Science.gov

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. 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.

  4. [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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. [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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. Precise genome editing by homologous recombination.

    PubMed

    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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

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

    PubMed

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

    2015-11-20

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

  7. 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.

  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. 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.

  11. 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.

  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. Modulation of meiotic homologous recombination by DNA helicases.

    PubMed

    Lorenz, Alexander

    2016-12-08

    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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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,

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  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. 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

    2017-03-17

    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, 2017.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases.

    PubMed

    Shin, Jimann; Chen, Jiakun; Solnica-Krezel, Lilianna

    2014-10-01

    Custom-designed nucleases afford a powerful reverse genetic tool for direct gene disruption and genome modification in vivo. Among various applications of the nucleases, homologous recombination (HR)-mediated genome editing is particularly useful for inserting heterologous DNA fragments, such as GFP, into a specific genomic locus in a sequence-specific fashion. However, precise HR-mediated genome editing is still technically challenging in zebrafish. Here, we establish a GFP reporter system for measuring the frequency of HR events in live zebrafish embryos. By co-injecting a TALE nuclease and GFP reporter targeting constructs with homology arms of different size, we defined the length of homology arms that increases the recombination efficiency. In addition, we found that the configuration of the targeting construct can be a crucial parameter in determining the efficiency of HR-mediated genome engineering. Implementing these modifications improved the efficiency of zebrafish knock-in generation, with over 10% of the injected F0 animals transmitting gene-targeting events through their germline. We generated two HR-mediated insertion alleles of sox2 and gfap loci that express either superfolder GFP (sfGFP) or tandem dimeric Tomato (tdTomato) in a spatiotemporal pattern that mirrors the endogenous loci. This efficient strategy provides new opportunities not only to monitor expression of endogenous genes and proteins and follow specific cell types in vivo, but it also paves the way for other sophisticated genetic manipulations of the zebrafish genome.

  16. Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

    PubMed Central

    Gaurivaud, Patrice; Souza, Leonardo C. A.; Virgílio, Andrea C. D.; Mariano, Anelise G.; Palma, Renê R.; Monteiro, Patrícia B.

    2002-01-01

    Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. PMID:12200328

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

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

    SciTech Connect

    Yoshino, Masayasu; Sagai, Tomoko; Shiroishi, Toshihiko

    1996-06-01

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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  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. 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.

  2. 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.

  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 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

  5. 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.

  6. 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.

  7. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  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 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. Recombining Low Homology, Functionally Rich Regions of Bacterial Subtilisins by Combinatorial Fragment Exchange

    PubMed Central

    Jones, D. Dafydd

    2011-01-01

    Combinatorial fragment exchange was utilised to recombine key structural and functional low homology regions of bacilli subtilisins to generate new active hybrid proteases with altered substrate profiles. Up to six different regions comprising mostly of loop residues from the commercially important subtilisin Savinase were exchanged with the structurally equivalent regions of six other subtilisins. The six additional subtilisins derive from diverse origins and included thermophilic and intracellular subtilisins as well as other academically and commercially relevant subtilisins. Savinase was largely tolerant to fragment exchange; rational replacement of all six regions with 5 of 6 donating subtilisin sequences preserved activity, albeit reduced compared to Savinase. A combinatorial approach was used to generate hybrid Savinase variants in which the sequences derived from all seven subtilisins at each region were recombined to generate new region combinations. Variants with different substrate profiles and with greater apparent activity compared to Savinase and the rational fragment exchange variants were generated with the substrate profile exhibited by variants dependent on the sequence combination at each region. PMID:21915310

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. [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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  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. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. A role for the p53 tumour suppressor in regulating the balance between homologous recombination and non-homologous end joining

    PubMed Central

    Moureau, Sylvie; Luessing, Janna; Harte, Emma Christina; Voisin, Muriel

    2016-01-01

    Loss of p53, a transcription factor activated by cellular stress, is a frequent event in cancer. The role of p53 in tumour suppression is largely attributed to cell fate decisions. Here, we provide evidence supporting a novel role for p53 in the regulation of DNA double-strand break (DSB) repair pathway choice. 53BP1, another tumour suppressor, was initially identified as p53 Binding Protein 1, and has been shown to inhibit DNA end resection, thereby stimulating non-homologous end joining (NHEJ). Yet another tumour suppressor, BRCA1, reciprocally promotes end resection and homologous recombination (HR). Here, we show that in both human and mouse cells, the absence of p53 results in impaired 53BP1 focal recruitment to sites of DNA damage induced by ionizing radiation. This effect is largely independent of cell cycle phase and the extent of DNA damage. In p53-deficient cells, diminished localization of 53BP1 is accompanied by a reciprocal increase in BRCA1 recruitment to DSBs. Consistent with these findings, we demonstrate that DSB repair via NHEJ is abrogated, while repair via homology-directed repair (HDR) is stimulated. Overall, we propose that in addition to its role as an ‘effector’ protein in the DNA damage response, p53 plays a role in the regulation of DSB repair pathway choice. PMID:27655732

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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 directly reflects the previously observed developmental pattern of the RPE, namely that cells in the regions most distal from the optic nerve are proliferating most vigorously. The numbers and positions of RPE cells displaying the transformed (pigmented) phenotype strongly advocate the proposal that dividing cells are at highest risk to deletions induced by carcinogens.

  9. Genetic variations in the homologous recombination repair pathway genes modify risk of glioma.

    PubMed

    Zhang, Haishi; Liu, Yanhong; Zhou, Keke; Zhou, Chengcheng; Zhou, Renke; Cheng, Chunxia; Wei, Qingyi; Lu, Daru; Zhou, Liangfu

    2016-01-01

    Accumulative epidemiological evidence suggests that single nucleotide polymorphisms (SNPs) in genes involved in homologous recombination (HR) DNA repair pathway play an important role in glioma susceptibility. However, the effects of such SNPs on glioma risk remain unclear. We used a used a candidate pathway-based approach to elucidate the relationship between glioma risk and 12 putative functional SNPs in genes involved in the HR pathway. Genotyping was conducted on 771 histologically-confirmed glioma patients and 752 cancer-free controls from the Chinese Han population. Odds ratios (OR) were calculated both for each SNP individually and for grouped analyses, examining the effects of the numbers of adverse alleles on glioma risk, and evaluated their potential gene-gene interactions using the multifactor dimensionality reduction (MDR). In the single-locus analysis, two variants, the NBS1 rs1805794 (OR 1.42, 95% CI 1.15-1.76, P = 0.001), and RAD54L rs1048771 (OR 1.61, 95% CI 1.17-2.22, P = 0.002) were significantly associated with glioma risk. When we examined the joint effects of the risk-conferring alleles of these three SNPs, we found a significant trend indicating that the risk increases as the number of adverse alleles increase (P = 0.005). Moreover, the MDR analysis suggested a significant three-locus interaction model involving NBS1 rs1805794, MRE11 rs10831234, and ATM rs227062. These results suggested that these variants of the genes involved in the HR pathway may contribute to glioma susceptibility.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

  14. MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks.

    PubMed

    Medová, Michaela; Aebersold, Daniel M; Zimmer, Yitzhak

    2012-02-01

    Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal-Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct-repeat direct-repeat green fluorescent protein ((DR)-GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose-dependent manner HR. Using cells that express MET-mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET-dependent. Furthermore, our data also suggest that decline in HR-dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51-BRCA2 complex, which is crucial for error-free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    2006-04-21

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

  18. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    PubMed

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  19. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

    PubMed

    Ogiwara, Hideaki; Kohno, Takashi

    2012-01-01

    Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  20. Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice.

    PubMed

    Nishizawa-Yokoi, Ayako; Nonaka, Satoko; Saika, Hiroaki; Kwon, Yong-Ik; Osakabe, Keishi; Toki, Seiichi

    2012-12-01

    Evidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive. Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70, OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA. Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70, OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents. Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice.

  1. 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

  2. The requirement for recombination factors differs considerably between different pathways of homologous double-strand break repair in somatic plant cells.

    PubMed

    Roth, Nadine; Klimesch, Jacqueline; Dukowic-Schulze, Stefanie; Pacher, Michael; Mannuss, Anja; Puchta, Holger

    2012-12-01

    In recent years, multiple factors involved in DNA double-strand break (DSB) repair have been characterised in Arabidopsis thaliana. Using homologous sequences in somatic cells, DSBs are mainly repaired by two different pathways: synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA). By applying recombination substrates in which recombination is initiated by the induction of a site-specific DSB by the homing endonuclease I-SceI, we were able to characterise the involvement of different factors in both pathways. The nucleases MRE11 and COM1, both involved in DSB end processing, were not required for either SDSA or SSA in our assay system. Both SDSA and SSA were even more efficient without MRE11, in accordance with the fact that a loss of MRE11 might negatively affect the efficiency of non-homologous end joining. Loss of the classical recombinase RAD51 or its two paralogues RAD51C and XRCC3, as well as the SWI2/SNF2 remodelling factor RAD54, resulted in a drastic deficiency in SDSA but had hardly any influence on SSA, confirming that a strand exchange reaction is only required for SDSA. The helicase FANCM, which is postulated to be involved in the stabilisation of recombination intermediates, is surprisingly not only needed for SDSA but to a lesser extent also for SSA. Both SSA and SDSA were affected only weakly when the SMC6B protein, implicated in sister chromatid recombination, was absent, indicating that SSA and SDSA are in most cases intrachromatid recombination reactions.

  3. RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria.

    PubMed

    Gupta, Richa; Shuman, Stewart; Glickman, Michael S

    2015-10-01

    Mycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation of adnAB or recO individually causes partial impairment of HR, but loss of adnAB and recO in combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNA in vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis of recF and recR in mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCE This study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284-2295, 2013, http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair.

  4. Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining.

    PubMed

    Grabarz, Anastazja; Barascu, Aurélia; Guirouilh-Barbat, Josée; Lopez, Bernard S

    2012-01-01

    A DNA double strand break (DSB) is a highly toxic lesion, which can generate genetic instability and profound genome rearrangements. However, DSBs are required to generate diversity during physiological processes such as meiosis or the establishment of the immune repertoire. Thus, the precise regulation of a complex network of processes is necessary for the maintenance of genomic stability, allowing genetic diversity but protecting against genetic instability and its consequences on oncogenesis. Two main strategies are employed for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by single-stranded DNA (ssDNA) resection and requires sequence homology with an intact partner, while NHEJ requires neither resection at initiation nor a homologous partner. Thus, resection is an pivotal step at DSB repair initiation, driving the choice of the DSB repair pathway employed. However, an alternative end-joining (A-EJ) pathway, which is highly mutagenic, has recently been described; A-EJ is initiated by ssDNA resection but does not require a homologous partner. The choice of the appropriate DSB repair system, for instance according the cell cycle stage, is essential for genome stability maintenance. In this context, controlling the initial events of DSB repair is thus an essential step that may be irreversible, and the wrong decision should lead to dramatic consequences. Here, we first present the main DSB repair mechanisms and then discuss the importance of the choice of the appropriate DSB repair pathway according to the cell cycle phase. In a third section, we present the early steps of DSB repair i.e., DSB signaling, chromatin remodeling, and the regulation of ssDNA resection. In the last part, we discuss the competition between the different DSB repair mechanisms. Finally, we conclude with the importance of the fine tuning of this network for genome stability maintenance and for tumor protection in fine.

