effects on homologous recombination that are consistent with separation-of-function mutations. The rad59-K166A allele alters an amino acid in a conserved α-helical domain, and, like the rad59 null allele diminishes association of Rad52 with double-strand breaks. The rad59-K174A and rad59-F180A alleles alter amino acids in the same domain and have genetically similar effects on homologous recombination. The rad59-Y92A allele alters a conserved amino acid in a separate domain, has genetically distinct effects on homologous recombination, and does not diminish association of Rad52 with double-strand breaks. In this study, rad59 mutant strains were crossed with a rad27 null mutant to examine the effects of the rad59 alleles on the link between viability, growth and the stimulation of homologous recombination in replication-defective cells. Like the rad59 null allele, rad59-K166A was synthetically lethal in combination with rad27. The rad59-K174A and rad59-F180A alleles were not synthetically lethal in combination with rad27, had effects on growth that coincided with decreased ectopic gene conversion, but did not affect mutation, unequal sister-chromatid recombination, or loss of heterozygosity. The rad59-Y92A allele was not synthetically lethal when combined with rad27, stimulated ectopic gene conversion and heteroallelic recombination independently from rad27, and was mutually epistatic with srs2. Unlike rad27, the stimulatory effect of rad59-Y92A on homologous recombination was not accompanied by effects on growth rate, cell cycle distribution, mutation, unequal sister-chromatid recombination, or loss of heterozygosity. The synthetic lethality conferred by rad59 null and rad59-K166A alleles correlates with their inhibitory effect on association

  16. Multiple branches of the meiotic recombination pathway contribute independently to homolog pairing and stable juxtaposition during meiosis in budding yeast

    PubMed Central

    Peoples-Holst, Tamara L.; Burgess, Sean M.

    2005-01-01

    A unique aspect of meiosis is the segregation of homologous chromosomes at the meiosis I division. Homologs are physically connected prior to segregation by crossing over between nonsister chromatids. Crossovers arise from the repair of induced double-strand breaks (DSBs). In many organisms, more DSBs are formed than crossovers in a given nucleus. It has been previously suggested that repair of DSBs to noncrossover recombination products aids homolog alignment. Here we explore how two modes of the meiotic recombination pathway (crossover and noncrossover) and meiotic telomere reorganization contribute to the pairing and close juxtaposition of homologous chromosomes in budding yeast. We found that intermediates in the DSB repair pathway leading to both crossover and noncrossover recombination products contribute independently to close, stable homolog juxtaposition (CSHJ), a measurable state of homolog pairing. Analysis of the ndj1Δ mutant indicates that the effect of meiotic telomere reorganization on CSHJ is exerted through recombination intermediates at interstitial chromosomal loci, perhaps through the noncrossover branch of the DSB repair pathway. We suggest that transient, early DSB-initiated interactions, including those that give rise to noncrossovers, are important for homolog recognition and juxtaposition. PMID:15805472

  17. Polynucleotide phosphorylase is implicated in homologous recombination and DNA repair in Escherichia coli.

    PubMed

    Carzaniga, Thomas; Sbarufatti, Giulia; Briani, Federica; Dehò, Gianni

    2017-04-04

    Polynucleotide phosphorylase (PNPase, encoded by pnp) is generally thought of as an enzyme dedicated to RNA metabolism. The pleiotropic effects of PNPase deficiency is imputed to altered processing and turnover of mRNAs and small RNAs, which in turn leads to aberrant gene expression. However, it has long since been known that this enzyme may also catalyze template-independent polymerization of dNDPs into ssDNA and the reverse phosphorolytic reaction. Recently, PNPase has been implicated in DNA recombination, repair, mutagenesis and resistance to genotoxic agents in diverse bacterial species, raising the possibility that PNPase may directly, rather than through control of gene expression, participate in these processes. In this work we present evidence that in Escherichia coli PNPase enhances both homologous recombination upon P1 transduction and error prone DNA repair of double strand breaks induced by zeocin, a radiomimetic agent. Homologous recombination does not require PNPase phosphorolytic activity and is modulated by its RNA binding domains whereas error prone DNA repair of zeocin-induced DNA damage is dependent on PNPase catalytic activity and cannot be suppressed by overexpression of RNase II, the other major enzyme (encoded by rnb) implicated in exonucleolytic RNA degradation. Moreover, E. coli pnp mutants are more sensitive than the wild type to zeocin. This phenotype depends on PNPase phosphorolytic activity and is suppressed by rnb, thus suggesting that zeocin detoxification may largely depend on RNA turnover. Our data suggest that PNPase may participate both directly and indirectly through regulation of gene expression to several aspects of DNA metabolism such as recombination, DNA repair and resistance to genotoxic agents.

  18. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. PARP-mediated repair, homologous recombination, and back-up non-homologous end joining-like repair of single-strand nicks.

    PubMed

    Metzger, Michael J; Stoddard, Barry L; Monnat, Raymond J

    2013-07-01

    Double-strand breaks (DSBs) in chromosomal DNA can induce both homologous recombination (HR) and non-homologous end-joining (NHEJ). Recently we showed that single-strand nicks induce HR with a significant reduction in toxicity and mutagenic effects associated with NHEJ. To further investigate the differences and similarities of DSB- and nick-induced repair, we used an integrated reporter system in human cells to measure HR and NHEJ produced by the homing endonuclease I-AniI and a designed 'nickase' variant that nicks the same target site, focusing on the PARP and HR repair pathways. PARP inhibitors, which block single-strand break repair, increased the rate of nick-induced HR up to 1.7-fold but did not affect DSB-induced HR or mutNHEJ. Additionally, expression of the PALB2 WD40 domain in trans acted as a dominant-negative inhibitor of both DSB- and nick-induced HR, sensitized cells to PARP inhibition, and revealed an alternative mutagenic repair pathway for nicks. Thus, while both DSB- and nick-induced HR use a common pathway, their substrates are differentially processed by cellular factors. These results also suggest that the synthetic lethality of PARP and BRCA may be due to repair of nicks through an error prone, NHEJ-like mechanism that is active when both PARP and HR pathways are blocked.

  20. Impact of two DNA repair pathways, homologous recombination and non-homologous end joining, on bacterial spore inactivation under simulated martian environmental conditions

    NASA Astrophysics Data System (ADS)

    Moeller, Ralf; Schuerger, Andrew C.; Reitz, Günther; Nicholson, Wayne L.

    2011-09-01

    Spores of Bacillus subtilis were used as a model system to study the impact of the two major DNA double-strand break (DSB) repair mechanisms [homologous recombination (HR) and non-homologous end-joining (NHEJ)] on the survivability of air-dried mono- and multilayers of bacterial spores under a simulated martian environment; i.e., an environment with low temperature (-10 °C), pure CO 2 atmosphere (99.99% CO 2), 200-1100 nm UV-VIS-NIR radiation, and 0.69 kPa pressure. Spores in multilayers exhibited low inactivation rates compared to monolayers, mainly due to shadowing effects of overlying spores. Simulated martian UV irradiation reduced dramatically spore viability, whereas when shielded from martian UV radiation, spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to simulated martian environmental conditions than were wild-type spores. In addition, NHEJ-deficient spores were consistently more sensitive than HR-deficient spores to simulated Mars environmental conditions, suggesting that DSBs were an important type of DNA damage. The results indicated that both HR and NHEJ provide an efficient set of DNA repair pathways ensuring spore survival after exposure to simulated martian environmental conditions.

  1. SPAR1/RTEL1 maintains genomic stability by suppressing homologous recombination

    PubMed Central

    Barber, Louise J.; Youds, Jillian L.; Ward, Jordan D.; McIlwraith, Michael J.; O’Neil, Nigel J.; Petalcorin, Mark I.R.; Martin, Julie S.; Collis, Spencer J.; Cantor, Sharon B.; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C.; Rose, Ann M.; Boulton, Simon J.

    2013-01-01

    SUMMARY Inappropriate homologous recombination (HR) can cause gross chromosomal rearrangements that in mammalian cells may lead to tumorigenesis. In yeast, the Srs2 protein is an anti-recombinase that eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has proven to be elusive. In this work, we identify C. elegans SPAR-1 as a functional analogue of Srs2 and describe its vertebrate counterpart, SPAR1/RTEL1, which is required for genome stability and tumour avoidance. We find that spar-1 mutant worms and SPAR1 knockdown human cells share characteristic phenotypes with yeast srs2 mutants, including inviability upon deletion of the sgs1/BLM homologue, hyper-recombination, and DNA damage sensitivity. In vitro, purified human SPAR1 antagonises HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control following deregulation of SPAR1/RTEL1 may be a critical event that drives genome instability and cancer. PMID:18957201

  2. Homologous recombination induced by doxazosin mesylate and saw palmetto in the Drosophila wing-spot test.

    PubMed

    Gabriel, Katiane Cella; Dihl, Rafael Rodrigues; Lehmann, Mauricio; Reguly, Maria Luiza; Richter, Marc François; Andrade, Heloisa Helena Rodrigues de

    2013-03-01

    Benign prostatic hyperplasia (BPH) is the most common tumor in men over 40 years of age. Acute urinary retention (AUR) is regarded as the most serious hazard of untreated BPH. α-Blockers, such as doxazosin mesylate, and 5-α reductase inhibitors, such as finasteride, are frequently used because they decrease both AUR and the need for BPH-related surgery. An extract of the fruit from American saw palmetto plant has also been used as an alternative treatment for BPH. The paucity of information available concerning the genotoxic action of these compounds led us to assess their activity as inducers of different types of DNA lesions using the somatic mutation and recombination test in Drosophila melanogaster. Finasteride did not induce gene mutation, chromosomal mutation or mitotic recombination, which means it was nongenotoxic in our experimental conditions. On the other hand, doxazosin mesylate and saw palmetto induced significant increases in spot frequencies in trans-heterozygous flies. In order to establish the actual role played by mitotic recombination and by mutation in the genotoxicity observed, the balancer-heterozygous flies were also analyzed, showing no increment in the total spot frequencies in relation to the negative control, for both drugs. Doxazosin mesylate and saw palmetto were classified as specific inducers of homologous recombination in Drosophila proliferative cells, an event linked to the loss of heterozygosity.

  3. Efficient generation of targeted and controlled mutational events in porcine cells using nuclease-directed homologous recombination.

    PubMed

    Butler, James R; Santos, Rafael M N; Martens, Gregory R; Ladowski, Joseph M; Wang, Zheng-Yu; Li, Ping; Tector, Matthew; Tector, A Joseph

    2017-05-15

    Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5' to a nuclease cut site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by Sanger sequence, restriction enzyme digest and flow cytometry. Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic

  4. Exploiting the homologous recombination DNA repair network for targeted cancer therapy.

    PubMed

    Peng, Guang; Lin, Shiaw-Yih

    2011-02-10

    Genomic instability is a characteristic of cancer cells. In order to maintain genomic integrity, cells have evolved a complex DNA repair system to detect, signal and repair a diversity of DNA lesions. Homologous recombination (HR)-mediated DNA repair represents an error-free repair mechanism to maintain genomic integrity and ensure high-fidelity transmission of genetic information. Deficiencies in HR repair are of tremendous importance in the etiology of human cancers and at the same time offer great opportunities for designing targeted therapeutic strategies. The increase in the number of proteins identified as being involved in HR repair has dramatically shifted our concept of the proteins involved in this process: traditionally viewed as existing in a linear and simple pathway, today they are viewed as existing in a dynamic and interconnected network. Moreover, exploration of the targets within this network that can be modulated by small molecule drugs has led to the discovery of many effective kinase inhibitors, such as ATM, ATR, DNA-PK, CHK1, and CHK2 inhibitors. In preclinical studies, these inhibitors have been shown to sensitize cancer cells to chemotherapy and radiation therapy. The most exciting discovery in the field of HR repair is the identification of the synthetic lethality relationship between poly (ADP-ribose) polymerase (PARP) inhibitors and HR deficiency. The promises of clinical applications of PARP inhibitors and the concept of synthetic lethality also bring challenges into focus. Future research directions in the area of HR repair include determining how to identify the patients most likely to benefit from PARP inhibitors and developing strategies to overcome resistance to PARP inhibitors.

  5. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

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

    PubMed

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

    2014-06-01

    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.

  7. Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination.

    PubMed

    Lee, Mi Ok; Bornelöv, Susanne; Andersson, Leif; Lamont, Susan J; Chen, Junfeng; Womack, James E

    2016-11-29

    Defensins constitute an evolutionary conserved family of cationic antimicrobial peptides that play a key role in host innate immune responses to infection. Defensin genes generally reside in complex genomic regions that are prone to structural variation, and defensin genes exhibit extensive copy number variation in humans and in other species. Copy number variation of defensin genes was examined in inbred lines of Leghorn and Fayoumi chickens, and a duplication of defensin7 was discovered in the Fayoumi breed. Analysis of junction sequences confirmed the occurrence of a simple tandem duplication of defensin7 with sequence identity at the junction, suggesting nonallelic homologous recombination between defensin7 and defensin6 The duplication event generated two chimeric promoters that are best explained by gene conversion followed by homologous recombination. Expression of defensin7 was not elevated in animals with two genes despite both genes being transcribed in the tissues examined. Computational prediction of promoter regions revealed the presence of several putative transcription factor binding sites generated by the duplication event. These data provide insight into the evolution and possible function of large gene families and specifically, the defensins.

  8. Duplication of chicken defensin7 gene generated by gene conversion and homologous recombination

    PubMed Central

    Lee, Mi Ok; Bornelöv, Susanne; Andersson, Leif; Lamont, Susan J.; Chen, Junfeng; Womack, James E.

    2016-01-01

    Defensins constitute an evolutionary conserved family of cationic antimicrobial peptides that play a key role in host innate immune responses to infection. Defensin genes generally reside in complex genomic regions that are prone to structural variation, and defensin genes exhibit extensive copy number variation in humans and in other species. Copy number variation of defensin genes was examined in inbred lines of Leghorn and Fayoumi chickens, and a duplication of defensin7 was discovered in the Fayoumi breed. Analysis of junction sequences confirmed the occurrence of a simple tandem duplication of defensin7 with sequence identity at the junction, suggesting nonallelic homologous recombination between defensin7 and defensin6. The duplication event generated two chimeric promoters that are best explained by gene conversion followed by homologous recombination. Expression of defensin7 was not elevated in animals with two genes despite both genes being transcribed in the tissues examined. Computational prediction of promoter regions revealed the presence of several putative transcription factor binding sites generated by the duplication event. These data provide insight into the evolution and possible function of large gene families and specifically, the defensins. PMID:27849592

  9. TopBP1 associates with NBS1 and is involved in homologous recombination repair

    SciTech Connect

    Morishima, Ken-ichi; Sakamoto, Shuichi; Kobayashi, Junya; Izumi, Hideki; Suda, Tetsuji; Matsumoto, Yoshiyuki; Tauchi, Hiroshi; Ide, Hiroshi; Komatsu, Kenshi; Matsuura, Shinya

    2007-11-03

    TopBP1 is involved in DNA replication and DNA damage checkpoint. Recent studies have demonstrated that TopBP1 is a direct positive effecter of ATR. However, it is not known how TopBP1 recognizes damaged DNA. Here, we show that TopBP1 formed nuclear foci after exposure to ionizing radiation, but such TopBP1 foci were abolished in Nijmegen breakage syndrome cells. We also show that TopBP1 physically associated with NBS1 in vivo. These results suggested that NBS1 might regulate TopBP1 recruitment to the sites of DNA damage. TopBP1-depleted cells showed hypersensitivity to Mitomycin C and ionizing radiation, an increased frequency of sister-chromatid exchange level, and a reduced frequency of DNA double-strand break induced homologous recombination repair. Together, these results suggested that TopBP1 might be a mediator of DNA damage signaling from NBS1 to ATR and promote homologous recombination repair.