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

    PubMed

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

    2015-09-29

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

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

    PubMed Central

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

    2015-01-01

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

  7. Disruption of ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum.

    PubMed

    Gandía, Mónica; Xu, Shaomei; Font, Cristina; Marcos, Jose F

    2016-03-01

    The dominant mechanism to repair double-stranded DNA breaks in filamentous fungi is the non-homologous end joining (NHEJ) pathway, and not the homologous recombination (HR) pathway that operates in the mutation of genes by replacement of target DNA for selection cassettes. The key to improve HR frequency is the inactivation of the NHEJ pathway by eliminating components of its Ku70/80 heterodimeric complex. We have obtained ku70 mutants of Penicillium digitatum, the main citrus postharvest pathogen. The increased efficiency of HR in Δku70 strains was demonstrated by the generation of mutants in two different chitin synthase genes (PdchsII and PdchsV). P. digitatum Δku70 strains showed no differences from the parental strain in vegetative growth, asexual development or virulence to citrus fruit, when experiments were conducted at the optimal temperature of 24°C. However, growth of Δku70 strains at temperatures higher than 24°C demonstrated a detrimental effect in axenic growth and conidia production. These observations are in agreement with previous studies describing differences between ku70 mutants and their parental strains in some fungal species, and must be taken into account for future applications of the Δku approach to increase HR efficiency in fungi.

  8. Integration of a transfected gene into the genome of Babesia bovis occurs by legitimate homologous recombination mechanisms.

    PubMed

    Suarez, Carlos E; Johnson, Wendell C; Herndon, David R; Laughery, Jacob M; Davis, William C

    2015-08-01

    This study examines the patterns of gene integration of gfp-bsd upon stable transfection into the T3Bo strain of Babesia bovis using a plasmid designed to integrate homologous sequences of the parasite's two identical ef-1α A and B genes. While the transfected BboTf-149-6 cell line displayed two distinct patterns of gene integration, clonal lines derived from this strain by cell sorting contained only single gfp-bsd insertions. Whole genome sequencing of two selected clonal lines, E9 and C6, indicated two distinct patterns of gfp-bsd insertion occurring by legitimate homologous recombination mechanisms: one into the expected ef-1α orf B, and another into the ef-1α B promoter. The data suggest that expression of the ef-1α orf B is not required for development of B. bovis in cultured erythrocyte stages. Use of legitimate homologous recombination mechanisms in transfected B. bovis supports the future use of transfection methods for developing efficient gene function assignment experiments using gene knockout techniques.

  9. Transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes DNA damage and triggers homologous recombination repair in mammalian cells.

    PubMed

    Stoimenov, Ivaylo; Gottipati, Ponnari; Schultz, Niklas; Helleday, Thomas

    2011-01-10

    Transcription, replication and homologous recombination are intrinsically connected and it is well established that an increase of transcription is associated with an increase in homologous recombination. Here, we have studied how homologous recombination is affected during transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that prevents activating phosphorylations of the RNA Pol II C-terminal domain. We identify that DRB triggers an increase in homologous recombination within the hprt gene as well as increasing RAD51 foci formation in mammalian cells. Furthermore, we find that DRB-induced transcriptional stress is associated with formation of the nuclear foci of the phosphorylated form of H2AX (γH2AX). We accounted that about 72% of RAD51 foci co-localized with the observed γH2AX foci. Interestingly, we find that XRCC3 mutated, homologous recombination defective cells are hypersensitive to the toxic effect of DRB and fail to form RAD51 foci. In conclusion, we show that DRB-induced transcription inhibition is associated with the formation of a lesion that triggers RAD51-dependent homologous recombination repair, required for survival under transcriptional stress.

  10. A role for the malignant brain tumour (MBT) domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

    PubMed

    Johnson, Nicholas M; Lemmens, Bennie B L G; Tijsterman, Marcel

    2013-01-01

    Malignant brain tumour (MBT) domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR) for the repair of DNA double-strand breaks (DSBs). lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

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

    PubMed

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

    2007-07-01

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

  12. Homologous recombination is a primary pathway to repair DNA double-strand breaks generated during DNA rereplication.

    PubMed

    Truong, Lan N; Li, Yongjiang; Sun, Emily; Ang, Katrina; Hwang, Patty Yi-Hwa; Wu, Xiaohua

    2014-10-17

    Re-initiation of DNA replication at origins within a given cell cycle would result in DNA rereplication, which can lead to genome instability and tumorigenesis. DNA rereplication can be induced by loss of licensing control at cellular replication origins, or by viral protein-driven multiple rounds of replication initiation at viral origins. DNA double-strand breaks (DSBs) are generated during rereplication, but the mechanisms of how these DSBs are repaired to maintain genome stability and cell viability are poorly understood in mammalian cells. We generated novel EGFP-based DSB repair substrates, which specifically monitor the repair of rereplication-associated DSBs. We demonstrated that homologous recombination (HR) is an important mechanism to repair rereplication-associated DSBs, and sister chromatids are used as templates for such HR-mediated DSB repair. Micro-homology-mediated non-homologous end joining (MMEJ) can also be used but to a lesser extent compared to HR, whereas Ku-dependent classical non-homologous end joining (C-NHEJ) has a minimal role to repair rereplication-associated DSBs. In addition, loss of HR activity leads to severe cell death when rereplication is induced. Therefore, our studies identify HR, the most conservative repair pathway, as the primary mechanism to repair DSBs upon rereplication.

  13. Rad51 and Rad52 Are Involved in Homologous Recombination of Replicating Herpes Simplex Virus DNA

    PubMed Central

    Tang, Ka-Wei; Norberg, Peter; Holmudden, Martin; Elias, Per; Liljeqvist, Jan-Åke

    2014-01-01

    Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells. PMID:25365323

  14. Rad51 and Rad52 are involved in homologous recombination of replicating herpes simplex virus DNA.

    PubMed

    Tang, Ka-Wei; Norberg, Peter; Holmudden, Martin; Elias, Per; Liljeqvist, Jan-Åke

    2014-01-01

    Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.

  15. Temperate phages acquire DNA from defective prophages by relaxed homologous recombination: the role of Rad52-like recombinases.

    PubMed

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

    2014-03-01

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

  16. Homologous recombinational repair factors are recruited and loaded onto the viral DNA genome in Epstein-Barr virus replication compartments.

    PubMed

    Kudoh, Ayumi; Iwahori, Satoko; Sato, Yoshitaka; Nakayama, Sanae; Isomura, Hiroki; Murata, Takayuki; Tsurumi, Tatsuya

    2009-07-01

    Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

  17. Heterogeneous duplications in patients with Pelizaeus-Merzbacher disease suggest a mechanism of coupled homologous and nonhomologous recombination.

    PubMed

    Woodward, Karen J; Cundall, Maria; Sperle, Karen; Sistermans, Erik A; Ross, Mark; Howell, Gareth; Gribble, Susan M; Burford, Deborah C; Carter, Nigel P; Hobson, Donald L; Garbern, James Y; Kamholz, John; Heng, Henry; Hodes, M E; Malcolm, Sue; Hobson, Grace M

    2005-12-01

    We describe genomic structures of 59 X-chromosome segmental duplications that include the proteolipid protein 1 gene (PLP1) in patients with Pelizaeus-Merzbacher disease. We provide the first report of 13 junction sequences, which gives insight into underlying mechanisms. Although proximal breakpoints were highly variable, distal breakpoints tended to cluster around low-copy repeats (LCRs) (50% of distal breakpoints), and each duplication event appeared to be unique (100 kb to 4.6 Mb in size). Sequence analysis of the junctions revealed no large homologous regions between proximal and distal breakpoints. Most junctions had microhomology of 1-6 bases, and one had a 2-base insertion. Boundaries between single-copy and duplicated DNA were identical to the reference genomic sequence in all patients investigated. Taken together, these data suggest that the tandem duplications are formed by a coupled homologous and nonhomologous recombination mechanism. We suggest repair of a double-stranded break (DSB) by one-sided homologous strand invasion of a sister chromatid, followed by DNA synthesis and nonhomologous end joining with the other end of the break. This is in contrast to other genomic disorders that have recurrent rearrangements formed by nonallelic homologous recombination between LCRs. Interspersed repetitive elements (Alu elements, long interspersed nuclear elements, and long terminal repeats) were found at 18 of the 26 breakpoint sequences studied. No specific motif that may predispose to DSBs was revealed, but single or alternating tracts of purines and pyrimidines that may cause secondary structures were common. Analysis of the 2-Mb region susceptible to duplications identified proximal-specific repeats and distal LCRs in addition to the previously reported ones, suggesting that the unique genomic architecture may have a role in nonrecurrent rearrangements by promoting instability.

  18. Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi

    PubMed Central

    Chiurillo, Miguel A.; Moraes Barros, Roberto R.; Souza, Renata T.; Marini, Marjorie M.; Antonio, Cristiane R.; Cortez, Danielle R.; Curto, María Á.; Lorenzi, Hernán A.; Schijman, Alejandro G.; Ramirez, José L.; da Silveira, José F.

    2016-01-01

    Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families. PMID:28066363

  19. Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi.

    PubMed

    Chiurillo, Miguel A; Moraes Barros, Roberto R; Souza, Renata T; Marini, Marjorie M; Antonio, Cristiane R; Cortez, Danielle R; Curto, María Á; Lorenzi, Hernán A; Schijman, Alejandro G; Ramirez, José L; da Silveira, José F

    2016-01-01

    Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.

  20. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

    PubMed

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-11-02

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

  1. Genes encoding homologous antigens in taeniid cestode parasites: Implications for development of recombinant vaccines produced in Escherichia coli.

    PubMed

    Gauci, Charles; Lightowlers, Marshall W

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

  2. BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.

    PubMed

    Snouwaert, J N; Gowen, L C; Latour, A M; Mohn, A R; Xiao, A; DiBiase, L; Koller, B H

    1999-12-20

    BRCA1 is a nuclear phosphoprotein that has been classified as a tumor suppressor based on the fact that women carrying a mutated copy of the BRCA1 gene are at increased risk of developing breast and ovarian cancer. The association of BRCA1 with RAD51 has led to the hypothesis that BRCA1 is involved in DNA repair. We describe here the generation and analysis of murine embryonic stem (ES) cell lines in which both copies of the murine homologue of the human BRCA1 gene have been disrupted by gene targeting. We show that exogenous DNA introduced into these BRCA1 deficient cells by electroporation is randomly integrated into the genome at a significantly higher rate than in wild type ES cells. In contrast, integration of exogenous DNA by homologous recombination occurs in BRCA1 deficient cells at a significantly lower rate than in wild type controls. When BRCA1 expression is re-established at 5-10% of normal levels by introduction of a Brca1 transgene into BRCA1 deficient ES cells, the frequency of random integration is reduced to wild type levels, although the frequency of homologous recombination is not significantly improved. These results suggest that BRCA1 plays a role in determining the response of cells to double stranded DNA breaks.

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

    PubMed Central

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

    2016-01-01

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

  4. Extracellular signal-related kinase positively regulates ataxia telangiectasia mutated, homologous recombination repair, and the DNA damage response.

    PubMed

    Golding, Sarah E; Rosenberg, Elizabeth; Neill, Steven; Dent, Paul; Povirk, Lawrence F; Valerie, Kristoffer

    2007-02-01

    The accurate joining of DNA double-strand breaks by homologous recombination repair (HRR) is critical to the long-term survival of the cell. The three major mitogen-activated protein (MAP) kinase (MAPK) signaling pathways, extracellular signal-regulated kinase (ERK), p38, and c-Jun-NH(2)-kinase (JNK), regulate cell growth, survival, and apoptosis. To determine the role of MAPK signaling in HRR, we used a human in vivo I-SceI-based repair system. First, we verified that this repair platform is amenable to pharmacologic manipulation and show that the ataxia telangiectasia mutated (ATM) kinase is critical for HRR. The ATM-specific inhibitor KU-55933 compromised HRR up to 90% in growth-arrested cells, whereas this effect was less pronounced in cycling cells. Then, using well-characterized MAPK small-molecule inhibitors, we show that ERK1/2 and JNK signaling are important positive regulators of HRR in growth-arrested cells. On the other hand, inhibition of the p38 MAPK pathway generated an almost 2-fold stimulation of HRR. When ERK1/2 signaling was stimulated by oncogenic RAF-1, an approximately 2-fold increase in HRR was observed. KU-55933 partly blocked radiation-induced ERK1/2 phosphorylation, suggesting that ATM regulates ERK1/2 signaling. Furthermore, inhibition of MAP/ERK kinase (MEK)/ERK signaling resulted in severely reduced levels of phosphorylated (S1981) ATM foci but not gamma-H2AX foci, and suppressed ATM phosphorylation levels >85% throughout the cell cycle. Collectively, these results show that MAPK signaling positively and negatively regulates HRR in human cells. More specifically, ATM-dependent signaling through the RAF/MEK/ERK pathway is critical for efficient HRR and for radiation-induced ATM activation, suggestive of a regulatory feedback loop between ERK and ATM.

  5. Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells

    PubMed Central

    Hartlerode, Andrea J.; Rajendran, Anbazhagan; Manis, John P.

    2016-01-01

    A proportion of homologous recombination (HR) events in mammalian cells resolve by “long tract” gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer. PMID:27832076

  6. Independent intrachromosomal recombination events underlie the pericentric inversions of chimpanzee and gorilla chromosomes homologous to human chromosome 16

    PubMed Central

    Goidts, Violaine; Szamalek, Justyna M.; de Jong, Pieter J.; Cooper, David N.; Chuzhanova, Nadia; Hameister, Horst; Kehrer-Sawatzki, Hildegard

    2005-01-01

    Analyses of chromosomal rearrangements that have occurred during the evolution of the hominoids can reveal much about the mutational mechanisms underlying primate chromosome evolution. We characterized the breakpoints of the pericentric inversion of chimpanzee chromosome 18 (PTR XVI), which is homologous to human chromosome 16 (HSA 16). A conserved 23-kb inverted repeat composed of satellites, LINE and Alu elements was identified near the breakpoints and could have mediated the inversion by bringing the chromosomal arms into close proximity with each other, thereby facilitating intrachromosomal recombination. The exact positions of the breakpoints may then have been determined by local DNA sequence homologies between the inversion breakpoints, including a 22-base pair direct repeat. The similarly located pericentric inversion of gorilla (GGO) chromosome XVI, was studied by FISH and PCR analysis. The p- and q-arm breakpoints of the inversions in PTR XVI and GGO XVI were found to occur at slightly different locations, consistent with their independent origin. Further, FISH studies of the homologous chromosomal regions in macaque and orangutan revealed that the region represented by HSA BAC RP11-696P19, which spans the inversion breakpoint on HSA 16q11-12, was derived from the ancestral primate chromosome homologous to HSA 1. After the divergence of orangutan from the other great apes ∼12 million years ago (Mya), a duplication of the corresponding region occurred followed by its interchromosomal transposition to the ancestral chromosome 16q. Thus, the most parsimonious interpretation is that the gorilla and chimpanzee homologs exhibit similar but nonidentical derived pericentric inversions, whereas HSA 16 represents the ancestral form among hominoids. PMID:16140991

  7. Independent intrachromosomal recombination events underlie the pericentric inversions of chimpanzee and gorilla chromosomes homologous to human chromosome 16.

    PubMed

    Goidts, Violaine; Szamalek, Justyna M; de Jong, Pieter J; Cooper, David N; Chuzhanova, Nadia; Hameister, Horst; Kehrer-Sawatzki, Hildegard

    2005-09-01

    Analyses of chromosomal rearrangements that have occurred during the evolution of the hominoids can reveal much about the mutational mechanisms underlying primate chromosome evolution. We characterized the breakpoints of the pericentric inversion of chimpanzee chromosome 18 (PTR XVI), which is homologous to human chromosome 16 (HSA 16). A conserved 23-kb inverted repeat composed of satellites, LINE and Alu elements was identified near the breakpoints and could have mediated the inversion by bringing the chromosomal arms into close proximity with each other, thereby facilitating intrachromosomal recombination. The exact positions of the breakpoints may then have been determined by local DNA sequence homologies between the inversion breakpoints, including a 22-base pair direct repeat. The similarly located pericentric inversion of gorilla (GGO) chromosome XVI, was studied by FISH and PCR analysis. The p- and q-arm breakpoints of the inversions in PTR XVI and GGO XVI were found to occur at slightly different locations, consistent with their independent origin. Further, FISH studies of the homologous chromosomal regions in macaque and orangutan revealed that the region represented by HSA BAC RP11-696P19, which spans the inversion breakpoint on HSA 16q11-12, was derived from the ancestral primate chromosome homologous to HSA 1. After the divergence of orangutan from the other great apes approximately 12 million years ago (Mya), a duplication of the corresponding region occurred followed by its interchromosomal transposition to the ancestral chromosome 16q. Thus, the most parsimonious interpretation is that the gorilla and chimpanzee homologs exhibit similar but nonidentical derived pericentric inversions, whereas HSA 16 represents the ancestral form among hominoids.

  8. Multiscale analysis of nonlinear systems using computational homology

    SciTech Connect

    Konstantin Mischaikow, Rutgers University /Georgia Institute of Technology, Michael Schatz, Georgia Institute of Technology, William Kalies, Florida Atlantic University, Thomas Wanner,George Mason University

    2010-05-19

    - We extended our previous work on studying the time evolution of patterns associated with phase separation in conserved concentration fields. (6) Probabilistic Homology Validation - work on microstructure characterization is based on numerically studying the homology of certain sublevel sets of a function, whose evolution is described by deterministic or stochastic evolution equations. (7) Computational Homology and Dynamics - Topological methods can be used to rigorously describe the dynamics of nonlinear systems. We are approaching this problem from several perspectives and through a variety of systems. (8) Stress Networks in Polycrystals - we have characterized stress networks in polycrystals. This part of the project is aimed at developing homological metrics which can aid in distinguishing not only microstructures, but also derived mechanical response fields. (9) Microstructure-Controlled Drug Release - This part of the project is concerned with the development of topological metrics in the context of controlled drug delivery systems, such as drug-eluting stents. We are particularly interested in developing metrics which can be used to link the processing stage to the resulting microstructure, and ultimately to the achieved system response in terms of drug release profiles. (10) Microstructure of Fuel Cells - we have been using our computational homology software to analyze the topological structure of the void, metal and ceramic components of a Solid Oxide Fuel Cell.

  9. Multiscale analysis of nonlinear systems using computational homology

    SciTech Connect

    Konstantin Mischaikow; Michael Schatz; William Kalies; Thomas Wanner

    2010-05-24

    - We extended our previous work on studying the time evolution of patterns associated with phase separation in conserved concentration fields. (6) Probabilistic Homology Validation - work on microstructure characterization is based on numerically studying the homology of certain sublevel sets of a function, whose evolution is described by deterministic or stochastic evolution equations. (7) Computational Homology and Dynamics - Topological methods can be used to rigorously describe the dynamics of nonlinear systems. We are approaching this problem from several perspectives and through a variety of systems. (8) Stress Networks in Polycrystals - we have characterized stress networks in polycrystals. This part of the project is aimed at developing homological metrics which can aid in distinguishing not only microstructures, but also derived mechanical response fields. (9) Microstructure-Controlled Drug Release - This part of the project is concerned with the development of topological metrics in the context of controlled drug delivery systems, such as drug-eluting stents. We are particularly interested in developing metrics which can be used to link the processing stage to the resulting microstructure, and ultimately to the achieved system response in terms of drug release profiles. (10) Microstructure of Fuel Cells - we have been using our computational homology software to analyze the topological structure of the void, metal and ceramic components of a Solid Oxide Fuel Cell.

  10. Expression of Talaromyces thermophilus lipase gene in Trichoderma reesei by homologous recombination at the cbh1 locus.

    PubMed

    Zhang, Xu; Xia, Liming

    2017-03-01

    CBH1 (cellobiohydrolase) comprises the majority of secreted proteins by Trichoderma reesei. For expression of Talaromyces thermophilus lipase gene in T. reesei, a self-designed CBH1 promoter was applied to drive the lipase gene expression cassette which was bracketed by flanking sequences of cbh1 gene for homologous recombination. Protoplast and Agrobacterium-mediated plasmid transformations were performed and compared, resultantly, transformation mediated by Agrobacterium was overall proved to be more efficient. Stable integration of lipase gene into chromosomal DNA of T. reesei transformants was verified by PCR. After shaking flask fermentation, lipase activity of transformant reached 375 IU mL(-1), whereas no cellobiohydrolase activity was detected. SDS-PAGE analysis further showed an obvious protein band about 39 kDa and no CBH1 band in fermentation broth, implying lipase gene was successfully extracellularly expressed in T. reesei via homologous recombination at cbh1 locus. This study herein would benefit genetic engineering of filamentous fungi and industrial application of thermo-alkaline lipase like in paper making and detergents addition.

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

    SciTech Connect

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

    2015-08-31

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

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

    DOE PAGES

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; ...

    2015-08-31

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

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

    PubMed Central

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

    2015-01-01

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

  14. Roles of the DNA binding proteins H-NS and StpA in homologous recombination and repair of bleomycin-induced damage in Escherichia coli.

    PubMed

    Shiraishi, Kouya; Ogata, Yasuyuki; Hanada, Katsuhiro; Kano, Yasunobu; Ikeda, Hideo

    2007-10-01

    The DNA binding protein H-NS promotes homologous recombination in Escherichia coli, but the role of its paralog StpA in this process remains unclear. Here we show that an hns mutant, but not an stpA mutant, are marginally defective in conjugational recombination and is sensitive to the double-strand-break-inducing agent bleomycin. Interestingly, the hns stpA double mutant is severely defective in homologous recombination and more bleomycin-sensitive than is the hns or stpA single mutant, indicating that the stpA mutation synergistically enhances the defects of homologous recombination and the increased bleomycin-sensitivity in the hns mutant. In addition, the transduction analysis in the hns stpA double mutant indicated that the stpA mutation also enhances the defect of recombination in the hns mutant. These results suggest that H-NS plays an important role in both homologous recombination and repair of bleomycin-induced damage, while StpA can substitute the H-NS function. The recombination analysis of hns single, stpA single, and hns stpA double mutants in the recBC sbcA and recBC sbcBC backgrounds suggested that the reduction of the hns single or hns stpA double mutants may not be due to the defect in a particular recombination pathway, but may be due to the defect in a common process of the pathways. The model for the functions of H-NS and StpA in homologous recombination and double-strand break repair is discussed.

  15. Homologous overexpression of a lipase from Burkholderia cepacia using the lambda Red recombinase system.

    PubMed

    Jia, Bin; Yang, Jiang-Ke; Liu, Wen-Shan; Li, Xu; Yan, Yun-Jun

    2010-04-01

    Red recombinase system of the lambda phage is widely used for recombination of short linear DNA fragments and genome. Using this system, we obtained T7 RNA polymerase (RNAP) substitution mutants in Burkholderia cepacia. To test the expression abilities of the T7 mutants, four different lipase expression vectors were transformed and the lipase activity of these recombinants was evaluated. Our results suggest that 500 nt homology between the unit and the genome is sufficient to generate mutations and this strategy enables the rapid establishment of mutant strains with efficiencies of 85%. After expression and purification, the highest purified lipase activity obtained was 3,990 U/l, nearly triple that of the wild-type organism.