  10. 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.

  11. Acetylation of PCNA Sliding Surface by Eco1 Promotes Genome Stability through Homologous Recombination.

    PubMed

    Billon, Pierre; Li, Jian; Lambert, Jean-Philippe; Chen, Yizhang; Tremblay, Véronique; Brunzelle, Joseph S; Gingras, Anne-Claude; Verreault, Alain; Sugiyama, Tomohiko; Couture, Jean-Francois; Côté, Jacques

    2017-01-05

    During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.

  12. Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination1[OPEN

    PubMed Central

    Lu, Pingli

    2017-01-01

    Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice. PMID:28049740

  13. ATM-dependent chromatin remodeler Rsf-1 facilitates DNA damage checkpoints and homologous recombination repair.

    PubMed

    Min, Sunwoo; Jo, Sujin; Lee, Ho-Soo; Chae, Sunyoung; Lee, Jong-Soo; Ji, Jae-Hoon; Cho, Hyeseong

    2014-01-01

    As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of γH2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas γH2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair.

  14. Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates

    PubMed Central

    Gyimesi, Máté; Pires, Ricardo H.; Billington, Neil; Sarlós, Kata; Kocsis, Zsuzsa S.; Módos, Károly; Kellermayer, Miklós S. Z.; Kovács, Mihály

    2013-01-01

    Homologous recombination (HR) is a key process in the repair of double-stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Bloom's syndrome RecQ-family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR-intermediate DNA structures, by using single-molecule visualization (electron microscopic and atomic force microscopic single-particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full-length BLM, we used a previously characterized truncated construct (BLM642–1290) as a monomeric control. Contrary to previous models proposing a ring-forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single-stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.—Gyimesi, M., Pires, R.H., Billington, N., Sarlós, K., Kocsis, Z.S. Módos, K., Kellermayer, M. S. Z., Kovács, M. Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates. PMID:24005907

  15. 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

  16. 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

  17. 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. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. XRCC3 is essential for proper double-strand break repair and homologous recombination in rice meiosis.

    PubMed

    Zhang, Bingwei; Wang, Mo; Tang, Ding; Li, Yafei; Xu, Meng; Gu, Minghong; Cheng, Zhukuan; Yu, Hengxiu

    2015-09-01

    RAD51 paralogues play important roles in the assembly and stabilization of RAD51 nucleoprotein filaments, which promote homologous pairing and strand exchange reactions in organisms ranging from yeast to vertebrates. XRCC3, a RAD51 paralogue, has been characterized in budding yeast, mouse, and Arabidopsis. In the present study, XRCC3 in rice was identified and characterized. The rice xrcc3 mutant exhibited normal vegetative growth but complete male and female sterility. Cytological investigations revealed that homologous pairing and synapsis were severely disrupted in the mutant. Meiotic chromosomes were frequently entangled from diplotene to metaphase I, resulting in chromosome fragmentation at anaphase I. The immunostaining signals from γH2AX were regular, implying that double-strand break (DSB) formation was normal in xrcc3 meiocytes. However, COM1 was not detected on early prophase I chromosomes, suggesting that the DSB end-processing system was destroyed in the mutant. Moreover, abnormal chromosome localization of RAD51C, DMC1, ZEP1, ZIP4, and MER3 was observed in xrcc3. Taken together, the results suggest that XRCC3 plays critical roles in both DSB repair and homologous chromosome recombination during rice meiosis.

  19. Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses.

    PubMed

    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.

  20. 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

    2015-08-06

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks.

    PubMed

    Oum, Ji-Hyun; Seong, Changhyun; Kwon, Youngho; Ji, Jae-Hoon; Sid, Amy; Ramakrishnan, Sreejith; Ira, Grzegorz; Malkova, Anna; Sung, Patrick; Lee, Sang Eun; Shim, Eun Yong

    2011-10-01

    Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

  2. Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

    PubMed

    Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

    2016-01-01

    Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

  3. DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination.

    PubMed

    Srivastava, Shikha; Dahal, Sumedha; Naidu, Sharanya J; Anand, Deepika; Gopalakrishnan, Vidya; Kooloth Valappil, Rajendran; Raghavan, Sathees C

    2017-01-24

    DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome.

  4. Homologous recombination in hybridoma cells: dependence on time and fragment length.

    PubMed Central

    Shulman, M J; Nissen, L; Collins, C

    1990-01-01

    Mutant hybridoma-myeloma cell lines that are defective in immunoglobulin production are expected to be useful for defining the molecular requirements of immunoglobulin gene expression. The analysis of such mutants would be greatly facilitated if they could be mapped by marker rescue, i.e., by identifying the segments of wild-type DNA that can restore the normal phenotype by homologous recombination with the mutant chromosomal immunoglobulin gene. To assess the feasibility of this type of mapping, we have measured the efficiency with which fragments of wild-type DNA recombine with a mutant hybridoma immunoglobulin gene and restore normal immunoglobulin production. We found that most if not all recombinants were detectable 2 days after DNA transfer and that the frequency of gene restoration increased with increasing length of the transferred mu gene fragments, between 1.2 and 9.5 kilobases. These results indicate that the available technology should be adequate to map mutations in the mu gene to within approximately 1 kilobase. Images PMID:2117699

  5. Structure of recombinant rat UBF by electron image analysis and homology modelling.

    PubMed Central

    Neil, K J; Ridsdale, R A; Rutherford, B; Taylor, L; Larson, D E; Glibetic, M; Rothblum, L I; Harauz, G

    1996-01-01

    We have studied the structure of recombinant rat UBF (rrUBF), an RNA polymerase I transcription factor, by electron microscopy and image analysis of single particles contrasted with methylamine tungstate. Recombinant rat UBF appeared to be a flat, U-shaped protein with a central region of low density. In the dominant projections, 2-fold mirror symmetry was seen, consistent with the dimerization properties of this molecule, and of dimensions in agreement with the length of DNA that rat UBF protects in footprinting studies. Electron microscopy of various rrUBF-DNA complexes confirmed that our recombinant protein was fully able to bind the 45S rDNA promoter, and that it caused substantial bends in the DNA. Upon extended incubation in a droplet covered by a lipid monolayer at the liquid-air interface, rrUBF formed long filamentous arrays with a railway track appearance. This structure was interpreted to consist of overlapping rrUBF dimers 3.5 nm apart, which value would represent the thickness of the protein. Our results show rrUBF to interact with and bend the promoter DNA into a roughly 10 nm diameter superhelix. Based on all these electron microscopical results, an atomic structure was predicted by homology modelling of the HMG fingers, and connected by energy minimized intervening segments. PMID:8628680

  6. Genetic dissection of Helicobacter pylori AddAB role in homologous recombination.

    PubMed

    Marsin, Stéphanie; Lopes, Anne; Mathieu, Aurélie; Dizet, Eléa; Orillard, Emilie; Guérois, Raphaël; Radicella, J Pablo

    2010-10-01

    Helicobacter pylori infects the stomach of about half of the world's human population, frequently causing chronic inflammation at the origin of several gastric pathologies. One of the most remarkable characteristics of the species is its remarkable genomic plasticity in which homologous recombination (HR) plays a critical role. Here, we analyzed the role of the H. pylori homologue of the AddAB recombination protein. Bioinformatics analysis of the proteins unveils the similarities and differences of the H. pylori AddAB complex with respect to the RecBCD and AddAB complexes from Escherichia coli and Bacillus subtilis, respectively. Helicobacter pylori mutants lacking functional addB or/and addA show the same level of sensitivity to DNA-damaging agents such as UV or irradiation and of deficiency in intrachromosomal RecA-dependent HR. Epistasis analyses of both DNA repair and HR phenotypes, using double and triple recombination mutants, demonstrate that, in H. pylori, AddAB and RecOR complexes define two separate presynaptic pathways with little functional overlap. However, neither of these complexes participates in the RecA-dependent process of transformation of these naturally competent bacteria.

  7. Stimulated recombination in open systems

    SciTech Connect

    Muoz-Cobo, J.L.; Verdu, G.; Jimenez, P.; Pea, J.

    1986-09-01

    In this comment we study the problem of the stimulated recombination in an open system from a stochastic point of view. We set up the bivariate master equation for the number of photons and ions inside the system. Then we perform a systematic expansion, with the system volume as an expansion parameter, and we obtain the fluctuations of the number of photons and ions around its macroscopic values in the linear noise approximation; the stationary solution is also investigated.

  8. Solubility of recombinant Src homology 2 domains expressed in E. coli can be predicted by TANGO.

    PubMed

    Andersen, Thorny Cecilie Bie; Lindsjø, Kjersti; Hem, Cecilie Dahl; Koll, Lise; Kristiansen, Per Eugen; Skjeldal, Lars; Andreotti, Amy H; Spurkland, Anne

    2014-01-14

    Signalling proteins often contain several well defined and conserved protein domains. Structural analyses of such domains by nuclear magnetic spectroscopy or X-ray crystallography may greatly inform the function of proteins. A limiting step is often the production of sufficient amounts of the recombinant protein. However, there is no particular way to predict whether a protein will be soluble when expressed in E.coli. Here we report our experience with expression of a Src homology 2 (SH2) domain. The SH2 domain of the SH2D2A protein (or T cell specific adapter protein, TSAd) forms insoluble aggregates when expressed as various GST-fusion proteins in Escherichia coli (E. coli). Alteration of the flanking sequences, or growth temperature influenced expression and solubility of TSAd-SH2, however overall yield of soluble protein remained low. The algorithm TANGO, which predicts amyloid fibril formation in eukaryotic cells, identified a hydrophobic sequence within the TSAd-SH2 domain with high propensity for beta-aggregation. Mutation to the corresponding amino acids of the related HSH2- (or ALX) SH2 domain increased the yield of soluble TSAd-SH2 domains. High beta-aggregation values predicted by TANGO correlated with low solubility of recombinant SH2 domains as reported in the literature. Solubility of recombinant proteins expressed in E.coli can be predicted by TANGO, an algorithm developed to determine the aggregation propensity of peptides. Targeted mutations representing corresponding amino acids in similar protein domains may increase solubility of recombinant proteins.

  9. Solubility of recombinant Src homology 2 domains expressed in E. coli can be predicted by TANGO

    PubMed Central

    2014-01-01

    Background Signalling proteins often contain several well defined and conserved protein domains. Structural analyses of such domains by nuclear magnetic spectroscopy or X-ray crystallography may greatly inform the function of proteins. A limiting step is often the production of sufficient amounts of the recombinant protein. However, there is no particular way to predict whether a protein will be soluble when expressed in E.coli. Here we report our experience with expression of a Src homology 2 (SH2) domain. Results The SH2 domain of the SH2D2A protein (or T cell specific adapter protein, TSAd) forms insoluble aggregates when expressed as various GST-fusion proteins in Escherichia coli (E. coli). Alteration of the flanking sequences, or growth temperature influenced expression and solubility of TSAd-SH2, however overall yield of soluble protein remained low. The algorithm TANGO, which predicts amyloid fibril formation in eukaryotic cells, identified a hydrophobic sequence within the TSAd-SH2 domain with high propensity for beta-aggregation. Mutation to the corresponding amino acids of the related HSH2- (or ALX) SH2 domain increased the yield of soluble TSAd-SH2 domains. High beta-aggregation values predicted by TANGO correlated with low solubility of recombinant SH2 domains as reported in the literature. Conclusions Solubility of recombinant proteins expressed in E.coli can be predicted by TANGO, an algorithm developed to determine the aggregation propensity of peptides. Targeted mutations representing corresponding amino acids in similar protein domains may increase solubility of recombinant proteins. PMID:24423197

  10. Homology and the hierarchy of biological systems.

    PubMed

    Sommer, Ralf J

    2008-07-01

    Homology is the similarity between organisms due to common ancestry. Introduced by Richard Owen in 1843 in a paper entitled "Lectures on comparative anatomy and physiology of the invertebrate animals", the concept of homology predates Darwin's "Origin of Species" and has been very influential throughout the history of evolutionary biology. Although homology is the central concept of all comparative biology and provides a logical basis for it, the definition of the term and the criteria of its application remain controversial. Here, I will discuss homology in the context of the hierarchy of biological organization. I will provide insights gained from an exemplary case study in evolutionary developmental biology that indicates the uncoupling of homology at different levels of biological organization. I argue that continuity and hierarchy are separate but equally important issues of homology. (c) 2008 Wiley Periodicals, Inc.

  11. Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae.

    PubMed

    Chopra-Dewasthaly, Rohini; Marenda, Marc; Rosengarten, Renate; Jechlinger, Wolfgang; Citti, Christine

    2005-12-01

    Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.

  12. 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.

  13. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

    PubMed

    Aymard, François; Bugler, Beatrix; Schmidt, Christine K; Guillou, Emmanuelle; Caron, Pierre; Briois, Sébastien; Iacovoni, Jason S; Daburon, Virginie; Miller, Kyle M; Jackson, Stephen P; Legube, Gaëlle

    2014-04-01

    Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

  14. Generating gene knockout rats by homologous recombination in embryonic stem cells

    PubMed Central

    Tong, Chang; Huang, Guanyi; Ashton, Charles; Li, Ping; Ying, Qi-Long

    2013-01-01

    We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats. PMID:21637202

  15. Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1

    PubMed Central

    Machida, Shinichi; Takaku, Motoki; Ikura, Masae; Sun, Jiying; Suzuki, Hidekazu; Kobayashi, Wataru; Kinomura, Aiko; Osakabe, Akihisa; Tachiwana, Hiroaki; Horikoshi, Yasunori; Fukuto, Atsuhiko; Matsuda, Ryo; Ura, Kiyoe; Tashiro, Satoshi; Ikura, Tsuyoshi; Kurumizaka, Hitoshi

    2014-01-01

    Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1. PMID:24798879

  16. Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1.