  16. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    PubMed

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  17. Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

    PubMed Central

    Chappell, William H.; Gautam, Dipendra; Ok, Suzan T.; Johnson, Bryan A.; Anacker, Daniel C.

    2015-01-01

    ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for

  18. The ATPase activity of Fml1 is essential for its roles in homologous recombination and DNA repair

    PubMed Central

    Nandi, Saikat; Whitby, Matthew C.

    2012-01-01

    In fission yeast, the DNA helicase Fml1, which is an orthologue of human FANCM, is a key component of the machinery that drives and governs homologous recombination (HR). During the repair of DNA double-strand breaks by HR, it limits the occurrence of potentially deleterious crossover recombinants, whereas at stalled replication forks, it promotes HR to aid their recovery. Here, we have mutated conserved residues in Fml1’s Walker A (K99R) and Walker B (D196N) motifs to determine whether its activities are dependent on its ability to hydrolyse ATP. Both Fml1K99R and Fml1D196N are proficient for DNA binding but totally deficient in DNA unwinding and ATP hydrolysis. In vivo both mutants exhibit a similar reduction in recombination at blocked replication forks as a fml1Δ mutant indicating that Fml1’s motor activity, fuelled by ATP hydrolysis, is essential for its pro-recombinogenic role. Intriguingly, both fml1K99R and fml1D196N mutants exhibit greater sensitivity to genotoxins and higher levels of crossing over during DSB repair than a fml1Δ strain. These data suggest that without its motor activity, the binding of Fml1 to its DNA substrate can impede alternative mechanisms of repair and crossover avoidance. PMID:22844101

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

    PubMed Central

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

    2015-01-01

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

  20. Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors.

    PubMed

    Deng, Xinzhu; Michaelson, David; Tchieu, Jason; Cheng, Jin; Rothenstein, Diana; Feldman, Regina; Lee, Sang-gyu; Fuller, John; Haimovitz-Friedman, Adriana; Studer, Lorenz; Powell, Simon; Fuks, Zvi; Hubbard, E Jane Albert; Kolesnick, Richard

    2015-01-01

    Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.

  1. Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: properties and uses.

    PubMed

    Kurosawa, Norio; Grogan, Dennis W

    2005-12-01

    In order to quantify recombination between exogenous DNA and the Sulfolobus acidocaldarius chromosome, we electroporated pyrE (uracil-auxtotrophic) recipient strains with functional pyrE sequences and counted Pyr+ transformants by direct plating. Certain culture and post-electroporation conditions increased the yield of Pyr+ recombinants from non-replicating pyrE plasmid, whereas cognate methylation of SuaI restriction sites in the plasmid decreased it. Recombination of linear DNAs with the S. acidocaldarius genome was proportional to the length of a limiting overlap, but even synthetic oligonucleotides produced reasonable numbers of recombinants with appropriate recipient strains. To investigate uses of this latter property, we electroporated an 18-bp pyrE deletion mutant with mixtures of synthetic oligonucleotides altering glycine-55 of the orotate phosphoribosyl transferase encoded by pyrE. Pyr+ transformants were recovered in which this codon was converted to each of the alternatives encoded by the oligonucleotide mixtures, thereby identifying five amino acid substitutions tolerated at this position of the thermostable enzyme.

  2. Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants.

    PubMed

    Yao, Youli; Kovalchuk, Igor

    2011-02-10

    In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.

  3. DNA annealing by Redβ is insufficient for homologous recombination and the additional requirements involve intra- and inter-molecular interactions

    PubMed Central

    Subramaniam, Sivaraman; Erler, Axel; Fu, Jun; Kranz, Andrea; Tang, Jing; Gopalswamy, Mohanraj; Ramakrishnan, Saminathan; Keller, Adrian; Grundmeier, Guido; Müller, Daniel; Sattler, Michael; Stewart, A. Francis

    2016-01-01

    Single strand annealing proteins (SSAPs) like Redβ initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redβ, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se. As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Redα-Redβ protein-protein interaction, which requires not only contacts in the C-terminal domain but also a region near the N-terminus. Because the nucleoprotein filament formed with C-terminally truncated Redβ has altered properties, the second C-terminal function could be due to an interaction required for functional filaments. Alternatively the second C-terminal function could indicate a requirement for a Redβ-host factor interaction. These data further advance the model for Red recombination and the proposition that Redβ and RAD52 SSAPs share ancestral and mechanistic roots. PMID:27708411

  4. Homologous recombination with linear DNA to insert antigenic protein in the flagellin: improvement of the Th1 immune response.

    PubMed

    Le Moigne, Vincent; Robreau, Georges; Mahana, Wahib

    2006-01-01

    Bacterial flagellin is a surface protein with numerous advantages for the presentation of exogenous peptides. However, the production of recombinant bacteria and the expression of fusion proteins is laborious and time consuming. Here, we present a simple way to produce modified bacteria. Partially deleted, non-functional, chromosomal flagellin gene (fliC ) was changed using homologous recombination by a functional linear fliC gene in which we introduced an exogenous oligonucleotide encoding for the peptide of interest. The modified fliC gene was produced by polymerase chain amplification. Linear amplicons were introduced into the non-motile E. coli by electroporation. The formation of functional flagellar filaments allowed the discrimination of motile transformants from non-motile, non-transformed cells. Thus antibiotic selection and gene expression inductors are not required since transformed bacteria can be easily isolated and used as a vector and adjuvant for immunization. To validate this hypothesis, we studied the immune response against the N-terminal peptide of Clostridium tyrobutyricum flagellin fragment. BALB/c mice were immunized either with the protein displayed as flagellin fusion protein on the surface of E. coli, with the recombinant protein in Freund's adjuvant (FA), or with the pcDNA3 vector bearing the DNA fragment encoding this protein. Immunization with the flagellin recombinant bacteria induced a strong Th1 response as measured by high level of IFN-gamma production and the lack of IL-4 production. The results indicate that the flagellar filament protein carrying a specific epitope can be a potent inducer of the Th1 cellular response.

  5. Cell biology of mitotic recombination.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2015-03-02

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.

  6. Cell Biology of Mitotic Recombination

    PubMed Central

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination. PMID:25731763

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

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

    2014-01-01

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

  9. Moonlighting at replication forks - a new life for homologous recombination proteins BRCA1, BRCA2 and RAD51.

    PubMed

    Kolinjivadi, Arun Mouli; Sannino, Vincenzo; de Antoni, Anna; Técher, Hervé; Baldi, Giorgio; Costanzo, Vincenzo

    2017-01-12

    Coordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double-strand break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA-damaging agents. Here, we examine the mechanistic aspects of HR-mediated fork protection, addressing the crosstalk between HR and replication proteins.

  10. Genetic instability is prevented by Mrc1-dependent spatio-temporal separation of replicative and repair activities of homologous recombination

    PubMed Central

    Prado, Félix

    2014-01-01

    Homologous recombination (HR) is required to protect and restart stressed replication forks. Paradoxically, the Mrc1 branch of the S phase checkpoints, which is activated by replicative stress, prevents HR repair at breaks and arrested forks. Indeed, the mechanisms underlying HR can threaten genome integrity if not properly regulated. Thus, understanding how cells avoid genetic instability associated with replicative stress, a hallmark of cancer, is still a challenge. Here I discuss recent results that support a model by which HR responds to replication stress through replicative and repair activities that operate at different stages of the cell cycle (S and G2, respectively) and in distinct subnuclear structures. Remarkably, the replication checkpoint appears to control this scenario by inhibiting the assembly of HR repair centers at stressed forks during S phase, thereby avoiding genetic instability. PMID:24615940

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

    PubMed

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

    2016-02-16

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

  12. MicroRNAs down-regulate homologous recombination in the G1 phase of cycling cells to maintain genomic stability

    PubMed Central

    Choi, Young Eun; Pan, Yunfeng; Park, Eunmi; Konstantinopoulos, Panagiotis; De, Subhajyoti; D'Andrea, Alan; Chowdhury, Dipanjan

    2014-01-01

    Homologous recombination (HR)-mediated repair of DNA double-strand break (DSB)s is restricted to the post-replicative phases of the cell cycle. Initiation of HR in the G1 phase blocks non-homologous end joining (NHEJ) impairing DSB repair. Completion of HR in G1 cells can lead to the loss-of-heterozygosity (LOH), which is potentially carcinogenic. We conducted a gain-of-function screen to identify miRNAs that regulate HR-mediated DSB repair, and of these miRNAs, miR-1255b, miR-148b*, and miR-193b* specifically suppress the HR-pathway in the G1 phase. These miRNAs target the transcripts of HR factors, BRCA1, BRCA2, and RAD51, and inhibiting miR-1255b, miR-148b*, and miR-193b* increases expression of BRCA1/BRCA2/RAD51 specifically in the G1-phase leading to impaired DSB repair. Depletion of CtIP, a BRCA1-associated DNA end resection protein, rescues this phenotype. Furthermore, deletion of miR-1255b, miR-148b*, and miR-193b* in independent cohorts of ovarian tumors correlates with significant increase in LOH events/chromosomal aberrations and BRCA1 expression. DOI: http://dx.doi.org/10.7554/eLife.02445.001 PMID:24843000

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

    PubMed Central

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

    2016-01-01

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

  14. Transcription-associated recombination is independent of XRCC2 and mechanistically separate from homology-directed DNA double-strand break repair.

    PubMed

    Savolainen, Linda; Helleday, Thomas

    2009-02-01

    It has previously been shown that transcription greatly enhances recombination in mammalian cells. However, the proteins involved in catalysing this process and the recombination pathways involved in transcription-associated recombination (TAR) are still unknown. It is well established that both the BRCA2 protein and the RAD51 paralog protein XRCC2 are required for homologous recombination. Here, we show that the BRCA2 protein is also required for TAR, while the XRCC2 protein is not involved. Expression of the XRCC2 gene in XRCC2 mutated irs1 cells restores the defect in homologous recombination repair of an I-SceI-induced DNA double-strand break, while TAR is unaffected. Interestingly, the XRCC2-deficient irs1 cells are also proficient in recombination induced at slowed replication forks, suggesting that TAR is mechanistically linked with this recombination pathway. In conclusion, we show that TAR depends on BRCA2 but is independent of XRCC2, and that this recombination pathway is separate from that used to repair a two-ended DNA double-strand break.

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

    PubMed Central

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

    2016-01-01

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

  16. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer

    PubMed Central

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-01-01

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT+/− cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT+/− rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species. PMID:26522387

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

    SciTech Connect

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

    2007-04-11

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

  18. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting.

    PubMed

    Fu, Jun; Bian, Xiaoying; Hu, Shengbaio; Wang, Hailong; Huang, Fan; Seibert, Philipp M; Plaza, Alberto; Xia, Liqiu; Müller, Rolf; Stewart, A Francis; Zhang, Youming

    2012-05-01

    Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.

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

    PubMed

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

    2014-06-01

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

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

    PubMed Central

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

    2010-01-01

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

  1. Live imaging of induced and controlled DNA double-strand break formation reveals extremely low repair by homologous recombination in human cells.

    PubMed

    Shahar, O D; Raghu Ram, E V S; Shimshoni, E; Hareli, S; Meshorer, E; Goldberg, M

    2012-07-26

    DNA double-strand breaks (DSBs), the most hazardous DNA lesions, may result in genomic instability, a hallmark of cancer cells. The main DSB repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). In mammalian cells, NHEJ, which can lead to inaccurate repair, predominates. HR repair (HRR) is considered accurate and is restricted to S, G2 and M phases of the cell cycle. Despite its importance, many aspects regarding HRR remain unknown. Here, we developed a novel inducible on/off switch cell system that enables, for the first time, to induce a DSB in a rapid and reversible manner in human cells. By limiting the duration of DSB induction, we found that non-persistent endonuclease-induced DSBs are rarely repaired by HR, whereas persistent DSBs result in the published HRR frequencies (non-significant HR frequency versus frequency of ∼10%, respectively). We demonstrate that these DSBs are repaired by an accurate repair mechanism, which is distinguished from HRR (most likely, error-free NHEJ). Notably, our data reveal that HRR frequencies of endonuclease-induced DSBs in human cells are >10-fold lower than what was previously estimated by prevailing methods, which resulted in recurrent DSB formation. Our findings suggest a role for HRR mainly in repairing challenging DSBs, in contrast to uncomplicated lesions that are frequently repaired by NHEJ. Preventing HR from repairing DSBs in the complex and repetitive human genome probably has an essential role in maintaining genomic stability.

  2. An expert system for processing sequence homology data

    SciTech Connect

    Sonnhammer, E.L.L.; Durbin, R.

    1994-12-31

    When confronted with the task of finding homology to large numbers of sequences, database searching tools such as Blast and Fasta generate prohibitively large amounts of information. An automatic way of making most of the decisions a trained sequence analyst would make was developed by means of a rule-based expert system combined with an algorithm to avoid non-informative biased residue composition matches. The results found relevant by the system are presented in a very concise and clear way, so that the homology can be assessed with minimum effort. The expert system, HSPcrunch, was implemented to process the output of the programs in the BLAST suite. HSPcrunch embodies rules on detecting distant similarities when pairs of weak matches are consistent with a larger gaped alignment, i.e. when Blast has broken a longer gaped alignment up into smaller ungaped ones. This way, more distant similarities can be detected with no or little side-effects of more spurious matches. The rules for how small the gaps must be to be considered significant have been derived empirically. Currently a set of rules are used that operate on two different scoring levels, one for very weak matches that have very small gaps and one for medium weak matches that have slightly larger gaps. This set of rules proved to be robust for most cases and gives high fidelity separation between real homologies and spurious matches, One of the most important rules for reducing the amount of output is to limit the number of overlapping matches to the same region of the query sequence. This way, a region with many high-scoring matches will not dominate the output and hide weaker but relevant matches to other regions. This is particularly valuable for multi-domain queries.

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

    PubMed

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

    2015-12-28

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-05-20

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

  6. The expression of a recombinant cry1Ac gene with subtilisin-like protease CDEP2 gene in acrystalliferous Bacillus thuringiensis by Red/ET homologous recombination.

    PubMed

    Xia, Liqiu; Zeng, Zhi; Ding, Xuezhi; Huang, Fan

    2009-10-01

    A novel cDNA encoding the subtilisin-like serine protease gene CDEP2 was isolated from Beauveria bassiana by reverse transcription polymerase chain reaction (RT-PCR). It contained an 1137 bp ORF that predicted a protein of 379 amino acids with M = 38863 Da and pI = 8.21. In an attempt to improve insecticidal activity, the CDEP2 gene and the cry1Ac gene from Bacillus thuringiensis were co-fused into the vector pHT315 as pHAc-CDEP2 plasmid by Red/ET homologous recombination. The co-fusion gene was attempted under the control of the native cry1Ac promoter. Plasmid pHAc-CDEP2 was electro-transformed into the B. thuringiensis subsp. kurstaki Cry(-)B. Analyzed by SDS-PAGE and Western blotting, the transformant Cry(-)B-pHAc-CDEP2 strain produced a 130 kDa Cry1Ac protein and 39 kDa CDEP2 protein. The 50% lethal concentration values (LC(50)) of Cry(-)B-pHAc-CDEP2 strain (8.5 microl/ml) to Helicoverpa armigera third instars larvae was clearly higher than the Cry(-)B-pHAc strain (16.7 microl/ml) at 72 h.

  7. Physiological homology between Drosophila melanogaster and vertebrate cardiovascular systems

    PubMed Central

    Choma, Michael A.; Suter, Melissa J.; Vakoc, Benjamin J.; Bouma, Brett E.; Tearney, Guillermo J.

    2011-01-01

    SUMMARY The physiology of the Drosophila melanogaster cardiovascular system remains poorly characterized compared with its vertebrate counterparts. Basic measures of physiological performance remain unknown. It also is unclear whether subtle physiological defects observed in the human cardiovascular system can be reproduced in D. melanogaster. Here we characterize the cardiovascular physiology of D. melanogaster in its pre-pupal stage by using high-speed dye angiography and optical coherence tomography. The heart has vigorous pulsatile contractions that drive intracardiac, aortic and extracellular-extravascular hemolymph flow. Several physiological measures, including weight-adjusted cardiac output, body-length-adjusted aortic velocities and intracardiac shear forces, are similar to those in the closed vertebrate cardiovascular systems, including that of humans. Extracellular-extravascular flow in the pre-pupal D. melanogaster circulation drives convection-limited fluid transport. To demonstrate homology in heart dysfunction, we showed that, at the pre-pupal stage, a troponin I mutant, held-up2 (hdp2), has impaired systolic and diastolic heart wall velocities. Impaired heart wall velocities occur in the context of a non-dilated phenotype with a mildly depressed fractional shortening. We additionally derive receiver operating characteristic curves showing that heart wall velocity is a potentially powerful discriminator of systolic heart dysfunction. Our results demonstrate physiological homology and support the use of D. melanogaster as an animal model of complex cardiovascular disease. PMID:21183476

  8. Molecular epidemiologic evidence of homologous recombination in infectious bursal disease viruses.

    PubMed

    Jackwood, Daral J

    2012-09-01

    Nucleotide and predicted amino acid sequences of the infectious bursal disease virus (IBDV) surface protein VP2 have been used to identify strains of the virus and place them into phylogenetic groups. The amino acids across the hypervariable sequence region of VP2 (hvVP2) vary, but typically variant viruses have amino acids 222T, 249K, 286I, and 318D and classic viruses have 222P, 249Q, 286T, and 318G. A molecular epidemiologic study was conducted from 2001 to 2011 in commercial chickens (Gallus gallus) from Mexico, Colombia, and Venezuela. Although many IBDVs were identified, most had the typical variant or classic amino acid sequences across the hvVP2 region. Four viruses identified in 2004, one in 2006, and 10 in 2011 from Mexico had the amino acids 222T, 249Q, 286T, and 318D. Six samples from Venezuela in 2001, one sample from Colombia in 2001, two samples from Venezuela in 2004, and one sample from Venezuela in 2005 had the amino acids 222P, 249K, 286I, and 318G. These combinations of classic and variant amino acid sequence markers had not been identified previously in any IBDV strains. The VP2 amino acid sequences in the P(BC) and P(HI) loop structures of the Venezuela and Colombia viruses were similar to most classic viruses, whereas their minor P(DE) and P(FG) loop sequences were typical of Delaware variant strains. The Mexico viruses had VP2 P(BC) loop sequences that were typical of variant IBDV strains, but their minor PDE and PFG loop structures contained amino acids that were similar but not identical to classic strains. The P(HI) loop sequences of the Mexico viruses had 318D that is typical of a Delaware variant virus, but the other amino acids in this loop structure distinguished them from all other IBDV strains. The data suggest that one or more recombination events may have occurred to create this type of sequence diversity. Because of importation regulations, immunologic studies could not be conducted in the United States to determine the

  9. Homologous recombination mediated by the mycobacterial AdnAB helicase without end resection by the AdnAB nucleases

    PubMed Central

    Gupta, Richa; Unciuleac, Mihaela-Carmen; Shuman, Stewart; Glickman, Michael S.

    2017-01-01

    Current models of bacterial homologous recombination (HR) posit that extensive resection of a DNA double-strand break (DSB) by a multisubunit helicase–nuclease machine (e.g. RecBCD, AddAB or AdnAB) generates the requisite 3′ single-strand DNA substrate for RecA-mediated strand invasion. AdnAB, the helicase–nuclease implicated in mycobacterial HR, consists of two subunits, AdnA and AdnB, each composed of an N-terminal ATPase domain and a C-terminal nuclease domain. DSB unwinding by AdnAB in vitro is stringently dependent on the ATPase activity of the ‘lead’ AdnB motor translocating on the 3′ ssDNA strand, but not on the putative ‘lagging’ AdnA ATPase. Here, we queried genetically which activities of AdnAB are pertinent to its role in HR and DNA damage repair in vivo by inactivating each of the four catalytic domains. Complete nuclease-dead AdnAB enzyme can sustain recombination in vivo, as long as its AdnB motor is intact and RecO and RecR are available. We conclude that AdnAB's processive DSB unwinding activity suffices for AdnAB function in HR. Albeit not excluding the agency of a backup nuclease, our findings suggest that mycobacterial HR can proceed via DSB unwinding and protein capture of the displaced 3′-OH single strand, without a need for extensive end-resection. PMID:27899634

  10. Multifactorial resistance of Bacillus subtilis spores to high-energy proton radiation: role of spore structural components and the homologous recombination and non-homologous end joining DNA repair pathways.

    PubMed

    Moeller, Ralf; Reitz, Günther; Li, Zuofeng; Klein, Stuart; Nicholson, Wayne L

    2012-11-01

    The space environment contains high-energy charged particles (e.g., protons, neutrons, electrons, α-particles, heavy ions) emitted by the Sun and galactic sources or trapped in the radiation belts. Protons constitute the majority (87%) of high-energy charged particles. Spores of Bacillus species are one of the model systems used for astro- and radiobiological studies. In this study, spores of different Bacillus subtilis strains were used to study the effects of high energetic proton irradiation on spore survival. Spores of the wild-type B. subtilis strain [mutants deficient in the homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways and mutants deficient in various spore structural components such as dipicolinic acid (DPA), α/β-type small, acid-soluble spore protein (SASP) formation, spore coats, pigmentation, or spore core water content] were irradiated as air-dried multilayers on spacecraft-qualified aluminum coupons with 218 MeV protons [with a linear energy transfer (LET) of 0.4 keV/μm] to various final doses up to 2500 Gy. Spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to proton radiation than wild-type spores, indicating that both HR and NHEJ DNA repair pathways are needed for spore survival. Spores lacking DPA, α/β-type SASP, or with increased core water content were also significantly more sensitive to proton radiation, whereas the resistance of spores lacking pigmentation or spore coats was essentially identical to that of the wild-type spores. Our results indicate that α/β-type SASP, core water content, and DPA play an important role in spore resistance to high-energy proton irradiation, suggesting their essential function as radioprotectants of the spore interior.

  11. Multifactorial Resistance of Bacillus subtilis Spores to High-Energy Proton Radiation: Role of Spore Structural Components and the Homologous Recombination and Non-Homologous End Joining DNA Repair Pathways

    PubMed Central

    Reitz, Günther; Li, Zuofeng; Klein, Stuart; Nicholson, Wayne L.