    PubMed

    Machida, Shinichi; Takaku, Motoki; Ikura, Masae; Sun, Jiying; Suzuki, Hidekazu; Kobayashi, Wataru; Kinomura, Aiko; Osakabe, Akihisa; Tachiwana, Hiroaki; Horikoshi, Yasunori; Fukuto, Atsuhiko; Matsuda, Ryo; Ura, Kiyoe; Tashiro, Satoshi; Ikura, Tsuyoshi; Kurumizaka, Hitoshi

    2014-05-06

    Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.

  17. Biochemical analysis of the human ENA/VASP-family proteins, MENA, VASP and EVL, in homologous recombination.

    PubMed

    Takaku, Motoki; Ueno, Hiroyuki; Kurumizaka, Hitoshi

    2011-06-01

    MENA, VASP and EVL are members of the ENA/VASP family of proteins and are involved in cytoplasmic actin remodeling. Previously, we found that EVL directly interacts with RAD51, an essential protein in the homologous recombinational repair of double-strand breaks (DSBs) and stimulates the RAD51-mediated recombination reactions in vitro. The EVL-knockdown MCF7 cells exhibited a clear reduction in RAD51-foci formation, suggesting that EVL may function in the DSB repair pathway through RAD51-mediated homologous recombination. However, the DSB repair defects were less significant in the EVL-knockdown cells, implying that two EVL paralogues, MENA and VASP, may complement the EVL function in human cells. Therefore, in the present study, we purified human MENA, VASP and EVL as recombinant proteins, and compared their biochemical activities in vitro. We found that all three proteins commonly exhibited the RAD51 binding, DNA binding and DNA-annealing activities. Stimulation of the RAD51-mediated homologous pairing was also observed with all three proteins. In addition, surface plasmon resonance analyses revealed that MENA, VASP and EVL mutually interacted. These results support the ideas that the ENA/VASP-family proteins are functionally redundant in homologous recombination, and that all three may be involved in the DSB repair pathway in humans.

  18. Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus

    PubMed Central

    Guo, Jin-Chao; Tang, Yan-Dong; Zhao, Kuan; Wang, Tong-Yun; Liu, Ji-Ting; Gao, Jia-Cong; Chang, Xiao-Bo; Cui, Hong-Yu; Tian, Zhi-Jun; Cai, Xue-Hui; An, Tong-Qing

    2016-01-01

    Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses. PMID:28066407

  19. Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

    PubMed Central

    Grose, William E.; Clark, K. Reed; Griffin, Danielle; Malik, Vinod; Shontz, Kimberly M.; Montgomery, Chrystal L.; Lewis, Sarah; Brown, Robert H.; Janssen, Paul M. L.; Mendell, Jerry R.; Rodino-Klapac, Louise R.

    2012-01-01

    The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes. PMID:22720081

  20. 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

    2006-01-05

    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.

  1. Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair.

    PubMed Central

    Schürer, K Anke; Rudolph, Christian; Ulrich, Helle D; Kramer, Wilfried

    2004-01-01

    The MPH1 gene from Saccharomyces cerevisiae, encoding a member of the DEAH family of proteins, had been identified by virtue of the spontaneous mutator phenotype of respective deletion mutants. Genetic analysis suggested that MPH1 functions in a previously uncharacterized DNA repair pathway that protects the cells from damage-induced mutations. We have now analyzed genetic interactions of mph1 with a variety of mutants from different repair systems with respect to spontaneous mutation rates and sensitivities to different DNA-damaging agents. The dependence of the mph1 mutator phenotype on REV3 and REV1 and the synergy with mutations in base and nucleotide excision repair suggest an involvement of MPH1 in error-free bypass of lesions. However, although we observed an unexpected partial suppression of the mph1 mutator phenotype by rad5, genetic interactions with other mutations in postreplicative repair imply that MPH1 does not belong to this pathway. Instead, mutations from the homologous recombination pathway were found to be epistatic to mph1 with respect to both spontaneous mutation rates and damage sensitivities. Determination of spontaneous mitotic recombination rates demonstrated that mph1 mutants are not deficient in homologous recombination. On the contrary, in an sgs1 background we found a pronounced hyperrecombination phenotype. Thus, we propose that MPH1 is involved in a branch of homologous recombination that is specifically dedicated to error-free bypass. PMID:15126389

  2. 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.

  3. A phosphorylation-deubiquitination cascade regulates the BRCA2-RAD51 axis in homologous recombination.

    PubMed

    Luo, Kuntian; Li, Lei; Li, Yunhui; Wu, Chenming; Yin, Yujiao; Chen, Yuping; Deng, Min; Nowsheen, Somaira; Yuan, Jian; Lou, Zhenkun

    2016-12-01

    Homologous recombination (HR) is one of the major DNA double-strand break (DSB) repair pathways in mammalian cells. Defects in HR trigger genomic instability and result in cancer predisposition. The defining step of HR is homologous strand exchange directed by the protein RAD51, which is recruited to DSBs by BRCA2. However, the regulation of the BRCA2-RAD51 axis remains unclear. Here we report that ubiquitination of RAD51 hinders RAD51-BRCA2 interaction, while deubiquitination of RAD51 facilitates RAD51-BRCA2 binding and RAD51 recruitment and thus is critical for proper HR. Mechanistically, in response to DNA damage, the deubiquitinase UCHL3 is phosphorylated and activated by ATM. UCHL3, in turn, deubiquitinates RAD51 and promotes the binding between RAD51 and BRCA2. Overexpression of UCHL3 renders breast cancer cells resistant to radiation and chemotherapy, while depletion of UCHL3 sensitizes cells to these treatments, suggesting a determinant role of UCHL3 in cancer therapy. Overall, we identify UCHL3 as a novel regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination cascade dynamically regulates the BRCA2-RAD51 pathway. © 2016 Luo et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells.

    PubMed

    Takahashi, Akihisa; Mori, Eiichiro; Nakagawa, Yosuke; Kajihara, Atsuhisa; Kirita, Tadaaki; Pittman, Douglas L; Hasegawa, Masatoshi; Ohnishi, Takeo

    2016-11-13

    Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock. B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock. Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR. The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

  5. DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

    PubMed Central

    Li, Lei; Germain, Devon R.; Poon, Ho-Yin; Hildebrandt, Matthew R.; Monckton, Elizabeth A.; McDonald, Darin; Hendzel, Michael J.

    2016-01-01

    Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR. PMID:27550810

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

    PubMed

    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.

  7. Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation

    PubMed Central

    Groth, Petra; Orta, Manuel Luís; Elvers, Ingegerd; Majumder, Muntasir Mamun; Lagerqvist, Anne; Helleday, Thomas

    2012-01-01

    Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7–9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair. PMID:22505579

  8. Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation.

    PubMed

    Groth, Petra; Orta, Manuel Luís; Elvers, Ingegerd; Majumder, Muntasir Mamun; Lagerqvist, Anne; Helleday, Thomas

    2012-08-01

    Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7-9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair.

  9. Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination

    PubMed Central

    Taylor, Martin R.G.; Špírek, Mário; Chaurasiya, Kathy R.; Ward, Jordan D.; Carzaniga, Raffaella; Yu, Xiong; Egelman, Edward H.; Collinson, Lucy M.; Rueda, David; Krejci, Lumir; Boulton, Simon J.

    2015-01-01

    Summary Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, “open,” and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex. PMID:26186187

  10. 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

  11. Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination.

    PubMed

    Taylor, Martin R G; Špírek, Mário; Chaurasiya, Kathy R; Ward, Jordan D; Carzaniga, Raffaella; Yu, Xiong; Egelman, Edward H; Collinson, Lucy M; Rueda, David; Krejci, Lumir; Boulton, Simon J

    2015-07-16

    Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  12. 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

  13. Intertypic modular exchanges of genomic segments by homologous recombination at universally conserved segments in human adenovirus species D.

    PubMed

    Gonzalez, Gabriel; Koyanagi, Kanako O; Aoki, Koki; Kitaichi, Nobuyoshi; Ohno, Shigeaki; Kaneko, Hisatoshi; Ishida, Susumu; Watanabe, Hidemi

    2014-08-15

    Human adenovirus species D (HAdV-D), which is composed of clinically and epidemiologically important pathogens worldwide, contains more taxonomic "types" than any other species of the genus Mastadenovirus, although the mechanisms accounting for the high level of diversity remain to be disclosed. Recent studies of known and new types of HAdV-D have indicated that intertypic recombination between distant types contributes to the increasing diversity of the species. However, such findings raise the question as to how homologous recombination events occur between diversified types since homologous recombination is suppressed as nucleotide sequences diverge. In order to address this question, we investigated the distribution of the recombination boundaries in comparison with the landscape of intergenomic sequence conservation assessed according to the synonymous substitution rate (dS). The results revealed that specific genomic segments are conserved between even the most distantly related genomes; we call these segments "universally conserved segments" (UCSs). These findings suggest that UCSs facilitate homologous recombination, resulting in intergenomic segmental exchanges of UCS-flanking genomic regions as recombination modules. With the aid of such a mechanism, the haploid genomes of HAdV-Ds may have been reshuffled, resulting in chimeric genomes out of diversified repertoires in the HAdV-D population analogous to the MHC region reshuffled via crossing over in vertebrates. In addition, some HAdVs with chimeric genomes may have had the opportunity to avoid host immune responses thereby causing epidemics. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. 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.

  15. CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica.

    PubMed

    Schwartz, Cory; Frogue, Keith; Ramesh, Adithya; Misa, Joshua; Wheeldon, Ian

    2017-08-19

    In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica. By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of eight out of nine targeted genes to enhance HR. Strains with nonhomologous end-joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting of KU70 and KU80, and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR in Y. lipolytica without permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies. © 2017 Wiley Periodicals, Inc.

  16. The mismatch repair system reduces meiotic homeologous recombination and stimulates recombination-dependent chromosome loss.

    PubMed Central

    Chambers, S R; Hunter, N; Louis, E J; Borts, R H

    1996-01-01

    Efficient genetic recombination requires near-perfect homology between participating molecules. Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. The effects of chromosomal divergence in diploids of Saccharomyces cerevisiae in which one copy of chromosome III is derived from a closely related species, Saccharomyces paradoxus, have been examined. Meiotic recombination between the diverged chromosomes is decreased by 25-fold. Spore viability is reduced with an observable increase in the number of tetrads with only two or three viable spores. Asci with only two viable spores are disomic for chromosome III, consistent with meiosis I nondisjunction of the homeologs. Asci with three viable spores are highly enriched for recombinants relative to tetrads with four viable spores. In 96% of the class with three viable spores, only one spore possesses a recombinant chromosome III, suggesting that the recombination process itself contributes to meiotic death. This phenomenon is dependent on the activities of the mismatch repair genes PMS1 and MSH2. A model of mismatch-stimulated chromosome loss is proposed to account for this observation. As expected, crossing over is increased in pms1 and msh2 mutants. Furthermore, genetic exchange in pms1 msh2 double mutants is affected to a greater extent than in either mutant alone, suggesting that the two proteins act independently to inhibit homeologous recombination. All mismatch repair-deficient strains exhibited reductions in the rate of chromosome III nondisjunction. PMID:8887641

  17. Role of non-homologous end joining in V(D)J recombination.

    PubMed

    Malu, Shruti; Malshetty, Vidyasagar; Francis, Dailia; Cortes, Patricia

    2012-12-01

    The pathway of V(D)J recombination was discovered almost three decades ago. Yet it continues to baffle scientists because of its inherent complexity and the multiple layers of regulation that are required to efficiently generate a diverse repertoire of T and B cells. The non-homologous end-joining (NHEJ) DNA repair pathway is an integral part of the V(D)J reaction, and its numerous players perform critical functions in generating this vast diversity, while ensuring genomic stability. In this review, we summarize the efforts of a number of laboratories including ours in providing the mechanisms of V(D)J regulation with a focus on the NHEJ pathway. This involves discovering new players, unraveling unknown roles for known components, and understanding how deregulation of these pathways contributes to generation of primary immunodeficiencies. A long-standing interest of our laboratory has been to elucidate various mechanisms that control RAG activity. Our recent work has focused on understanding the multiple protein-protein interactions and protein-DNA interactions during V(D)J recombination, which allow efficient and regulated generation of the antigen receptors. Exploring how deregulation of this process contributes to immunodeficiencies also continues to be an important area of research for our group.

  18. A barrier to homologous recombination between sympatric strains of the cooperative soil bacterium Myxococcus xanthus.

    PubMed

    Wielgoss, Sébastien; Didelot, Xavier; Chaudhuri, Roy R; Liu, Xuan; Weedall, Gareth D; Velicer, Gregory J; Vos, Michiel

    2016-10-01

    The bacterium Myxococcus xanthus glides through soil in search of prey microbes, but when food sources run out, cells cooperatively construct and sporulate within multicellular fruiting bodies. M. xanthus strains isolated from a 16 × 16-cm-scale patch of soil were previously shown to have diversified into many distinct compatibility types that are distinguished by the failure of swarming colonies to merge upon encounter. We sequenced the genomes of 22 isolates from this population belonging to the two most frequently occurring multilocus sequence type (MLST) clades to trace patterns of incipient genomic divergence, specifically related to social divergence. Although homologous recombination occurs frequently within the two MLST clades, we find an almost complete absence of recombination events between them. As the two clades are very closely related and live in sympatry, either ecological or genetic barriers must reduce genetic exchange between them. We find that the rate of change in the accessory genome is greater than the rate of amino-acid substitution in the core genome. We identify a large genomic tract that consistently differs between isolates that do not freely merge and therefore is a candidate region for harbouring gene(s) responsible for self/non-self discrimination.

  19. LINC complexes promote homologous recombination in part through inhibition of nonhomologous end joining.

    PubMed

    Lawrence, Katherine S; Tapley, Erin C; Cruz, Victor E; Li, Qianyan; Aung, Kayla; Hart, Kevin C; Schwartz, Thomas U; Starr, Daniel A; Engebrecht, JoAnne

    2016-12-19

    The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks. © 2016 Lawrence et al.