    2012-01-01

    Abstract The space environment contains high-energy charged particles (e.g., protons, neutrons, electrons, α-particles, heavy ions) emitted by the Sun and galactic sources or trapped in the radiation belts. Protons constitute the majority (87%) of high-energy charged particles. Spores of Bacillus species are one of the model systems used for astro- and radiobiological studies. In this study, spores of different Bacillus subtilis strains were used to study the effects of high energetic proton irradiation on spore survival. Spores of the wild-type B. subtilis strain [mutants deficient in the homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways and mutants deficient in various spore structural components such as dipicolinic acid (DPA), α/β-type small, acid-soluble spore protein (SASP) formation, spore coats, pigmentation, or spore core water content] were irradiated as air-dried multilayers on spacecraft-qualified aluminum coupons with 218 MeV protons [with a linear energy transfer (LET) of 0.4 keV/μm] to various final doses up to 2500 Gy. Spores deficient in NHEJ- and HR-mediated DNA repair were significantly more sensitive to proton radiation than wild-type spores, indicating that both HR and NHEJ DNA repair pathways are needed for spore survival. Spores lacking DPA, α/β-type SASP, or with increased core water content were also significantly more sensitive to proton radiation, whereas the resistance of spores lacking pigmentation or spore coats was essentially identical to that of the wild-type spores. Our results indicate that α/β-type SASP, core water content, and DPA play an important role in spore resistance to high-energy proton irradiation, suggesting their essential function as radioprotectants of the spore interior. Key Words: Bacillus—Spores—DNA repair—Protection—High-energy proton radiation. Astrobiology 12, 1069–1077. PMID:23088412

  12. Non-catalytic Roles For XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-20

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

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

    PubMed Central

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

    2016-01-01

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

  15. Bi-allelic alterations in DNA repair genes underpin homologous recombination DNA repair defects in breast cancer.

    PubMed

    Mutter, Robert W; Riaz, Nadeem; Ng, Charlotte K Y; Delsite, Rob; Piscuoglio, Salvatore; Edelweiss, Marcia; Martelotto, Luciano G; Sakr, Rita A; King, Tari A; Giri, Dilip D; Drobnjak, Maria; Brogi, Edi; Bindra, Ranjit; Bernheim, Giana; Lim, Raymond S; Blecua, Pedro; Desrichard, Alexis; Higginson, Dan; Towers, Russell; Jiang, Ruomu; Lee, William; Weigelt, Britta; Reis-Filho, Jorge S; Powell, Simon N

    2017-03-15

    Homologous recombination (HR) DNA repair deficient (HRD) breast cancers have been shown to be sensitive to DNA repair targeted therapies. Burgeoning evidence suggests that sporadic breast cancers, lacking germline BRCA1/BRCA2 mutations, may also be HRD. We developed a functional ex vivo RAD51-based test to identify HRD primary breast cancers. An integrated approach examining methylation, gene expression and whole-exome sequencing was employed to ascertain the etiology of HRD. Functional HRD breast cancers displayed genomic features of lack of competent HR, including large-scale state transitions and specific mutational signatures. Somatic and/or germline genetic alterations resulting in bi-allelic loss-of-function of HR genes underpinned functional HRD in 89% of cases, and were observed in only one of the 15 HR-proficient samples tested. These findings indicate the importance of a comprehensive genetic assessment of bi-allelic alterations in the HR pathway to deliver a precision medicine-based approach to select patients for therapies targeting tumor-specific DNA repair defects.

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

    PubMed

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

    2014-01-01

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

  17. TP53 mutations, tetraploidy and homologous recombination repair defects in early stage high-grade serous ovarian cancer.

    PubMed

    Chien, Jeremy; Sicotte, Hugues; Fan, Jian-Bing; Humphray, Sean; Cunningham, Julie M; Kalli, Kimberly R; Oberg, Ann L; Hart, Steven N; Li, Ying; Davila, Jaime I; Baheti, Saurabh; Wang, Chen; Dietmann, Sabine; Atkinson, Elizabeth J; Asmann, Yan W; Bell, Debra A; Ota, Takayo; Tarabishy, Yaman; Kuang, Rui; Bibikova, Marina; Cheetham, R Keira; Grocock, Russell J; Swisher, Elizabeth M; Peden, John; Bentley, David; Kocher, Jean-Pierre A; Kaufmann, Scott H; Hartmann, Lynn C; Shridhar, Viji; Goode, Ellen L

    2015-08-18

    To determine early somatic changes in high-grade serous ovarian cancer (HGSOC), we performed whole genome sequencing on a rare collection of 16 low stage HGSOCs. The majority showed extensive structural alterations (one had an ultramutated profile), exhibited high levels of p53 immunoreactivity, and harboured a TP53 mutation, deletion or inactivation. BRCA1 and BRCA2 mutations were observed in two tumors, with nine showing evidence of a homologous recombination (HR) defect. Combined Analysis with The Cancer Genome Atlas (TCGA) indicated that low and late stage HGSOCs have similar mutation and copy number profiles. We also found evidence that deleterious TP53 mutations are the earliest events, followed by deletions or loss of heterozygosity (LOH) of chromosomes carrying TP53, BRCA1 or BRCA2. Inactivation of HR appears to be an early event, as 62.5% of tumours showed a LOH pattern suggestive of HR defects. Three tumours with the highest ploidy had little genome-wide LOH, yet one of these had a homozygous somatic frame-shift BRCA2 mutation, suggesting that some carcinomas begin as tetraploid then descend into diploidy accompanied by genome-wide LOH. Lastly, we found evidence that structural variants (SV) cluster in HGSOC, but are absent in one ultramutated tumor, providing insights into the pathogenesis of low stage HGSOC.

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

    SciTech Connect

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

    2006-12-22

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

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

    PubMed

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

    2016-02-17

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

  20. The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast

    PubMed Central

    Aronica, Lucia; Kasparek, Torben; Ruchman, David; Marquez, Yamile; Cipak, Lubos; Cipakova, Ingrid; Anrather, Dorothea; Mikolaskova, Barbora; Radtke, Maximilian; Sarkar, Sovan; Pai, Chen-Chun; Blaikley, Elizabeth; Walker, Carol; Shen, Kuo-Fang; Schroeder, Renee; Barta, Andrea; Forsburg, Susan L.; Humphrey, Timothy C.

    2016-01-01

    The formation of RNA–DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability. PMID:26682798

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

    PubMed

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

    2016-02-01

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

  2. Excess Polθ functions in response to replicative stress in homologous recombination-proficient cancer cells

    PubMed Central

    de Rugy, T. Goullet; Bashkurov, M.; Datti, A.; Betous, R.; Guitton-Sert, L.; Cazaux, C.; Durocher, D.

    2016-01-01

    ABSTRACT DNA polymerase theta (Polθ) is a specialized A-family DNA polymerase that functions in processes such as translesion synthesis (TLS), DNA double-strand break repair and DNA replication timing. Overexpression of POLQ, the gene encoding Polθ, is a prognostic marker for an adverse outcome in a wide range of human cancers. While increased Polθ dosage was recently suggested to promote survival of homologous recombination (HR)-deficient cancer cells, it remains unclear whether POLQ overexpression could be also beneficial to HR-proficient cancer cells. By performing a short interfering (si)RNA screen in which genes encoding druggable proteins were knocked down in Polθ-overexpressing cells as a means to uncover genetic vulnerabilities associated with POLQ overexpression, we could not identify genes that were essential for viability in Polθ-overexpressing cells in normal growth conditions. We also showed that, upon external DNA replication stress, Polθ expression promotes cell survival and limits genetic instability. Finally, we report that POLQ expression correlates with the expression of a set of HR genes in breast, lung and colorectal cancers. Collectively, our data suggest that Polθ upregulation, besides its importance for survival of HR-deficient cancer cells, may be crucial also for HR-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes. PMID:27612511

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

    PubMed

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

    2016-02-18

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

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

    SciTech Connect

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

    2010-06-29

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

  5. Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae.

    PubMed

    Gay, Glen; Wagner, Drew T; Keatinge-Clay, Adrian T; Gay, Darren C

    2014-11-01

    The ability to rapidly customize an expression vector of choice is a valuable tool for any researcher involved in high-throughput molecular cloning for protein overexpression. Unfortunately, it is common practice to amend or neglect protein targets if the gene that encodes the protein of interest is incompatible with the multiple-cloning region of a preferred expression vector. To address this issue, a method was developed to quickly exchange the multiple-cloning region of the popular expression plasmid pET-28 with a ligation-independent cloning cassette, generating pGAY-28. This cassette contains dual inverted restriction sites that reduce false positive clones by generating a linearized plasmid incapable of self-annealing after a single restriction-enzyme digest. We also establish that progressively cooling the vector and insert leads to a significant increase in ligation-independent transformation efficiency, demonstrated by the incorporation of a 10.3 kb insert into the vector. The method reported to accomplish plasmid reconstruction is uniquely versatile yet simple, relying on the strategic placement of primers combined with homologous recombination of PCR products in yeast.

  6. Endogenous levels of Rad51 and Brca2 are required for homologous recombination and regulated by homeostatic re-balancing.

    PubMed

    Magwood, Alissa C; Malysewich, Michael J; Cealic, Iulia; Mundia, Maureen M; Knapp, Jennifer; Baker, Mark D

    2013-12-01

    Stable expression of Rad51 siRNA was used to generate mouse hybridoma cell lines in which endogenous Rad51 levels were depleted by as much as 60%. Stable Rad51 knockdowns feature reduced homologous recombination responses. The relative ease with which stable Rad51 knockdowns were recovered was surprising, given the embryonic lethality of Rad51 ablation. Interestingly, Rad51-depleted hybridoma cell lines are characterized by reduced levels of p53 protein. Completely unexpected, was the finding that Rad51-depleted hybridoma cell lines are also reduced for the breast cancer susceptibility 2 (Brca2) protein. Additionally, hybridoma cell lines that are siRNA depleted for mouse Brca2 show a corresponding reduction in Rad51 and p53 proteins. Furthermore, cellular levels of Rad51, Brca2 and p53 can be elevated in these cell lines by ectopic expression of wild-type human Rad51 and wild-type human BRCA2. In marked contrast, hybridoma cell lines that are siRNA depleted for mouse p53 feature relatively normal Rad51 and Brca2 levels. These results suggest that cellular levels of Brca2 and Rad51 are mutually dependent on each other, and that low levels of these proteins provide selective pressure for reduction of p53, which permits cell growth.

  7. PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination.

    PubMed

    Clarke, Thomas L; Sanchez-Bailon, Maria Pilar; Chiang, Kelly; Reynolds, John J; Herrero-Ruiz, Joaquin; Bandeiras, Tiago M; Matias, Pedro M; Maslen, Sarah L; Skehel, J Mark; Stewart, Grant S; Davies, Clare C

    2017-02-21

    Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization.