  20. Roles for APRIN (PDS5B) in homologous recombination and in ovarian cancer prediction

    PubMed Central

    Couturier, Anthony M.; Fleury, Hubert; Patenaude, Anne-Marie; Bentley, Victoria L.; Rodrigue, Amélie; Coulombe, Yan; Niraj, Joshi; Pauty, Joris; Berman, Jason N.; Dellaire, Graham; Di Noia, Javier M.; Mes-Masson, Anne-Marie; Masson, Jean-Yves

    2016-01-01

    APRIN (PDS5 cohesin associated factor B) interacts with both the cohesin complex and the BRCA2 tumor suppressor. How APRIN influences cohesion and DNA repair processes is not well understood. Here, we show that APRIN is recruited to DNA damage sites. We find that APRIN interacts directly with RAD51, PALB2 and BRCA2. APRIN stimulates RAD51-mediated DNA strand invasion. APRIN also binds DNA with an affinity for D-loop structures and single-strand (ss) DNA. APRIN is a new homologous recombination (HR) mediator as it counteracts the RPA inhibitory effect on RAD51 loading to ssDNA. We show that APRIN strongly improves the annealing of complementary-strand DNA and that it can stimulate this process in synergy with BRCA2. Unlike cohesin constituents, its depletion has no impact on class switch recombination, supporting a specific role for this protein in HR. Furthermore, we show that low APRIN expression levels correlate with a better survival in ovarian cancer patients and that APRIN depletion sensitizes cells to the PARP inhibitor Olaparib in xenografted zebrafish. Our findings establish APRIN as an important and specific actor of HR, with cohesin-independent functions. PMID:27924011

  1. A recombinant capsid protein from Dengue-2 induces protection in mice against homologous virus.

    PubMed

    Lazo, Laura; Hermida, Lisset; Zulueta, Aída; Sánchez, Jorge; López, Carlos; Silva, Ricardo; Guillén, Gerardo; Guzmán, María G

    2007-01-22

    In the present work, we study the immunogenicity and protective capacity of a recombinant capsid protein from Dengue-2 virus. The capsid gene was cloned under the T5 phage promoter and expressed in Escherichia coli. The recombinant protein was obtained mainly associated to the soluble fraction upon cellular disruption and exhibited a pattern of high aggregation, determined by gel filtration chromatography. The semipurified preparation was inoculated in mice and after three doses, no antiviral antibodies were induced. On the other hand, mice intracranially challenged with homologous lethal virus, exhibited statistically significant protection with respect to the control group. These results describe, for the first time, the protective capacity of the capsid protein of Dengue virus indicating the existence of a protector mechanism, which is totally independent of the antibodies. This lack of induction of antiviral antibodies makes the capsid protein an attractive vaccine candidate against dengue since eliminates the potential risk of the induction of antibody dependent enhancement associated to the current vaccines under study.

  2. Non-homologous end joining in class switch recombination: the beginning of the end

    PubMed Central

    Kotnis, Ashwin; Du, Likun; Liu, Chonghai; Popov, Sergey W.; Pan-Hammarström, Qiang

    2008-01-01

    Immunoglobulin class switch recombination (CSR) is initiated by a B-cell-specific factor, activation-induced deaminase, probably through deamination of deoxycytidine residues within the switch (S) regions. The initial lesions in the S regions are subsequently processed, resulting in the production of DNA double-strand breaks (DSBs). These breaks will then be recognized, edited and repaired, finally leading to the recombination of the two S regions. Two major repair pathways have been implicated in CSR, the predominant non-homologous end joining (NHEJ) and the alternative end-joining (A-EJ) pathways. The former requires not only components of the ‘classical’ NHEJ machinery, i.e. Ku70/Ku80, DNA-dependent protein kinase catalytic subunit, DNA ligase IV and XRCC4, but also a number of DNA-damage sensors or adaptors, such as ataxia–telangiectasia mutated, γH2AX, 53BP1, MDC1, the Mre11–Rad50–NBS1 complex and the ataxia telangiectasia and Rad3-related protein (ATR). The latter pathway is not well characterized yet and probably requires microhomologies. In this review, we will focus on the current knowledge of the predominant NHEJ pathway in CSR and will also give a perspective on the A-EJ pathway. PMID:19008195

  3. A barrier to homologous recombination between sympatric strains of the cooperative soil bacterium Myxococcus xanthus

    PubMed Central

    Wielgoss, Sébastien; Didelot, Xavier; Chaudhuri, Roy R; Liu, Xuan; Weedall, Gareth D; Velicer, Gregory J; Vos, Michiel

    2016-01-01

    The bacterium Myxococcus xanthus glides through soil in search of prey microbes, but when food sources run out, cells cooperatively construct and sporulate within multicellular fruiting bodies. M. xanthus strains isolated from a 16 × 16-cm-scale patch of soil were previously shown to have diversified into many distinct compatibility types that are distinguished by the failure of swarming colonies to merge upon encounter. We sequenced the genomes of 22 isolates from this population belonging to the two most frequently occurring multilocus sequence type (MLST) clades to trace patterns of incipient genomic divergence, specifically related to social divergence. Although homologous recombination occurs frequently within the two MLST clades, we find an almost complete absence of recombination events between them. As the two clades are very closely related and live in sympatry, either ecological or genetic barriers must reduce genetic exchange between them. We find that the rate of change in the accessory genome is greater than the rate of amino-acid substitution in the core genome. We identify a large genomic tract that consistently differs between isolates that do not freely merge and therefore is a candidate region for harbouring gene(s) responsible for self/non-self discrimination. PMID:27046334

  4. DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination.

    PubMed

    Srivastava, Shikha; Dahal, Sumedha; Naidu, Sharanya J; Anand, Deepika; Gopalakrishnan, Vidya; Kooloth Valappil, Rajendran; Raghavan, Sathees C

    2017-04-01

    DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon. Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon. However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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

    PubMed Central

    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

    2016-01-01

    DNA repair by homologous recombination (HR)1 is highly suppressed in G1 cells2,3 to ensure that mitotic recombination occurs solely between sister chromatids4. Although many HR 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 in order to constrain BRCA2 function to the S/G2 phases. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein5, in complex with CUL3-RBX16. 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 HR in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR/Cas9-based gene targeting assay. We conclude that the mechanism prohibiting HR in G1 minimally consists of the suppression of DNA end resection coupled to a multi-step block to BRCA2 recruitment to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce HR in G1 cells with defined factors could spur the development of gene targeting applications in non-dividing cells. PMID:26649820

  6. A Heritable Recombination system for synthetic Darwinian evolution in yeast.

    PubMed

    Romanini, Dante W; Peralta-Yahya, Pamela; Mondol, Vanessa; Cornish, Virginia W

    2012-12-21

    Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology.

  7. Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae.

    PubMed

    Spugnesi, Laura; Balia, Cristina; Collavoli, Anita; Falaschi, Elisabetta; Quercioli, Valentina; Caligo, Maria Adelaide; Galli, Alvaro

    2013-03-01

    The tumour-suppressor gene BRCA2 has been demonstrated to be involved in maintenance of genome integrity by affecting DNA double-strand break repair and homologous recombination. Protein-truncating mutations in BRCA2 predispose women to early onset breast and ovarian cancers and account for 15-30% of familial breast cancer risk. In contrast, the human cancer risk due to missense mutations, intronic variants, and in-frame deletions and insertions in the BRCA2 gene, called unclassified variants, has not been determined. Here, we want to define if the yeast Saccharomyces cerevisiae is a good model to study the role of BRCA2 in DNA recombination and repair and to characterise the unclassified BRCA2 missense variants. Therefore, we expressed the wild-type BRCA2 in yeast and determined the effect of BRCA2 on yeast homologous recombination, methyl methanesulphonate (MMS)-induced Rad51 and Rad52 foci and MMS sensitivity. The expression of BRCA2 induces a high increase in both intra- and inter-recombination events and confers a higher MMS resistance as compared with the negative control. This may suggest that BRCA2 gets involved in DNA repair pathways in yeast. Moreover, the expression of BRCA2 did not affect the number of cells carrying Rad51 or Rad52 nuclear foci. Finally, we aimed to investigate if yeast could be reliable system to set up a functional assay to distinguish a mutated protein from a neutral polymorphism. Therefore, we have expressed two neutral (M1915T and A2951T) and one pathogenic variant (G2748D) in yeast and checked the effect on recombination. The neutral M1915T variant increased intra-chromosomal recombination by almost 2-fold and the other neutral A2975T variant increased intra-chromosomal recombination 2.5-fold as compared with the control. On the other end, the pathogenic variant G2748D did not increase intra- and inter-chromosomal recombination in yeast and, consequently, confers a phenotype very different from the wild-type BRCA2. Moreover, we

  8. 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.

  9. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    PubMed

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  10. A highly efficient site-specific integration strategy using combination of homologous recombination and the ΦC31 integrase.

    PubMed

    Ou, Hailong; Huang, Ying; Ma, Qingwen; Ren, Zhaorui; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2013-09-20

    The introduction of double-strand breaks (DSBs) at target sites could greatly enhance homologous recombination, and engineered nucleases, such as zinc finger and transcription activator-like effector nucleases, have been successfully developed for making such breaks. In this study, we present a highly efficient site-specific integration strategy based on homologous recombination and ΦC31 integrase. An attB sequence was introduced at the homologous arm of an insertion targeting vector. DSBs at the target locus and donor were then simultaneously generated by the ΦC31 integrase when co-transfected with the donor vector, consequently stimulating homologous recombination. The results demonstrated that our strategy is feasible and the efficiency at the BF4 target site, which we previously identified in the bovine genome, was as high as 93%. The frequency at another site (BF10) was almost two-fold greater in comparison to the vector without homologous arms. This technology requires no sophisticated nuclease design efforts, and the off-target effect is reduced by ΦC31 integrase compared to the use of engineered nucleases, thereby offering a simple and safe way to effectively express a donor gene at a desired locus. This development has great potential value, especially in transgenesis or gene therapy applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Cross-talk between nucleotide excision and homologous recombination DNA repair pathways in the mechanism of action of antitumor trabectedin.

    PubMed

    Herrero, Ana B; Martín-Castellanos, Cristina; Marco, Esther; Gago, Federico; Moreno, Sergio

    2006-08-15

    Trabectedin (Yondelis) is a potent antitumor drug that has the unique characteristic of killing cells by poisoning the DNA nucleotide excision repair (NER) machinery. The basis for the NER-dependent toxicity has not yet been elucidated but it has been proposed as the major determinant for the drug's cytotoxicity. To study the in vivo mode of action of trabectedin and to explore the role of NER in its cytotoxicity, we used the fission yeast Schizosaccharomyces pombe as a model system. Treatment of S. pombe wild-type cells with trabectedin led to cell cycle delay and activation of the DNA damage checkpoint, indicating that the drug causes DNA damage in vivo. DNA damage induced by the drug is mostly caused by the NER protein, Rad13 (the fission yeast orthologue to human XPG), and is mainly repaired by homologous recombination. By constructing different rad13 mutants, we show that the DNA damage induced by trabectedin depends on a 46-amino acid region of Rad13 that is homologous to a DNA-binding region of human nuclease FEN-1. More specifically, an arginine residue in Rad13 (Arg961), conserved in FEN1 (Arg314), was found to be crucial for the drug's cytotoxicity. These results lead us to propose a model for the action of trabectedin in eukaryotic cells in which the formation of a Rad13/DNA-trabectedin ternary complex, stabilized by Arg961, results in cell death.

  12. Extended recombinant bacterial ghost system.

    PubMed

    Lubitz, W; Witte, A; Eko, F O; Kamal, M; Jechlinger, W; Brand, E; Marchart, J; Haidinger, W; Huter, V; Felnerova, D; Stralis-Alves, N; Lechleitner, S; Melzer, H; Szostak, M P; Resch, S; Mader, H; Kuen, B; Mayr, B; Mayrhofer, P; Geretschläger, R; Haslberger, A; Hensel, A

    1999-08-20

    Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri

  13. Production systems for recombinant antibodies.

    PubMed

    Schirrmann, Thomas; Al-Halabi, Laila; Dübel, Stefan; Hust, Michael

    2008-05-01

    Recombinant antibodies are the fastest growing class of therapeutic proteins. Furthermore, antibodies are key detection reagents in research and diagnostics. The increasing demand for antibodies with regards to amount and quality resulted in the development of a variety of recombinant production systems employing gram-negative and gram-positive bacteria, yeast and filamentous fungi, insect cell lines as well as mammalian cell lines. More recently, antibodies were also successfully produced in transgenic plants and animals. Currently, the production of recombinant antibodies for therapy is performed in mammalian cell lines to reduce the risk of immunogenicity caused by non-human post-translational modifications, in particular glycosylation. However, novel strategies already allow human-like glycosylation patterns in yeast, insect cell lines and transgenic plants. Furthermore, therapeutic strategies not requiring glycosylation of the Fc portion have been conceived, most prominently using bispecific antibodies or scFv fusion proteins, which can be produced in bacteria. Here, we review all current antibody production systems considering their advantages and limitations with respect to intended applications.

  14. Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS)

    PubMed Central

    Liu, Zhenyi; Obenauf, Anna C.; Speicher, Michael R.; Kopan, Raphael

    2009-01-01

    Manipulating the mouse genome is a widespread technology with important applications in many biological fields ranging from cancer research to developmental biology. Likewise, correlations between copy number variations (CNVs) and human diseases are emerging. We have developed the reference-query pyrosequencing (RQPS) method, which is based on quantitative pyrosequencing and uniquely designed probes containing single nucleotide variations (SNVs), to offer a simple and affordable genotyping solution capable of identifying homologous recombinants independent of the homology arm size, determining the micro-amplification status of endogenous human loci, and quantifying virus/transgene copy number in experimental or commercial species. In addition, we also present a simple pyrosequencing-based protocol that could be used for the enrichment of homologous recombinant embryonic stem (ES) cells. PMID:19797679

  15. Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA.

    PubMed

    Klymiuk, Nikolai; Fezert, Pauline; Wünsch, Annegret; Kurome, Mayuko; Kessler, Barbara; Wolf, Eckhard

    2014-05-10

    Site-specific nucleases have become powerful tools for genome editing by the introduction of end-joining-mediated mutations, but it is unclear to which extent induced double strand breaks will also facilitate homologous recombination with exogenous DNA. This question is, however, of particular importance for somatic cells, which have to be modified for the generation of large animal models, but, on the other hand, have also been described to be reluctant to recombination-based DNA repair. Here, we examined zinc-finger nucleases for their potential to introduce modifications in pig somatic cells via end-joining or recombination. We found that co-transfection with nuclease-encoding plasmids resulted in a dramatic boost of recombination with different targeting vectors, suggesting a much more prominent role of this repair pathway in somatic cells than was previously thought. Although recombination with any of the vectors even occurred on both alleles of the target gene, we found also evidence for distinct properties of the used vectors regarding their preference for mono-allelic or bi-allelic modification. Thus, we show that the combined usage of site-specific nucleases and targeting vectors does not only promote homologous recombination in somatic cells but might also resemble a promising tool for detailed examination of DNA repair pathways.