  8. A PALB2-interacting domain in RNF168 couples homologous recombination to DNA break-induced chromatin ubiquitylation

    PubMed Central

    Luijsterburg, Martijn S; Typas, Dimitris; Caron, Marie-Christine; Wiegant, Wouter W; van den Heuvel, Diana; Boonen, Rick A; Couturier, Anthony M; Mullenders, Leon H; Masson, Jean-Yves; van Attikum, Haico

    2017-01-01

    DNA double-strand breaks (DSB) elicit a ubiquitylation cascade that controls DNA repair pathway choice. This cascade involves the ubiquitylation of histone H2A by the RNF168 ligase and the subsequent recruitment of RIF1, which suppresses homologous recombination (HR) in G1 cells. The RIF1-dependent suppression is relieved in S/G2 cells, allowing PALB2-driven HR to occur. With the inhibitory impact of RIF1 relieved, it remains unclear how RNF168-induced ubiquitylation influences HR. Here, we uncover that RNF168 links the HR machinery to H2A ubiquitylation in S/G2 cells. We show that PALB2 indirectly recognizes histone ubiquitylation by physically associating with ubiquitin-bound RNF168. This direct interaction is mediated by the newly identified PALB2-interacting domain (PID) in RNF168 and the WD40 domain in PALB2, and drives DNA repair by facilitating the assembly of PALB2-containing HR complexes at DSBs. Our findings demonstrate that RNF168 couples PALB2-dependent HR to H2A ubiquitylation to promote DNA repair and preserve genome integrity. DOI: http://dx.doi.org/10.7554/eLife.20922.001 PMID:28240985

  9. Homologous Recombination and Translesion DNA Synthesis Play Critical Roles on Tolerating DNA Damage Caused by Trace Levels of Hexavalent Chromium

    PubMed Central

    Chen, Youjun; Zhou, Yi-Hui; Neo, Dayna; Clement, Jean; Takata, Minoru; Takeda, Shunichi; Sale, Julian; Wright, Fred A.; Swenberg, James A.; Nakamura, Jun

    2016-01-01

    Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19.2 μg/L or higher as well as in TK6 cells deficient in polymerase delta subunit 3 (POLD3) at 9.8 μg/L or higher. The genotoxicity of Cr(VI) decreased ~3000 times when the incubation time was reduced from three days to ten minutes. TK mutation rate also significantly decreased from 6 day to 1 day exposure to Cr(VI). The DNA damage response analysis suggest that DNA repair pathways, including the homologous recombination and REV1- and POLD3-mediated error-prone translesion synthesis pathways, are critical for the cells to tolerate to DNA damage caused by trace amount of Cr(VI). PMID:27907204

  10. A new subclass of intrinsic aminoglycoside nucleotidyltransferases, ANT(3")-II, is horizontally transferred among Acinetobacter spp. by homologous recombination

    PubMed Central

    Zhang, Gang; Leclercq, Sébastien Olivier; Tian, Jingjing; Wang, Chao; Ai, Guomin; Liu, Shuangjiang

    2017-01-01

    The emergence and spread of antibiotic resistance among Acinetobacter spp. have been investigated extensively. Most studies focused on the multiple antibiotic resistance genes located on plasmids or genomic resistance islands. On the other hand, the mechanisms controlling intrinsic resistance are still not well understood. In this study, we identified the novel subclass of aminoglycoside nucleotidyltransferase ANT(3")-II in Acinetobacter spp., which comprised numerous variants distributed among three main clades. All members of this subclass can inactivate streptomycin and spectinomycin. The three ant(3")-II genes, encoding for the three ANT(3")-II clades, are widely distributed in the genus Acinetobacter and always located in the same conserved genomic region. According to their prevalence, these genes are intrinsic in Acinetobacter baumannii, Acinetobacter pittii, and Acinetobacter gyllenbergii. We also demonstrated that the ant(3")-II genes are located in a homologous recombination hotspot and were recurrently transferred among Acinetobacter species. In conclusion, our findings demonstrated a novel mechanism of natural resistance in Acinetobacter spp., identified a novel subclass of aminoglycoside nucleotidyltransferase and provided new insight into the evolutionary history of intrinsic resistance genes. PMID:28152054

  11. BRCA2-dependent homologous recombination is required for repair of Arsenite-induced replication lesions in mammalian cells

    PubMed Central

    Ying, Songmin; Myers, Katie; Bottomley, Sarah; Helleday, Thomas; Bryant, Helen E.

    2009-01-01

    Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 µM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers. PMID:19553191

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

    PubMed

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

    2015-08-01

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

  13. Role for the mammalian Swi5-Sfr1 complex in DNA strand break repair through homologous recombination.

    PubMed

    Akamatsu, Yufuko; Jasin, Maria

    2010-10-14

    In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR), a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5(-/-) and Sfr1(-/-) embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability. Importantly, the Swi5-Sfr1 complex plays a role in HR when Rad51 function is perturbed in vivo by expression of a BRC peptide from BRCA2. Swi5(-/-) and Sfr1(-/-) cells are selectively sensitive to agents that cause DNA strand breaks, in particular ionizing radiation, camptothecin, and the Parp inhibitor olaparib. Consistent with a role in HR, sister chromatid exchange induced by Parp inhibition is attenuated in Swi5(-/-) and Sfr1(-/-) cells, and chromosome aberrations are increased. Thus, Swi5-Sfr1 is a newly identified complex required for genomic integrity in mammalian cells with a specific role in the repair of DNA strand breaks.

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

    PubMed Central

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

    2016-01-01

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

  15. Homologous recombination and maintenance of genome integrity: cancer and aging through the prism of human RecQ helicases.

    PubMed

    Ouyang, Karen J; Woo, Leslie L; Ellis, Nathan A

    2008-01-01

    Homologous recombination (HR) is a genetic mechanism in somatic cells that repairs DNA double-strand breaks and restores productive DNA synthesis following disruption of replication forks. Although HR is indispensable for maintaining genome integrity, it must be tightly regulated to avoid harmful outcomes. HR-associated genomic instabilities arise in three human genetic disorders, Bloom syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS), which are caused by defects in three individual proteins of the RecQ family of helicases, BLM, WRN, and RECQL4, respectively. Cells derived from persons with these syndromes display varying types of genomic instability as evidenced by the presence of different kinds of chromosomal abnormalities and different sensitivities to DNA damaging agents. Persons with these syndromes exhibit a variety of developmental defects and are predisposed to a wide range of cancers. WS and RTS are further characterized by premature aging. Recent research has shown many connections between all three proteins and the regulation of excess HR. Here, we illustrate the elaborate networks of BLM, WRN, and RECQL4 in regulating HR, and the potential mechanistic linkages to cancer and aging.

  16. The role of BRCA1 in homologous recombination repair in response to replication stress: significance in tumorigenesis and cancer therapy

    PubMed Central

    2013-01-01

    Germ line mutations in breast cancer gene 1 (BRCA1) predispose women to breast and ovarian cancers. Although BRCA1 is involved in many important biological processes, the function of BRCA1 in homologous recombination (HR) mediated repair is considered one of the major mechanisms contributing to its tumor suppression activity, and the cause of hypersensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors when BRCA1 is defective. Mounting evidence suggests that the mechanism of repairing DNA double strand breaks (DSBs) by HR is different than the mechanism operating when DNA replication is blocked. Although BRCA1 has been recognized as a central component in HR, the precise role of BRCA1 in HR, particularly under replication stress, has remained largely unknown. Given the fact that DNA lesions caused by replication blockages are the primary substrates for HR in mitotic cells, functional analysis of BRCA1 in HR repair in the context of replication stress should benefit our understanding of the molecular mechanisms underlying tumorigenesis associated with BRCA1 deficiencies, as well as the development of therapeutic approaches for cancer patients carrying BRCA1 mutations or reduced BRCA1 expression. This review focuses on the current advances in this setting and also discusses the significance in tumorigenesis and cancer therapy. PMID:23388117

  17. Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11.

    PubMed

    Nichols, M D; DeAngelis, K; Keck, J L; Berger, J M

    1999-11-01

    In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 A resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

  18. mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved.

    PubMed

    McKim, K S; Hayashi-Hagihara, A

    1998-09-15

    Meiotic recombination requires the action of several gene products in both Saccharomyces cerevisiae and Drosophila melanogaster. Genetic studies in D. melanogaster have shown that the mei-W68 gene is required for all meiotic gene conversion and crossing-over. We cloned mei-W68 using a new genetic mapping method in which P elements are used to promote crossing-over at their insertion sites. This resulted in the high-resolution mapping of mei-W68 to a <18-kb region that contains a homolog of the S. cerevisiae spo11 gene. Molecular analysis of several mutants confirmed that mei-W68 encodes an spo11 homolog. Spo11 and MEI-W68 are members of a family of proteins similar to a novel type II topoisomerase. On the basis of this and other lines of evidence, Spo11 has been proposed to be the enzymatic activity that creates the double-strand breaks needed to initiate meiotic recombination. This raises the possibility that recombination in Drosophila is also initiated by double-strand breaks. Although these homologous genes are required absolutely for recombination in both species, their roles differ in other respects. In contrast to spo11, mei-W68 is not required for synaptonemal complex formation and does have a mitotic role.

  19. UV stimulation of chromosomal marker exchange in Sulfolobus acidocaldarius: implications for DNA repair, conjugation and homologous recombination at extremely high temperatures.

    PubMed Central

    Schmidt, K J; Beck, K E; Grogan, D W

    1999-01-01

    The hyperthermophilic archaeon Sulfolobus acidocaldarius exchanges and recombines chromosomal markers by a conjugational mechanism, and the overall yield of recombinants is greatly increased by previous exposure to UV light. This stimulation was studied in an effort to clarify its mechanism and that of marker exchange itself. A variety of experiments failed to identify a significant effect of UV irradiation on the frequency of cell pairing, indicating that subsequent steps are primarily affected, i.e., transfer of DNA between cells or homologous recombination. The UV-induced stimulation decayed rather quickly in parental cells during preincubation at 75 degrees, and the rate of decay depended on the incubation temperature. Preincubation at 75 degrees decreased the yield of recombinants neither from unirradiated parental cells nor from parental suspensions subsequently irradiated. We interpret these results as evidence that marker exchange is stimulated by recombinogenic DNA lesions formed as intermediates in the process of repairing UV photoproducts in the S. acidocaldarius chromosome. PMID:10430571

  20. Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae.

    PubMed

    Arazoe, Takayuki; Younomaru, Tetsuya; Ohsato, Shuichi; Kimura, Makoto; Arie, Tsutomu; Kuwata, Shigeru

    2014-03-01

    To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae.

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

    PubMed

    Morimatsu, Katsumi; Kowalczykowski, Stephen C

    2014-12-02

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

  2. Novel PLA2G6 mutations associated with an exonic deletion due to non-allelic homologous recombination in a patient with infantile neuroaxonal dystrophy

    PubMed Central

    Yamamoto, Toshiyuki; Shimojima, Keiko; Shibata, Takashi; Akiyama, Mari; Oka, Makio; Akiyama, Tomoyuki; Yoshinaga, Harumi; Kobayashi, Katsuhiro

    2015-01-01

    Novel PLA2G6 mutations associated with p.Asp283Asn and a unique intragenic deletion of exons 4 and 5 due to non-allelic homologous recombination were identified in a Japanese female patient with typical infantile neuroaxonal dystrophy. The patient showed progressive tetraplegia beginning at 9 months. An electroencephalogram showed a diffuse increase in fast waves, and brain magnetic resonance imaging showed progressive brain atrophy and T2 hypointensity in the globus pallidus. PMID:27081553

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

    PubMed Central

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

    2015-01-01

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

  4. Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair.

    PubMed

    Sonoda, Eiichiro; Zhao, Guang Yu; Kohzaki, Masaoki; Dhar, Pawan Kumar; Kikuchi, Koji; Redon, Christophe; Pilch, Duane R; Bonner, William M; Nakano, Atsushi; Watanabe, Masami; Nakayama, Tatsuo; Takeda, Shunichi; Takami, Yasunari

    2007-03-01

    One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.

  5. Promyelocytic leukemia nuclear bodies support a late step in DNA double-strand break repair by homologous recombination.