  16. Efficient detection of unpaired DNA requires a member of the rad54-like family of homologous recombination proteins.

    PubMed

    Samarajeewa, Dilini A; Sauls, Pegan A; Sharp, Kevin J; Smith, Zachary J; Xiao, Hua; Groskreutz, Katie M; Malone, Tyler L; Boone, Erin C; Edwards, Kevin A; Shiu, Patrick K T; Larson, Erik D; Hammond, Thomas M

    2014-11-01

    Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination. Copyright © 2014 by the Genetics Society of America.

  17. Ubiquitin-specific peptidase 20 regulates Rad17 stability, checkpoint kinase 1 phosphorylation and DNA repair by homologous recombination.

    PubMed

    Shanmugam, Ilanchezhian; Abbas, Mohammad; Ayoub, Farhan; Mirabal, Susan; Bsaili, Manal; Caulder, Erin K; Weinstock, David M; Tomkinson, Alan E; Hromas, Robert; Shaheen, Monte

    2014-08-15

    Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.

  18. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

    PubMed

    Cotta-de-Almeida, Vinicius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B

    2003-09-01

    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

  19. Homologous Recombination Defective Arabidopsis Mutants Exhibit Enhanced Sensitivity to Abscisic Acid

    PubMed Central

    Roy, Sujit; Das, Kali Pada

    2017-01-01

    Abscisic acid (ABA) acts as an important plant hormone in regulating various aspects of plant growth and developmental processes particularly under abiotic stress conditions. An increased ABA level in plant cells inhibits DNA replication and cell division, causing plant growth retardation. In this study, we have investigated the effects of ABA on the growth responses of some major loss-of-function mutants of DNA double-stand break (DSB) repair genes in Arabidopsis during seed germination and early stages of seedling growth for understanding the role of ABA in the induction of genome instability in plants. A comparative analysis of ABA sensitivity of wild-type Arabidopsis and the knockout mutant lines related to DSB sensors, including atatm, atatr, the non-homologous end joining (NHEJ) pathway genes, and mutants related to homologous recombination (HR) pathway genes showed relatively enhanced sensitivity of atatr and HR-related mutants to ABA treatment. The expression levels of HR-related genes were increased in wild-type Arabidopsis (Col-0) during seed germination and early stages of seedling growth. Immunoblotting experiments detected phosphorylation of histone H2AX in wild-type (Col-0) and DSB repair gene mutants after ABA treatment, indicating the activation of DNA damage response due to ABA treatment. Analyses of DSB repair kinetics using comet assay under neutral condition have revealed comparatively slower DSB repair activity in HR mutants. Overall, our results have provided comprehensive information on the possible effect of ABA on DNA repair machinery in plants and also indicated potential functional involvement of HR pathway in repairing ABA induced DNA damage in Arabidopsis. PMID:28046013

  20. 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. Copyright © 2014. Published by Elsevier B.V.

  1. 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. © 2014 UICC.

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

    PubMed Central

    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

    1996-01-01

    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

  3. 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.

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

    PubMed Central

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

    2014-01-01

    Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases1–3. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes4–8. 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 forks9. Although BRCA1 and BRCA2 affect replication fork processing10–12, direct evidence that BRCA genes regulate HR at stalled chromosomal replication forks is lacking due to a dearth of tools for studying this process. We report that the Escherichia coli Tus/Ter complex13–16 can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mammalian cells. Tus/Ter-induced HR entails processing of bidirectionally arrested forks. We find that the BRCA1 C-terminal tandem BRCT repeat and regions of BRCA1 encoded by exon 11—two BRCA1 elements implicated in tumor suppression—control Tus/Ter-induced HR. 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 restriction endonuclease-mediated chromosomal double strand break (DSB). Therefore, HR at stalled forks is regulated differently from HR at DSBs arising independently of a fork. We propose that aberrant long-tract HR at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells. PMID:24776801

  5. 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.

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  7. Generation of β-lactoglobulin-modified transgenic goats by homologous recombination.

    PubMed

    Zhu, Hongmei; Hu, Linyong; Liu, Jun; Chen, Huatao; Cui, Chenchen; Song, Yujie; Jin, Yaping; Zhang, Yong

    2016-12-01

    β-Lactoglobulin (BLG) is a dominant allergen present in the milk of goats and other ungulates, although it is not found in human breast milk. Thus, the presence of BLG restricts the consumption of goat's milk by humans. In the present study, we examined whether the disruption of the BLG gene in goats by homologous recombination (HR) reduced BLG content in goat's milk and decreased the allergic response to milk. In one approach, exon 2 of the BLG gene was efficiently targeted using HR with a BLG knockout vector. In a second approach to disrupt BLG gene expression and drive exogenous human α-lactalbumin (hLA) gene expression, two hLA knock-in constructs were used to target exons 1-4 of the BLG gene via HR, and expression of hLA was then confirmed in goat mammary epithelial cells in vitro. The recombinant clones from both approaches were then used for somatic cell nuclear transfer, generating two transgenic goats possessing a BLG knockout allele or site-specific hLA integration allele. Milk assays demonstrated a reduction in BLG levels in both the BLG knockout and hLA knock-in goats; furthermore, hLA was present in the hLA knock-in goat's milk. Allergenic analysis in mice indicated that the transgenic goat's milk was less allergenic than wild-type goat's milk. These results support the development of gene-targeted animals as an effective tool for reducing allergic reactions to milk and improving nutrition. © 2016 Federation of European Biochemical Societies.

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

    PubMed

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

    2016-07-02

    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.

  9. Ac insertion site affects the frequency of transposon-induced homologous recombination at the maize p1 locus.

    PubMed Central

    Xiao, Y L; Li, X; Peterson, T

    2000-01-01

    The maize p1 gene regulates the production of a red pigment in the kernel pericarp, cob, and other maize floral tissues. Insertions of the transposable element Ac can induce recombination between two highly homologous 5.2-kb direct repeat sequences that flank the p1 gene-coding region. Here, we tested the effects of the Ac insertion site and orientation on the induction of recombination at the p1 locus. A collection of unique p1 gene alleles was used, which carry Ac insertions at different sites in and near the p1 locus, outside of the direct repeats, within the direct repeat sequences, and between the direct repeats, in both orientations. Recombination was scored by the numbers of colorless pericarp sectors (somatic frequency) and heritable mutations (germinal frequency). In both the somatic and germinal tests, the frequency of homologous recombination is significantly higher when Ac is inserted between the direct repeats than when Ac is inserted either within or outside the repeats. In contrast, Ac orientation had no significant effect on recombination frequency. We discuss these results in terms of the possible mechanisms of transposon-induced recombination. PMID:11102391

  10. Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients.

    PubMed

    Reiter, L T; Hastings, P J; Nelis, E; De Jonghe, P; Van Broeckhoven, C; Lupski, J R

    1998-05-01

    The HNPP (hereditary neuropathy with liability to pressure palsies) deletion and CMT1A (Charcot-Marie-Tooth disease type 1A) duplication are the reciprocal products of homologous recombination events between misaligned flanking CMT1A-REP repeats on chromosome 17p11. 2-p12. A 1.7-kb hotspot for homologous recombination was previously identified wherein the relative risk of an exchange event is 50 times higher than in the surrounding 98.7% identical sequence shared by the CMT1A-REPs. To refine the region of exchange further, we designed a PCR strategy to amplify the recombinant CMT1A-REP from HNPP patients as well as the proximal and distal CMT1A-REPs from control individuals. By comparing the sequences across recombinant CMT1A-REPs to that of the proximal and distal CMT1A-REPs, the exchange was mapped to a 557-bp region within the previously identified 1.7-kb hotspot in 21 of 23 unrelated HNPP deletion patients. Two patients had recombined sequences suggesting an exchange event closer to the mariner-like element previously identified near the hotspot. Five individuals also had interspersed patches of proximal or distal repeat specific DNA sequence indicating potential gene conversion during the exchange of genetic material. Our studies provide a direct observation of human meiotic recombination products. These results are consistent with the hypothesis that minimum efficient processing segments, which have been characterized in Escherichia coli, yeast, and cultured mammalian cells, may be required for efficient homologous meiotic recombination in humans.

  11. Overexpression of OsRecQl4 and/or OsExo1 enhances DSB-induced homologous recombination in rice.

    PubMed

    Kwon, Yong-Ik; Abe, Kiyomi; Osakabe, Keishi; Endo, Masaki; Nishizawa-Yokoi, Ayako; Saika, Hiroaki; Shimada, Hiroaki; Toki, Seiichi

    2012-12-01

    During homologous recombination (HR)-mediated DNA double-strand break (DSB) repair in eukaryotes, an initial step is the creation of a 3'-single-stranded DNA (ssDNA) overhang via resection of a 5' end. Rad51 polymerizes on this ssDNA to search for a homologous sequence, and the gapped sequence is then repaired using an undamaged homologous DNA strand as template. Recent studies in eukaryotes indicate that resection of the DSB site is promoted by the cooperative action of RecQ helicase family proteins: Bloom helicase (BLM) in mammals or Sgs1 in yeast, and exonuclease 1 (Exo1). However, the role of RecQ helicase and exonuclease during the 5'-resection process of HR in plant cells has not yet been defined. Here, we demonstrate that overexpression of rice proteins OsRecQl4 (BLM counterpart) and/or OsExo1 (Exo1 homolog) can enhance DSB processing, as evaluated by recombination substrate reporter lines in rice. These results could be applied to construct an efficient gene targeting system in rice.

  12. Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) promotes non-homologous end joining and inhibits homologous recombination repair upon DNA damage.

    PubMed

    Tang, Mengfan; Li, Yujing; Zhang, Xiya; Deng, Tingting; Zhou, Zhifen; Ma, Wenbin; Songyang, Zhou

    2014-12-05

    Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) has been shown to be involved in gene silencing and DNA damage. However, the exact mechanisms of how SMCHD1 participates in DNA damage remains largely unknown. Here we present evidence that SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 led to aberrant γH2AX foci accumulation and compromised cell survival upon DNA damage, demonstrating the critical role of SMCHD1 in DNA damage repair. Following DNA damage induction, SMCHD1 depletion resulted in reduced 53BP1 foci and increased BRCA1 foci, as well as less efficient non-homologous end joining (NHEJ) and elevated levels of homologous recombination (HR). Taken together, these results suggest an important function of SMCHD1 in promoting NHEJ and repressing HR repair in response to DNA damage. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. 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

  14. Methotrexate induces DNA damage and inhibits homologous recombination repair in choriocarcinoma cells

    PubMed Central

    Xie, Lisha; Zhao, Tiancen; Cai, Jing; Su, You; Wang, Zehua; Dong, Weihong

    2016-01-01

    Objective The objective of this study was to investigate the mechanism of sensitivity to methotrexate (MTX) in human choriocarcinoma cells regarding DNA damage response. Methods Two choriocarcinoma cancer cell lines, JAR and JEG-3, were utilized in this study. An MTX-sensitive osteosarcoma cell line MG63, an MTX-resistant epithelial ovarian cancer cell line A2780 and an MTX-resistant cervical adenocarcinoma cell line Hela served as controls. Cell viability assay was carried out to assess MTX sensitivity of cell lines. MTX-induced DNA damage was evaluated by comet assay. Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of BRCA1, BRCA2, RAD51 and RAD52. The protein levels of γH2AX, RAD 51 and p53 were analyzed by Western blot. Results Remarkable DNA strand breaks were observed in MTX-sensitive cell lines (JAR, JEG-3 and MG63) but not in MTX-resistant cancer cells (A2780 and Hela) after 48 h of MTX treatment. Only in the choriocarcinoma cells, the expression of homologous recombination (HR) repair gene RAD51 was dramatically suppressed by MTX in a dose- and time-dependent manner, accompanied with the increase in p53. Conclusion The MTX-induced DNA strand breaks accompanied by deficiencies in HR repair may contribute to the hypersensitivity to chemotherapy in choriocarcinoma. PMID:27895503

  15. Phosphoglycerate mutase 1 regulates dNTP pool and promotes homologous recombination repair in cancer cells

    PubMed Central

    Qu, Jia; Sun, Wenyi; Zhong, Jie; Lv, Hao; Zhu, Mingrui; Xu, Jun; Jin, Nan; Tan, Minjia; Geng, Meiyu

    2017-01-01

    Glycolytic enzymes are known to play pivotal roles in cancer cell survival, yet their molecular mechanisms remain poorly understood. Phosphoglycerate mutase 1 (PGAM1) is an important glycolytic enzyme that coordinates glycolysis, pentose phosphate pathway, and serine biosynthesis in cancer cells. Herein, we report that PGAM1 is required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs) caused by DNA-damaging agents. Mechanistically, PGAM1 facilitates DSB end resection by regulating the stability of CTBP-interacting protein (CtIP). Knockdown of PGAM1 in cancer cells accelerates CtIP degradation through deprivation of the intracellular deoxyribonucleotide triphosphate pool and associated activation of the p53/p73 pathway. Enzymatic inhibition of PGAM1 decreases CtIP protein levels, impairs HR repair, and hence sensitizes BRCA1/2-proficient breast cancer to poly(ADP-ribose) polymerase (PARP) inhibitors. Together, this study identifies a metabolically dependent function of PGAM1 in promoting HR repair and reveals a potential therapeutic opportunity for PGAM1 inhibitors in combination with PARP inhibitors. PMID:28122957

  16. DNA replication and homologous recombination factors: acting together to maintain genome stability.

    PubMed

    Aze, Antoine; Zhou, Jin Chuan; Costa, Alessandro; Costanzo, Vincenzo

    2013-10-01

    Genome duplication requires the coordinated action of multiple proteins to ensure a fast replication with high fidelity. These factors form a complex called the Replisome, which is assembled onto the DNA duplex to promote its unwinding and to catalyze the polymerization of two new strands. Key constituents of the Replisome are the Cdc45-Mcm2-7-GINS helicase and the And1-Claspin-Tipin-Tim1 complex, which coordinate DNA unwinding with polymerase alpha-, delta-, and epsilon- dependent DNA polymerization. These factors encounter numerous obstacles, such as endogenous DNA lesions leading to template breakage and complex structures arising from intrinsic features of specific DNA sequences. To overcome these roadblocks, homologous recombination DNA repair factors, such as Rad51 and the Mre11-Rad50-Nbs1 complex, are required to ensure complete and faithful replication. Consistent with this notion, many of the genes involved in this process result in lethal phenotypes when inactivated in organisms with complex and large genomes. Here, we summarize the architectural and functional properties of the Replisome and propose a unified view of DNA replication and repair processes.

  17. Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species

    PubMed Central

    Rauwolf, U; Greiner, S; Mráček, J; Rauwolf, M; Golczyk, H; Mohler, V; Herrmann, R G; Meurer, J

    2011-01-01

    Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms—a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity. PMID:21448231

  18. Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination.

    PubMed

    Soniat, Michael M; Myler, Logan R; Schaub, Jeffrey M; Kim, Yoori; Gallardo, Ignacio F; Finkelstein, Ilya J

    2017-01-01

    Homologous recombination (HR) is a universally conserved DNA double-strand break repair pathway. Single-molecule fluorescence imaging approaches have revealed new mechanistic insights into nearly all aspects of HR. These methods are especially suited for studying protein complexes because multicolor fluorescent imaging can parse out subassemblies and transient intermediates that associate with the DNA substrates on the millisecond to hour timescales. However, acquiring single-molecule datasets remains challenging because most of these approaches are designed to measure one molecular reaction at a time. The DNA curtains platform facilitates high-throughput single-molecule imaging by organizing arrays of DNA molecules on the surface of a microfluidic flowcell. Here, we describe a second-generation UV lithography-based protocol for fabricating flowcells for DNA curtains. This protocol greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large datasets from individual single-molecule experiments. Drawing on our recent studies of human HR, we also provide an overview of how DNA curtains can be used for observing facilitated protein diffusion, processive enzyme translocation, and nucleoprotein filament dynamics on single-stranded DNA. Together, these protocols and case studies form a comprehensive introduction for other researchers that may want to adapt DNA curtains for high-throughput single-molecule studies of DNA replication, transcription, and repair. © 2017 Elsevier Inc. All rights reserved.

  19. 2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity

    PubMed Central

    Sulkowski, Parker L.; Corso, Christopher D.; Robinson, Nathaniel D.; Scanlon, Susan E.; Purshouse, Karin R.; Bai, Hanwen; Liu, Yanfeng; Sundaram, Ranjini K.; Hegan, Denise C.; Fons, Nathan R.; Breuer, Gregory A.; Song, Yuanbin; Mishra-Gorur, Ketu; De Feyter, Henk; de Graaf, Robin A.; Surovtseva, Yulia V.; Kachman, Maureen; Halene, Stephanie; Günel, Murat; Glazer, Peter M.; Bindra, Ranjit S.

    2017-01-01

    2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations, whereas the latter is produced under pathologic processes such as hypoxia. Here, we report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. This “BRCAness” phenotype of IDH mutant cells can be completely reversed by treatment with small molecule inhibitors of the mutant IDH1 enzyme, and, conversely, it can be entirely recapitulated by treatment with either 2HG enantiomer alone in cells with intact IDH1/2 proteins. We demonstrate IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR-deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability. PMID:28148839

  20. 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

  1. BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation.

    PubMed

    Isono, Mayu; Niimi, Atsuko; Oike, Takahiro; Hagiwara, Yoshihiko; Sato, Hiro; Sekine, Ryota; Yoshida, Yukari; Isobe, Shin-Ya; Obuse, Chikashi; Nishi, Ryotaro; Petricci, Elena; Nakada, Shinichiro; Nakano, Takashi; Shibata, Atsushi

    2017-01-10

    BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR.

  2. Targeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination.

    PubMed

    Martinez, Susan F; Renodon-Cornière, Axelle; Nomme, Julian; Eveillard, Damien; Fleury, Fabrice; Takahashi, Masayuki; Weigel, Pierre

    2010-12-01

    Human Rad51 (HsRad51), a key element of the homologous recombination repair pathway, is related to the resistance of cancer cells to chemo- and radio-therapies. This protein is thus a good target for the development of anti-cancer treatments. We have searched for new inhibitors directed against HsRad51 using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach. We have selected three aptamers displaying strong effects on strand exchange activity. Analysis by circular dichroism shows that they are highly structured DNA molecules. Our results also show that they affect the first step of the strand exchange reaction by promoting the dissociation of DNA from the ATP/HsRad51/DNA complex. Moreover, these inhibitors bind only weakly to RecA, a prokaryotic ortholog of HsRad51. Both the specificity and the efficiency of their inhibition of recombinase activity offer an analytical tool based on molecular recognition and the prospect of developing new therapeutic agents.

  3. Correction of sickle cell disease by homologous recombination in embryonic stem cells.

    PubMed

    Wu, Li-Chen; Sun, Chiao-Wang; Ryan, Thomas M; Pawlik, Kevin M; Ren, Jinxiang; Townes, Tim M

    2006-08-15

    Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing antisickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (beta(S)) with a normal copy of the gene (beta(A)). We recently derived embryonic stem (ES) cells from a novel knock-in mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. In this paper, we demonstrate the replacement of the human beta(S)-globin gene with a human beta(A)-globin gene and the derivation of mice from these cells. The animals produce high levels of normal human hemoglobin (HbA) and the pathology associated with SCD is corrected. Hematologic values are restored to normal levels and organ pathology is ameliorated. These experiments provide a foundation for similar studies in human ES cells derived from sickle cell patients. Although efficient methods for production of human ES cells by somatic nuclear transfer must be developed, the data in this paper demonstrate that sickle cell disease can be corrected without the risk of insertional mutagenesis.

  4. 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.

  5. BRCA1-directed, enhanced and aberrant homologous recombination: mechanism and potential treatment strategies.

    PubMed

    Dever, Seth M; White, E Railey; Hartman, Matthew C T; Valerie, Kristoffer

    2012-02-15

    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 ubiquitinligase 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.

  6. Real-time analysis of double-strand DNA break repair by homologous recombination.

    PubMed

    Hicks, Wade M; Yamaguchi, Miyuki; Haber, James E

    2011-02-22

    The ability to induce synchronously a single site-specific double-strand break (DSB) in a budding yeast chromosome has made it possible to monitor the kinetics and genetic requirements of many molecular steps during DSB repair. Special attention has been paid to the switching of mating-type genes in Saccharomyces cerevisiae, a process initiated by the HO endonuclease by cleaving the MAT locus. A DSB in MATa is repaired by homologous recombination--specifically, by gene conversion--using a heterochromatic donor, HMLα. Repair results in the replacement of the a-specific sequences (Ya) by Yα and switching from MATa to MATα. We report that MAT switching requires the DNA replication factor Dpb11, although it does not require the Cdc7-Dbf4 kinase or the Mcm and Cdc45 helicase components. Using Southern blot, PCR, and ChIP analysis of samples collected every 10 min, we extend previous studies of this process to identify the times for the loading of Rad51 recombinase protein onto the DSB ends at MAT, the subsequent strand invasion by the Rad51 nucleoprotein filament into the donor sequences, the initiation of new DNA synthesis, and the removal of the nonhomologous Y sequences. In addition we report evidence for the transient displacement of well-positioned nucleosomes in the HML donor locus during strand invasion.

  7. Berberine Radiosensitizes Human Esophageal Cancer Cells by Downregulating Homologous Recombination Repair Protein RAD51

    PubMed Central

    Liu, Zhaojian; Wang, Yu; Zhao, Minnan; Hao, Chunyan; Feng, Shuai; Guo, Haiyang; Xu, Bing; Yang, Qifeng; Gong, Yaoqin; Shao, Changshun

    2011-01-01

    Background Esophageal squamous cell carcinomas (ESCC) have poor prognosis. While combined modality of chemotherapy and radiotherapy increases survival, most patients die within five years. Development of agents that confer cancer cell-specific chemo- and radiosensitivity may improve the therapy of ESCC. We here reported the discovery of berberine as a potent radiosensitizing agent on ESCC cells. Principal Findings Berberine at low concentrations (<15 µM) substantially radiosensitized ESCC cells. X-ray induced DNA double-strand breaks (DSBs) persist longer in ESCC cells pretreated with berberine. Berberine pretreatment led to a significant downregulation of RAD51, a key player in homologous recombination repair, in ESCC cells, but not in non-malignant human cells. Downregulation of RAD51 by RNA interference similarly radiosensitized the cancer cells, and, conversely, introduction of exogenous RAD51 was able to significantly counteract the radiosensitizing effect of berberine, thus establishing RAD51 as a key determinant in radiation sensitivity. We also observed that RAD51 was commonly overexpressed in human ESCC tissues, suggesting that it is necessary to downregulate RAD51 to achieve high radio- or chemotherapeutic efficacy of ESCC in clinic, because overexpression of RAD51 is known to confer radio- and chemoresistance. Conclusions/Significance Berberine can effectively downregulate RAD51 in conferring radiosensitivity on esophageal cancer cells. Its clinical application as an adjuvant in chemotherapy and radiotherapy of esophageal cancers should be explored. PMID:21858113

  8. ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination

    PubMed Central

    Zhao, Bailin; Zhang, Dapeng; Li, Chengmin; Yuan, Zheng; Yu, Fangzhi; Zhong, Shangwei; Jiang, Guibin; Yang, Yun-Gui; Le, X Chris; Weinfeld, Michael; Zhu, Ping; Wang, Hailin

    2017-01-01

    Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites. This finding was confirmed by a nuclease protection assay and electron microscopy (EM) imaging. We further found that these RecA-unsaturated filaments promote strand exchange in vitro and HR in vivo. RecA mutants (P67D and P67E), which only form RecA-unsaturated nucleofilaments, were able to mediate HR in vitro and in vivo, but mutants favoring the formation of the saturated nucleofilaments failed to support HR. We thus present a new model for RecA-mediated HR in which RecA utilizes its intrinsic DNA binding-dependent ATPase activity to remodel the nucleofilaments to a less saturated form and thereby promote HR. PMID:28101376

  9. Triapine potentiates platinum-based combination therapy by disruption of homologous recombination repair

    PubMed Central

    Ratner, Elena S; Zhu, Yong-Lian; Penketh, Philip G; Berenblum, Julie; Whicker, Margaret E; Huang, Pamela H; Lee, Yashang; Ishiguro, Kimiko; Zhu, Rui; Sartorelli, Alan C; Lin, Z Ping

    2016-01-01

    Background: Platinum resistance may be attributable to inherent or acquired proficiency in homologous recombination repair (HRR) in epithelial ovarian cancer (EOC). The objective of this study was to evaluate the efficacy of the small molecule inhibitor triapine to disrupt HRR and sensitise BRCA wild-type EOC cells to platinum-based combination therapy in vitro and in vivo. Methods: The sensitivity of BRCA wild-type cancer cells to olaparib, cisplatin, carboplatin, doxorubicin, or etoposide in combination with triapine was evaluated by clonogenic survival assays. The effects of triapine on HRR activity in cells were measured with a DR-GFP reporter assay. The ability of triapine to enhance the effects of the carboplatin-doxil combination on EOC tumour growth delay was determined using a xenograft tumour mouse model. Results: Platinum resistance is associated with wild-type BRCA status. Triapine inhibits HRR activity and enhances the sensitivity of BRCA wild-type cancer cells to cisplatin, olaparib, and doxorubicin. However, sequential combination of triapine and cisplatin is necessary to achieve synergism. Moreover, triapine potentiates platinum-based combination therapy against BRCA wild-type EOC cells and produces significant delay of EOC tumour growth. Conclusions: Triapine promises to augment the clinical efficacy of platinum-based combination regimens for treatment of platinum-resistant EOC with wild-type BRCA and proficient HRR activity. PMID:26964031

  10. The RecU Holliday junction resolvase acts at early stages of homologous recombination

    PubMed Central

    Cañas, Cristina; Carrasco, Begoña; Ayora, Silvia; Alonso, Juan C.

    2008-01-01

    Homologous recombination is essential for DNA repair and generation of genetic diversity in all organisms. It occurs through a series of presynaptic steps where the substrate is presented to the recombinase (RecA in bacteria). Then, the recombinase nucleoprotein filament mediates synapsis by first promoting the formation of a D-loop and later of a Holliday junction (HJ) that is subsequently cleaved by the HJ resolvase. The coordination of the synaptic step with the late resolution step is poorly understood. Bacillus subtilis RecU catalyzes resolution of HJs, and biochemical evidence suggests that it might modulate RecA. We report here the isolation and characterization of two mutants of RecU (recU56 and recU71), which promote resolution of HJs, but do not promote RecA modulation. In vitro, the RecU mutant proteins (RecUK56A or RecUR71A) bind and cleave HJs and interact with RuvB. RecU interacts with RecA and inhibits its single-stranded DNA-dependent dATP hydrolysis, but RecUK56A and RecUR71A do not exert a negative effect on the RecA dATPase and fail to interact with it. Both activities are important in vivo since RecU mutants impaired only in RecA interaction are as sensitive to DNA damaging agents as a deletion mutant. PMID:18684995

  11. The RecU Holliday junction resolvase acts at early stages of homologous recombination.

    PubMed

    Cañas, Cristina; Carrasco, Begoña; Ayora, Silvia; Alonso, Juan C

    2008-09-01

    Homologous recombination is essential for DNA repair and generation of genetic diversity in all organisms. It occurs through a series of presynaptic steps where the substrate is presented to the recombinase (RecA in bacteria). Then, the recombinase nucleoprotein filament mediates synapsis by first promoting the formation of a D-loop and later of a Holliday junction (HJ) that is subsequently cleaved by the HJ resolvase. The coordination of the synaptic step with the late resolution step is poorly understood. Bacillus subtilis RecU catalyzes resolution of HJs, and biochemical evidence suggests that it might modulate RecA. We report here the isolation and characterization of two mutants of RecU (recU56 and recU71), which promote resolution of HJs, but do not promote RecA modulation. In vitro, the RecU mutant proteins (RecUK56A or RecUR71A) bind and cleave HJs and interact with RuvB. RecU interacts with RecA and inhibits its single-stranded DNA-dependent dATP hydrolysis, but RecUK56A and RecUR71A do not exert a negative effect on the RecA dATPase and fail to interact with it. Both activities are important in vivo since RecU mutants impaired only in RecA interaction are as sensitive to DNA damaging agents as a deletion mutant.

  12. Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells.

    PubMed

    Delabaere, Laetitia; Ertl, Henry A; Massey, Dashiell J; Hofley, Carolyn M; Sohail, Faraz; Bienenstock, Elisa J; Sebastian, Hans; Chiolo, Irene; LaRocque, Jeannine R

    2017-04-01

    Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  13. Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops

    PubMed Central

    Kar, Anirban; Willcox, Smaranda; Griffith, Jack D.

    2016-01-01

    The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex. The loops are very stable and some RNase H resistant TERRA remains at the t-loop, likely adding to their stability. Transcription of DNAs containing TTAGTG or TGAGTG repeats showed greatly reduced loop formation. While in the cell multiple pathways may lead to t-loop formation, the pathway revealed here does not depend on the shelterins but rather on the unique character of telomeric DNA when it is opened for transcription. Hence, telomeric sequences may have evolved to facilitate their ability to loop back on themselves. PMID:27608724

  14. 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.

  15. EXD2 promotes homologous recombination by facilitating DNA-end resection

    PubMed Central

    Baddock, Hannah T.; Deshpande, Rajashree; Gileadi, Opher; Paull, Tanya T.; McHugh, Peter J; Niedzwiedz, Wojciech

    2016-01-01

    Repair of DNA double strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A critical step in HR is MRN/CtIP-dependent end-resection that generates the 3′ single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN-complex to accelerate resection via its 3′-5′ exonuclease activity that efficiently processes dsDNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short and long-range DSB resection, and thus together with MRE11 is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair. PMID:26807646

  16. Roles of DNA helicases in the mediation and regulation of homologous recombination.

    PubMed

    Daley, James M; Niu, Hengyao; Sung, Patrick

    2013-01-01

    Homologous recombination (HR) is an evolutionarily conserved process that eliminates DNA double-strand breaks from chromosomes, repairs injured DNA replication forks, and helps orchestrate meiotic chromosome segregation. Recent studies have shown that DNA helicases play multifaceted roles in HR mediation and regulation. In particular, the S. cerevisiae Sgs1 helicase and its human ortholog BLM helicase are involved in not only the resection of the primary lesion to generate single-stranded DNA to prompt the assembly of the HR machinery, but they also function in somatic cells to suppress the formation of chromosome arm crossovers during HR. On the other hand, the S. cerevisiae Mph1 and Srs2 helicases, and their respective functional equivalents in other eukaryotes, suppress spurious HR events and favor the formation of noncrossovers via distinct mechanisms. Thus, the functional integrity of the HR process and HR outcomes are dependent upon these helicase enzymes. Since mutations in some of these helicases lead to cancer predisposition in humans and mice, studies on them have clear relevance to human health and disease.

  17. Isolation of Su(var)3-7 mutations by homologous recombination in Drosophila melanogaster.

    PubMed Central

    Seum, Carole; Pauli, Daniel; Delattre, Marion; Jaquet, Yannis; Spierer, Anne; Spierer, Pierre

    2002-01-01

    The Su(var)3-7 gene, a haplo-suppressor and triplo-enhancer of position-effect variegation (PEV), encodes a zinc finger heterochromatin-associated protein. To understand the role of this protein in heterochromatin and genomic silencing, mutations were generated by homologous recombination. The donor fragment contained a yellow(+) gene and 7.6 kb of the Su(var)3-7 gene inserted between two FRTs. The Su(var)3-7 sequence contained three stop codons flanking an I-SceI cut site located in the 5' half of the gene. Using two different screening approaches, we obtained an allelic series composed of three mutant alleles. The three mutations are dominant suppressors of PEV. One behaves as a null mutation and results in a maternal-effect recessive lethal phenotype that can be rescued by a zygotic paternal wild-type gene. A P transposon zygotically expressing a Su(var)3-7 full-length cDNA also rescues the mutant phenotype. One hypomorphic allele is viable and the pleiotropic phenotype showed by adult flies indicates that rapidly and late dividing cells seem the most affected by reduced amounts of Su(var)3-7 protein. All three mutants were characterized at the molecular level. Each expresses a portion of the Su(var)3-7 protein that is unable to enter the nucleus and bind chromatin. PMID:12136016

  18. Paclitaxel is necessary for improved survival in epithelial ovarian cancers with homologous recombination gene mutations

    PubMed Central

    Jean, Stephanie; Li, Jiaqi; Katsaros, Dionyssios; Wubbenhorst, Bradley; Maxwell, Kara N.; Fishbein, Lauren; McLane, Michael W.; Benedetto, Chiara; Canuto, Emilie Marion; Mitra, Nandita; Zhang, Lin; Nathanson, Katherine L.; Tanyi, Janos L.

    2016-01-01

    PURPOSE To investigate the impact of somatic mutations in homologous recombination (HR) genes on the chemotherapeutic response and survival of patients with epithelial ovarian cancer (EOC). EXPERIMENTAL DESIGN We performed targeted massively parallel sequencing of tumor DNA from 158 patients with EOC. We associated adjuvant chemotherapy and clinical outcome with mutations in selected genes, focusing on those encoding HR proteins. RESULTS HR mutations were found in 47 (30%) tumors. We did not detect an overall survival (OS) difference in advanced stage patients whose tumors had HR mutations compared to those without (median OS of 49.6 months (95% CI 29.9-57.7) vs. 43.3 months (95% CI 31.9-75.47), p = 0.87). However, when stratified by chemotherapy regimen, patients whose tumors had TP53 and HR mutations demonstrated a marked survival advantage when treated with platinum and paclitaxel vs. platinum +/− cyclophosphamide (median OS of 90 months (95% CI 50-NA) vs. 29.5 months (95% CI 17.7-50.5), p = 0.0005). CONCLUSIONS Previous studies demonstrating a survival advantage for EOC patients with somatic HR mutations have been conducted with almost universal use of both platinum and paclitaxel. Our study is the first to our knowledge to compare cohorts with somatic HR gene mutations treated with and without paclitaxel containing platinum regimens. The survival benefit attributed to the platinum sensitivity of HR deficient ovarian cancers may depend upon the combined use of paclitaxel. PMID:27191893

  19. Harmine suppresses homologous recombination repair and inhibits proliferation of hepatoma cells

    PubMed Central

    Zhang, Lei; Zhang, Fan; Zhang, Wenjun; Chen, Lu; Gao, Neng; Men, Yulong; Xu, Xiaojun; Jiang, Ying

    2015-01-01

    To avoid cell cycle arrest or apoptosis, rapidly proliferating cancer cells have to promote DNA double strand break (DSB) repair to fix replication stress induced DSBs. Therefore, developing drugs blocking homologous recombination (HR) and nonhomologous end joining (NHEJ) – 2 major DSB repair pathways – holds great potential for cancer therapy. Over the last few decades, much attention has been paid to explore drugs targeting DSB repair pathways for cancer therapy. Here, using 2 well-established reporters for analyzing HR and NHEJ efficiency, we found that both HR and NHEJ are elevated in hepatoma cell lines Hep3B and HuH7 compared with normal liver cell lines Chang liver and QSG-7701. Our further study found that Harmine, a natural compound, negatively regulates HR but not NHEJ by interfering Rad51 recruitment, resulting in severe cytotoxicity in hepatoma cells. Furthermore, NHEJ inhibitor Nu7441 markedly sensitizes Hep3B cells to the anti-proliferative effects of Harmine. Taken together, our study suggested that Harmine holds great promise as an oncologic drug and combination of Harmine with a NHEJ inhibitor might be an effective strategy for anti-cancer treatment. PMID:26382920

  20. Novel insights into RAD51 activity and regulation during homologous recombination and DNA replication

    PubMed Central

    Godin, Stephen K.; Sullivan, Meghan R.; Bernstein, Kara A.

    2016-01-01

    In this review we focus on new insights that challenge our understanding of homologous recombination (HR) and Rad51 regulation. Recent advances using high resolution microscopy and single molecule techniques have broadened our knowledge of Rad51 filament formation and strand invasion at double-strand break (DSB) sites and at replication forks, which are one of most physiologically relevant forms of HR from yeast to humans. Rad51 filament formation and strand invasion is regulated by many mediator proteins such as the Rad51 paralogues and the Shu complex, consisting of a Shu2/SWS1 family member and additional Rad51 paralogues. Importantly, a novel RAD-51 paralogue was discovered in C. elegans and its in vitro characterization has demonstrated a new function for the worm RAD-51 paralogues during HR. Conservation of the human RAD51 paralogues function during HR and repair of replicative damage demonstrate how the RAD51 mediators play a critical role in human health and genomic integrity. Together, these new findings provide a framework for understanding RAD51 and its mediators in DNA repair during multiple cellular contexts. PMID:27224545

  1. Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells.

    PubMed

    Sonoda, E; Sasaki, M S; Morrison, C; Yamaguchi-Iwai, Y; Takata, M; Takeda, S

    1999-07-01

    Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens. However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear. We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells. Spontaneous and mitomycin C-induced SCE levels were significantly reduced for chicken DT40 B cells lacking the key HR genes RAD51 and RAD54 but not for nonhomologous DNA end-joining (NHEJ)-defective KU70(-/-) cells. As measured by targeted integration efficiency, reconstitution of HR activity by expression of a human RAD51 transgene restored SCE levels to normal, confirming that HR is the mechanism responsible for SCE. Our findings show that HR uses the nascent sister chromatid to repair potentially lethal DNA lesions accompanying replication, which might explain the lethality or tumorigenic potential associated with defects in HR or HR-associated proteins.

  2. Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases.

    PubMed

    Sommer, Daniel; Peters, Annika; Wirtz, Tristan; Mai, Maren; Ackermann, Justus; Thabet, Yasser; Schmidt, Jürgen; Weighardt, Heike; Wunderlich, F Thomas; Degen, Joachim; Schultze, Joachim L; Beyer, Marc

    2014-01-01

    Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.

  3. Frequency of intrachromosomal homologous recombination induced by UV radiation in normally repairing and excision repair-deficient human cells

    SciTech Connect

    Tsujimura, T.; Maher, V.M.; McCormick, J.J. ); Godwin, A.R.; Liskay, R.M. )

    1990-02-01

    To investigate the role of DNA damage and nucleotide excision repair in intrachromosomal homologous recombination, a plasmid containing duplicated copies of the gene coding for hygromycin resistance was introduced into the genome of a repair-proficient human cell line, KMST-6, and two repair-deficient lines, XP2OS(SV) from xeroderma pigmentosum complementation group A and XP2YO(SV) from complementation group F. Neither hygromycin-resistance gene codes for a functional enzyme because each contains an insertion/deletion mutation at a unique site, but recombination between the two defective genes can yield hygromycin-resistant cells. The rates of spontaneous recombination in normal and xeroderma pigmentosum cell strains containing the recombination substrate were found to be similar. The frequency of UV-induced recombination was determined for three of these cell strains. At low doses, the group A cell strain and the group F cell strain showed a significant increase in frequency of recombinants. The repair-proficient cell strain required 10-to 20-fold higher doses of UV to exhibit comparable increases in frequency of recombinants. These results suggest that unexcised DNA damage, rather than the excision repair process per se, stimulates such recombination.

  4. Origins of sequence selectivity in homologous genetic recombination: insights from rapid kinetic probing of RecA-mediated DNA strand exchange.

    PubMed

    Lee, Andrew M; Xiao, Jie; Singleton, Scott F

    2006-07-07

    Despite intense effort over the past 30 years, the molecular determinants of sequence selectivity in RecA-mediated homologous recombination have remained elusive. Here, we describe when and how sequence homology is recognized between DNA strands during recombination in the context of a kinetic model for RecA-mediated DNA strand exchange. We characterized the transient intermediates of the reaction using pre-steady-state kinetic analysis of strand exchange using oligonucleotide substrates containing a single fluorescent G analog. We observed that the reaction system was sensitive to heterology between the DNA substrates; however, such a "heterology effect" was not manifest when functional groups were added to or removed from the edges of the base-pairs facing the minor groove of the substrate duplex. Hence, RecA-mediated recombination must occur without the involvement of a triple helix, even as a transient intermediate in the process. The fastest detectable reaction phase was accelerated when the structure or stability of the substrate duplex was perturbed by internal mismatches or the replacement of G.C by I.C base-pairs. These findings indicate that the sequence specificity in recombination is achieved by Watson-Crick pairing in the context of base-pair dynamics inherent to the extended DNA structure bound by RecA during strand exchange.

  5. 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

  6. Base pair switching by interconversion of sugar puckers in DNA extended by proteins of RecA-family: A model for homology search in homologous genetic recombination

    PubMed Central

    Nishinaka, Taro; Shinohara, Akira; Ito, Yutaka; Yokoyama, Shigeyuki; Shibata, Takehiko

    1998-01-01

    Escherichia coli RecA is a representative of proteins from the RecA family, which promote homologous pairing and strand exchange between double-stranded DNA and single-stranded DNA. These reactions are essential for homologous genetic recombination in various organisms. From NMR studies, we previously reported a novel deoxyribose-base stacking interaction between adjacent residues on the extended single-stranded DNA bound to RecA protein. In this study, we found that the same DNA structure was induced by the binding to Saccharomyces cerevisiae Rad51 protein, indicating that the unique DNA structure induced by the binding to RecA-homologs was conserved from prokaryotes to eukaryotes. On the basis of this structure, we have formulated the structure of duplex DNA within filaments formed by RecA protein and its homologs. Two types of molecular structures are presented. One is the duplex structure that has the N-type sugar pucker. Its helical pitch is ≈95 Å (18.6 bp/turn), corresponding to that of an active, or ATP-form of the RecA filament. The other is one that has the S-type sugar pucker. Its helical pitch is ≈64 Å (12.5 bp/turn), corresponding to that of an inactive, or ADP-form of the RecA filament. During this modeling, we found that the interconversion of sugar puckers between the N-type and the S-type rotates bases horizontally, while maintaining the deoxyribose-base stacking interaction. We propose that this base rotation enables base pair switching between double-stranded DNA and single-stranded DNA to take place, facilitating homologous pairing and strand exchange. A possible mechanism for strand exchange involving DNA rotation also is discussed. PMID:9736691

  7. Xer Site Specific Recombination: Double and Single Recombinase Systems

    PubMed Central

    Castillo, Fabio; Benmohamed, Amal; Szatmari, George

    2017-01-01

    The separation and segregation of newly replicated bacterial chromosomes can be constrained by the formation of circular chromosome dimers caused by crossing over during homologous recombination events. In Escherichia coli and most bacteria, dimers are resolved to monomers by site-specific recombination, a process performed by two Chromosomally Encoded tyrosine Recombinases (XerC and XerD). XerCD recombinases act at a 28 bp recombination site dif, which is located at the replication terminus region of the chromosome. The septal protein FtsK controls the initiation of the dimer resolution reaction, so that recombination occurs at the right time (immediately prior to cell division) and at the right place (cell division septum). XerCD and FtsK have been detected in nearly all sequenced eubacterial genomes including Proteobacteria, Archaea, and Firmicutes. However, in Streptococci and Lactococci, an alternative system has been found, composed of a single recombinase (XerS) genetically linked to an atypical 31 bp recombination site (difSL). A similar recombination system has also been found in 𝜀-proteobacteria such as Campylobacter and Helicobacter, where a single recombinase (XerH) acts at a resolution site called difH. Most Archaea contain a recombinase called XerA that acts on a highly conserved 28 bp sequence dif, which appears to act independently of FtsK. Additionally, several mobile elements have been found to exploit the dif/Xer system to integrate their genomes into the host chromosome in Vibrio cholerae, Neisseria gonorrhoeae, and Enterobacter cloacae. This review highlights the versatility of dif/Xer recombinase systems in prokaryotes and summarizes our current understanding of homologs of dif/Xer machineries. PMID:28373867

  8. [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.

  9. (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.

  10. Signs of Neutralization in a Redundant Gene Involved in Homologous Recombination in Wolbachia Endosymbionts

    PubMed Central

    Badawi, Myriam; Giraud, Isabelle; Vavre, Fabrice; Grève, Pierre; Cordaux, Richard

    2014-01-01

    Genomic reduction in bacterial endosymbionts occurs through large genomic deletions and long-term accumulation of mutations. The latter process involves successive steps including gene neutralization, pseudogenization, and gradual erosion until complete loss. Although many examples of pseudogenes at various levels of degradation have been reported, neutralization cases are scarce because of the transient nature of the process. Gene neutralization may occur due to relaxation of selection in nonessential genes, for example, those involved in redundant functions. Here, we report an example of gene neutralization in the homologous recombination (HR) pathway of Wolbachia, a bacterial endosymbiont of arthropods and nematodes. The HR pathway is often depleted in endosymbiont genomes, but it is apparently intact in some Wolbachia strains. Analysis of 12 major HR genes showed that they have been globally under strong purifying selection during the evolution of Wolbachia strains hosted by arthropods, supporting the evolutionary importance of the HR pathway for these Wolbachia genomes. However, we detected signs of recent neutralization of the ruvA gene in a subset of Wolbachia strains, which might be related to an ancestral, clade-specific amino acid change that impaired DNA-binding activity. Strikingly, RuvA is part of the RuvAB complex involved in branch migration, whose function overlaps with the RecG helicase. Although ruvA is experiencing neutralization, recG is under strong purifying selection. Thus, our high phylogenetic resolution suggests that we identified a rare example of targeted neutralization of a gene involved in a redundant function in an endosymbiont genome. PMID:25230723

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

    PubMed

    Beck, Carole; Boehler, Christian; 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

    2014-05-01

    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. © The Author(s) 2014. Published by Oxford University Press.

  12. Sirtuin 6 (SIRT6) rescues the decline of homologous recombination repair during replicative senescence

    PubMed Central

    Mao, Zhiyong; Tian, Xiao; Van Meter, Michael; Ke, Zhonghe; Gorbunova, Vera; Seluanov, Andrei

    2012-01-01

    Genomic instability is a hallmark of aging tissues. Genomic instability may arise from the inefficient or aberrant function of DNA double-stranded break (DSB) repair. DSBs are repaired by homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). HR is a precise pathway, whereas NHEJ frequently leads to deletions or insertions at the repair site. Here, we used normal human fibroblasts with a chromosomally integrated HR reporter cassette to examine the changes in HR efficiency as cells progress to replicative senescence. We show that HR declines sharply with increasing replicative age, with an up to 38-fold decrease in efficiency in presenescent cells relative to young cells. This decline is not explained by a reduction of the number of cells in S/G2/M stage as presenescent cells are actively dividing. Expression of proteins involved in HR such as Rad51, Rad51C, Rad52, NBS1, and Sirtuin 6 (SIRT6) diminished with cellular senescence. Supplementation of Rad51, Rad51C, Rad52, and NBS1 proteins, either individually or in combination, did not rescue the senescence-related decline of HR. However, overexpression of SIRT6 in “middle-aged” and presenescent cells strongly stimulated HR repair, and this effect was dependent on mono-ADP ribosylation activity of poly(ADP-ribose) polymerase (PARP1). These results suggest that in aging cells, the precise HR pathway becomes repressed giving way to a more error-prone NHEJ pathway. These changes in the processing of DSBs may contribute to age-related genomic instability and a higher incidence of cancer with age. SIRT6 activation provides a potential therapeutic strategy to prevent the decline in genome maintenance. PMID:22753495

  13. Correlation between gene expression and mutator phenotype predicts homologous recombination deficiency and outcome in ovarian cancer.

    PubMed

    Lu, Jianping; Wu, Di; Li, Chuanxing; Zhou, Meng; Hao, Dapeng

    2014-11-01

    New strategies are needed to predict response to platinum-based chemotherapy and outcome of ovarian cancers. We hypothesized that the mutator phenotype in the cancer genome represents the overuse of alternative DNA repair mechanisms, which might be a sign of homologous recombination (HR) deficiency and can be captured by gene expression. Multidimensional data of ovarian cancer patients and breast cancer patients from The Cancer Genome Atlas (TCGA) database were used for the development and validation of a potential clinical information-independent score that correlates with HR deficiency and predicts outcome. Correlation of the score with platinum response, outcome, and BRCA mutations was assessed. The score correlated with increased genomic mutation rate in both ovarian cancer and breast cancer cases that harbored a substantial subset of HR-deficient samples. Significantly improved outcomes were observed in the high-scoring group versus the low-scoring group in the TCGA dataset and in three large gene expression microarray datasets. A strong correlation was found between the score and the likelihood of achieving complete response to chemotherapy. The score was also found to be highly robust to noises in genomic mutations. Sixty-four patients harboring BRCA mutations were successfully divided into two groups based on scores, with the high-scoring group showing significantly improved outcomes compared with wild-type cases and the low-scoring group showing no significance in all the same analyses. The score was significantly correlated with the response to platinum therapy and outcome. Evaluation of the score as a prognostic tool in ovarian cancer patients is warranted. We develop a diagnostic signature for the HR-deficiency based on a novel hypothesis. HR-deficiency score is significantly correlated to platinum therapy and outcomes. HRDS was validated by its association with OS, PFS, DFS and CR in validation datasets. Evaluation of the score as a prognostic tool in

  14. Mutant IDH1-driven cellular transformation increases RAD51-mediated homologous recombination and temozolomide resistance.

    PubMed

    Ohba, Shigeo; Mukherjee, Joydeep; See, Wendy L; Pieper, Russell O

    2014-09-01

    Isocitrate dehydrogenase 1 (IDH1) mutations occur in most lower grade glioma and not only drive gliomagenesis but are also associated with longer patient survival and improved response to temozolomide. To investigate the possible causative relationship between these events, we introduced wild-type (WT) or mutant IDH1 into immortalized, untransformed human astrocytes, then monitored transformation status and temozolomide response. Temozolomide-sensitive parental cells exhibited DNA damage (γ-H2AX foci) and a prolonged G2 cell-cycle arrest beginning three days after temozolomide (100 μmol/L, 3 hours) exposure and persisting for more than four days. The same cells transformed by expression of mutant IDH1 exhibited a comparable degree of DNA damage and cell-cycle arrest, but both events resolved significantly faster in association with increased, rather than decreased, clonogenic survival. The increases in DNA damage processing, cell-cycle progression, and clonogenicity were unique to cells transformed by mutant IDH1, and were not noted in cells transformed by WT IDH1 or an oncogenic form (V12H) of Ras. Similarly, these effects were not noted following introduction of mutant IDH1 into Ras-transformed cells or established glioma cells. They were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic or pharmacologic suppression of the HR DNA repair protein RAD51. These results show that mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and facilitate repair of temozolomide-induced DNA damage and temozolomide resistance. The results also suggest that inhibitors of HR may be a viable means to enhance temozolomide response in IDH1-mutant glioma.

  15. Signs of neutralization in a redundant gene involved in homologous recombination in Wolbachia endosymbionts.

    PubMed

    Badawi, Myriam; Giraud, Isabelle; Vavre, Fabrice; Grève, Pierre; Cordaux, Richard

    2014-09-17

    Genomic reduction in bacterial endosymbionts occurs through large genomic deletions and long-term accumulation of mutations. The latter process involves successive steps including gene neutralization, pseudogenization, and gradual erosion until complete loss. Although many examples of pseudogenes at various levels of degradation have been reported, neutralization cases are scarce because of the transient nature of the process. Gene neutralization may occur due to relaxation of selection in nonessential genes, for example, those involved in redundant functions. Here, we report an example of gene neutralization in the homologous recombination (HR) pathway of Wolbachia, a bacterial endosymbiont of arthropods and nematodes. The HR pathway is often depleted in endosymbiont genomes, but it is apparently intact in some Wolbachia strains. Analysis of 12 major HR genes showed that they have been globally under strong purifying selection during the evolution of Wolbachia strains hosted by arthropods, supporting the evolutionary importance of the HR pathway for these Wolbachia genomes. However, we detected signs of recent neutralization of the ruvA gene in a subset of Wolbachia strains, which might be related to an ancestral, clade-specific amino acid change that impaired DNA-binding activity. Strikingly, RuvA is part of the RuvAB complex involved in branch migration, whose function overlaps with the RecG helicase. Although ruvA is experiencing neutralization, recG is under strong purifying selection. Thus, our high phylogenetic resolution suggests that we identified a rare example of targeted neutralization of a gene involved in a redundant function in an endosymbiont genome.

  16. 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

  17. Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib.

    PubMed

    Wilson, Andrew J; Sarfo-Kantanka, Kofi; Barrack, Toby; Steck, Alexandra; Saskowski, Jeanette; Crispens, Marta A; Khabele, Dineo

    2016-10-01

    Homologous recombination (HR) proficient ovarian cancers, including CCNE1 (cyclin E)-amplified tumors, are resistant to poly (ADP-ribose) polymerase inhibitors (PARPi). Histone deacetylase inhibitors (HDACi) are effective in overcoming tumor resistance to DNA damaging drugs. Our goal was to determine whether panobinostat, a newly FDA-approved HDACi, can sensitize cyclin E, HR-proficient ovarian cancer cells to the PARPi olaparib. Expression levels of CCNE1 (cyclin E), BRCA1, RAD51 and E2F1 in ovarian tumors and cell lines were extracted from The Cancer Genome Atlas (TCGA) and Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). In HR-proficient ovarian cancer cell line models (OVCAR-3, OVCAR-4, SKOV-3, and UWB1.289+BRCA1 wild-type), cell growth and viability were assessed by sulforhodamine B and xenograft assays. DNA damage and repair (pH2AX and RAD51 co-localization and DRGFP reporter activity) and apoptosis (cleaved PARP and cleaved caspase-3) were assessed by immunofluorescence and Western blot assays. TCGA and CCLE data revealed positive correlations (Spearman) between cyclin E E2F1, and E2F1 gene targets related to DNA repair (BRCA1 and RAD51). Panobinostat downregulated cyclin E and HR repair pathway genes, and reduced HR efficiency in cyclin E-amplified OVCAR-3 cells. Further, panobinostat synergized with olaparib in reducing cell growth and viability in HR-proficient cells. Similar co-operative effects were observed in xenografts, and on pharmacodynamic markers of HR repair, DNA damage and apoptosis. These results provide preclinical rationale for using HDACi to reduce HR in cyclin E-overexpressing and other types of HR-proficient ovarian cancer as a means of enhancing PARPi activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Suberoylanilide hydroxamic acid (SAHA) enhances olaparib activity by targeting homologous recombination DNA repair in ovarian cancer.

    PubMed

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

    2014-06-01

    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 in 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). 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. 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. 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. Copyright © 2014. Published by Elsevier Inc.

  19. 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

  20. Panobinostat sensitizes cyclin E high, homologous recombination-proficient ovarian cancer to olaparib

    PubMed Central

    Wilson, Andrew J.; Sarfo-Kantanka, Kofi; Barrack, Toby; Steck, Alexandra; Saskowski, Jeanette; Crispens, Marta A.; Khabele, Dineo

    2016-01-01

    Objective Homologous recombination (HR) proficient ovarian cancers, including CCNE1 (cyclin E)-amplified tumors, are resistant to poly (ADP-ribose) polymerase inhibitors (PARPi). Histone deacetylase inhibitors (HDACi) are effective in overcoming tumor resistance to DNA damaging drugs. Our goal was to determine whether panobinostat, a newly FDA-approved HDACi, can sensitize cyclin E, HR-proficient ovarian cancer cells to the PARPi olaparib. Methods Expression levels of CCNE1 (cyclin E), BRCA1, RAD51 and E2F1 in ovarian tumors and cell lines were extracted from The Cancer Genome Atlas (TCGA) and Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). In HR-proficient ovarian cancer cell line models (OVCAR-3, OVCAR-4, SKOV-3, and UWB1.289 + BRCA1 wild-type), cell growth and viability were assessed by sulforhodamine B and xenograft assays. DNA damage and repair (pH2AX and RAD51 co-localization and DRGFP reporter activity) and apoptosis (cleaved PARP and cleaved caspase-3) were assessed by immunofluorescence and Western blot assays. Results TCGA and CCLE data revealed positive correlations (Spearman) between cyclin E E2F1, and E2F1 gene targets related to DNA repair (BRCA1 and RAD51). Panobinostat downregulated cyclin E and HR repair pathway genes, and reduced HR efficiency in cyclin E-amplified OVCAR-3 cells. Further, panobinostat synergized with olaparib in reducing cell growth and viability in HR-proficient cells. Similar co-operative effects were observed in xenografts, and on pharmacodynamic markers of HR repair, DNA damage and apoptosis. Conclusions These results provide preclinical rationale for using HDACi to reduce HR in cyclin E-overexpressing and other types of HR-proficient ovarian cancer as a means of enhancing PARPi activity. PMID:27444036

  1. 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

  2. Poly(ADP-ribose) polymerase is hyperactivated in homologous recombination-defective cells.

    PubMed

    Gottipati, Ponnari; Vischioni, Barbara; Schultz, Niklas; Solomons, Joyce; Bryant, Helen E; Djureinovic, Tatjana; Issaeva, Natalia; Sleeth, Kate; Sharma, Ricky A; Helleday, Thomas

    2010-07-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers.

  3. Susceptibility of proliferating cells to benzo[a]pyrene-induced homologous recombination in mice.

    PubMed

    Bishop, A J; Kosaras, B; Carls, N; Sidman, R L; Schiestl, R H

    2001-04-01

    The pink-eyed unstable mutation, p(un), is the result of a 70 kb tandem duplication within the murine pink-eyed, p, gene. Deletion of one copy of the duplicated region by homologous deletion/recombination occurs spontaneously in embryos and results in pigmented spots in the fur and eye. Such deletion events are inducible by a variety of DNA damaging agents, as we have observed previously with both fur- and eye-spot assays. Here we describe a study of the effect of exposure to benzo[a]pyrene (B[a]P) at different times of development on reversion induction in the eye. Previously we, among others, have reported that the retinal pigment epithelium (RPE) displays a position effect variegation phenotype in the pattern of pink-eyed unstable reversions. Following an acute exposure to B[a]P or X-rays on the tenth day of gestation an increased frequency of reversion events was detected in a distinct region of the adult RPE. Examining exposure at different times of eye development reveals that both B[a]P and X-rays result in an increased frequency of reversion events, though the increase was only significant following B[a]P exposure, similar to our previous report limited to exposure on the tenth day of gestation. Examination of B[a]P-exposed RPE in the present study revealed distinct regions where the induced events lie and that the positions of these regions are found at increasing distances from the optic nerve the later the time of exposure. This position effect direc