    PubMed

    Yeung, Percy Luk; Denissova, Natalia G; Nasello, Cara; Hakhverdyan, Zhanna; Chen, J Don; Brenneman, Mark A

    2012-05-01

    The PML protein and PML nuclear bodies (PML-NB) are implicated in multiple cellular functions relevant to tumor suppression, including DNA damage response. In most cases of acute promyelocytic leukemia, the PML and retinoic acid receptor alpha (RARA) genes are translocated, resulting in expression of oncogenic PML-RARα fusion proteins. PML-NB fail to form normally, and promyelocytes remain in an undifferentiated, abnormally proliferative state. We examined the involvement of PML protein and PML-NB in homologous recombinational repair (HRR) of chromosomal DNA double-strand breaks. Transient overexpression of wild-type PML protein isoforms produced hugely enlarged or aggregated PML-NB and reduced HRR by ~2-fold, suggesting that HRR depends to some extent upon normal PML-NB structure. Knockdown of PML by RNA interference sharply attenuated formation of PML-NB and reduced HRR by up to 20-fold. However, PML-knockdown cells showed apparently normal induction of H2AX phosphorylation and RAD51 foci after DNA damage by ionizing radiation. These findings indicate that early steps in HRR, including recognition of DNA double-strand breaks, initial processing of ends, and assembly of single-stranded DNA/RAD51 nucleoprotein filaments, do not depend upon PML-NB. The HRR deficit in PML-depleted cells thus reflects inhibition of later steps in the repair pathway. Expression of PML-RARα fusion proteins disrupted PML-NB structure and reduced HRR by up to 10-fold, raising the possibility that defective HRR and resulting genomic instability may figure in the pathogenesis, progression and relapse of acute promyelocytic leukemia.

  6. Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

    SciTech Connect

    Nagasawa, H; Wilson, P F; Chen, D J; Thompson, L H; Bedford, J S; Little, J B

    2007-10-26

    We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells (Nagasawa et al., Radiat. Res. 164, 141-147, 2005). In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23-0.33 SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after {alpha}-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

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

    PubMed Central

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

    2014-01-01

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

  8. Structure-specific endonucleases Xpf and Mus81 play overlapping but essential roles in DNA repair by homologous recombination

    PubMed Central

    Kikuchi, Koji; Narita, Takeo; Thanh Van, Pham; Iijima, Junko; Hirota, Kouji; Keka, Islam Shamima; Mohiuddin; Okawa, Katsuya; Hori, Tetsuya; Fukagawa, Tatsuo; Essers, Jeroen; Kanaar, Roland; Whitby, Matthew C.; Sugasawa, Kaoru; Taniguchi, Yoshihito; Kitagawa, Katsumi; Takeda, Shunichi

    2013-01-01

    DNA double-strand breaks (DSBs) occur frequently during replication in sister chromatids, and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR hence plays pivotal roles in cellular proliferation and cellular tolerance to camptothecin. Mammalian cells carry several structure-specific endonucleases, such as Xpf-Ercc1 and Mus81-Eme1, in which Xpf and Mus81 are the essential subunits for enzymatic activity. Here we show the functional overlap between Xpf and Mus81 by conditionally inactivating Xpf in the chicken DT40 cell line, which has no Mus81 ortholog. Although mammalian cells deficient in either Xpf or Mus81 are viable, Xpf inactivation in DT40 cells was lethal, resulting in a marked increase in the number of spontaneous chromosome breaks. Similarly, inactivation of both Xpf and Mus81 in human HeLa cells and murine embryonic stem cells caused numerous spontaneous chromosome breaks. Furthermore, the phenotype of Xpf-deficient DT40 cells was reversed by ectopic expression of human Mus81-Eme1 or human Xpf-Ercc1 heterodimers. These observations indicate the functional overlap of Xpf-Ercc1 and Mus81-Eme1 in the maintenance of genomic DNA. Both Mus81-Eme1 and Xpf-Ercc1 contribute to the completion of HR as evidenced by the following data that the expression of Mus81-Eme1 or Xpf-Ercc1 diminished the number of camptothecin-induced chromosome breaks in Xpf-deficient DT40 cells, and preventing early steps in HR by deleting XRCC3 suppressed the inviability of Xpf-deficient DT40 cells. In summary, Xpf and Mus81 have a substantially overlapping function in completion of HR. PMID:23576554

  9. Artesunate enhances the therapeutic response of glioma cells to temozolomide by inhibition of homologous recombination and senescence

    PubMed Central

    Eich, Marcus; Kim, Ella; Kaina, Bernd

    2016-01-01

    Glioblastoma multiforme (GBM), a malignant brain tumor with a dismal prognosis, shows a high level of chemo- and radioresistance and, therefore, attempts to sensitize glioma cells are highly desired. Here, we addressed the question of whether artesunate (ART), a drug currently used in the treatment of malaria, enhances the killing response of glioblastoma cells to temozolomide (TMZ), which is the first-line therapeutic for GBM. We measured apoptosis, necrosis, autophagy and senescence, and the extent of DNA damage in glioblastoma cells. Further, we determined the tumor growth in nude mice. We show that ART enhances the killing effect of TMZ in glioblastoma cell lines and in glioblastoma stem-like cells. The DNA double-strand break level induced by TMZ was not clearly enhanced in the combined treatment regime. Also, we did not observe an attenuation of TMZ-induced autophagy, which is considered a survival mechanism. However, we observed a significant effect of ART on homologous recombination (HR) with downregulation of RAD51 protein expression and HR activity. Further, we found that ART is able to inhibit senescence induced by TMZ. Since HR and senescence are pro-survival mechanisms, its inhibition by ART appears to be a key node in enhancing the TMZ-induced killing response. Enhancement of the antitumor effect of TMZ by co-administration of ART was also observed in a mouse tumor model. In conclusion, the amelioration of TMZ-induced cell death upon ART co-treatment provides a rational basis for a combination regime of TMZ and ART in glioblastoma therapy. PMID:27626497

  10. Sgs1 RecQ helicase inhibits survival of Saccharomyces cerevisiae cells lacking telomerase and homologous recombination.

    PubMed

    Lee, Julia Y; Mogen, Jonathan L; Chavez, Alejandro; Johnson, F Brad

    2008-10-31

    In yeast telomerase mutants, the Sgs1 RecQ helicase slows the rate of senescence and also facilitates the appearance of certain types of survivors of critical telomere shortening via mechanisms dependent on Rad52-dependent homologous recombination (HR). Here we describe a third function for Sgs1 in telomerase-deficient cells, inhibition of survivors that grow independent of Rad52. Unlike tlc1 rad52 double mutants, which do not form survivors of telomere dysfunction, tlc1 rad52 sgs1 triple mutants readily generated survivors. After emerging from growth crisis, the triple mutants progressively lost telomeric and subtelomeric sequences, yet grew for more than 1 year. Analysis of cloned chromosome termini and of copy number changes of loci genome-wide using tiling arrays revealed terminal deletions extending up to 57 kb, as well as changes in Ty retrotransposon copy numbers. Amplification of the remaining terminal sequences generated large palindromes at some chromosome termini. Sgs1 helicase activity but not checkpoint function was essential for inhibiting the appearance of the survivors, and the continued absence of Sgs1 was required for the growth of the established survivors. Thus, in addition to facilitating the maintenance of telomere repeat sequences via HR-dependent mechanisms, a RecQ helicase can prevent the adoption of HR-independent mechanisms that stabilize chromosome termini without the use of natural telomere sequences. This provides a novel mechanism by which RecQ helicases may help maintain genome integrity and thus prevent age-related diseases and cancer.

  11. Introduction to 'Homology and convergence in nervous system evolution'.

    PubMed

    Strausfeld, Nicholas J; Hirth, Frank

    2016-01-05

    The origin of brains and central nervous systems (CNSs) is thought to have occurred before the Palaeozoic era 540 Ma. Yet in the absence of tangible evidence, there has been continued debate whether today's brains and nervous systems derive from one ancestral origin or whether similarities among them are due to convergent evolution. With the advent of molecular developmental genetics and genomics, it has become clear that homology is a concept that applies not only to morphologies, but also to genes, developmental processes, as well as to behaviours. Comparative studies in phyla ranging from annelids and arthropods to mammals are providing evidence that corresponding developmental genetic mechanisms act not only in dorso-ventral and anterior-posterior axis specification but also in segmentation, neurogenesis, axogenesis and eye/photoreceptor cell formation that appear to be conserved throughout the animal kingdom. These data are supported by recent studies which identified Mid-Cambrian fossils with preserved soft body parts that present segmental arrangements in brains typical of modern arthropods, and similarly organized brain centres and circuits across phyla that may reflect genealogical correspondence and control similar behavioural manifestations. Moreover, congruence between genetic and geological fossil records support the notion that by the 'Cambrian explosion' arthropods and chordates shared similarities in brain and nervous system organization. However, these similarities are strikingly absent in several sister- and outgroups of arthropods and chordates which raises several questions, foremost among them: what kind of natural laws and mechanisms underlie the convergent evolution of such similarities? And, vice versa: what are the selection pressures and genetic mechanisms underlying the possible loss or reduction of brains and CNSs in multiple lineages during the course of evolution? These questions were addressed at a Royal Society meeting to discuss

  12. Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated homology-independent knock-in system.

    PubMed

    Katoh, Yohei; Michisaka, Saki; Nozaki, Shohei; Funabashi, Teruki; Hirano, Tomoaki; Takei, Ryota; Nakayama, Kazuhisa

    2017-02-08

    The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore, the utilization of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here, we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system, and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and to increase the versatility of our knock-in system, we further constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.

  13. Fanconi anemia complementation group D2 (FANCD2) functions independently of BRCA2- and RAD51-associated homologous recombination in response to DNA damage.

    PubMed

    Ohashi, Akihiro; Zdzienicka, Malgorzata Z; Chen, Junjie; Couch, Fergus J

    2005-04-15

    The BRCA2 breast cancer tumor suppressor is involved in the repair of double strand breaks and broken replication forks by homologous recombination through its interaction with DNA repair protein Rad51. Cells defective in BRCA2.FANCD1 are extremely sensitive to mitomycin C (MMC) similarly to cells deficient in any of the Fanconi anemia (FA) complementation group proteins (FANC). These observations suggest that the FA pathway and the BRCA2 and Rad51 repair pathway may be linked, although a functional connection between these pathways in DNA damage signaling remains to be determined. Here, we systematically investigated the interaction between these pathways. We show that in response to DNA damage, BRCA2-dependent Rad51 nuclear focus formation was normal in the absence of FANCD2 and that FANCD2 nuclear focus formation and mono-ubiquitination appeared normal in BRCA2-deficient cells. We report that the absence of BRCA2 substantially reduced homologous recombination repair of DNA breaks, whereas the absence of FANCD2 had little effect. Furthermore, we established that depletion of BRCA2 or Rad51 had a greater effect on cell survival in response to MMC than depletion of FANCD2 and that depletion of BRCA2 in FANCD2 mutant cells further sensitized these cells to MMC. Our results suggest that FANCD2 mediates double strand DNA break repair independently of Rad51-associated homologous recombination.

  14. JMJD-5/KDM8 regulates H3K36me2 and is required for late steps of homologous recombination and genome integrity

    PubMed Central

    Zaghet, Nico; Ramalho, João J.; Vilstrup Johansen, Jens; Salcini, Anna Elisabetta

    2017-01-01

    The eukaryotic genome is organized in a three-dimensional structure called chromatin, constituted by DNA and associated proteins, the majority of which are histones. Post-translational modifications of histone proteins greatly influence chromatin structure and regulate many DNA-based biological processes. Methylation of lysine 36 of histone 3 (H3K36) is a post-translational modification functionally relevant during early steps of DNA damage repair. Here, we show that the JMJD-5 regulates H3K36 di-methylation and it is required at late stages of double strand break repair mediated by homologous recombination. Loss of jmjd-5 results in hypersensitivity to ionizing radiation and in meiotic defects, and it is associated with aberrant retention of RAD-51 at sites of double strand breaks. Analyses of jmjd-5 genetic interactions with genes required for resolving recombination intermediates (rtel-1) or promoting the resolution of RAD-51 double stranded DNA filaments (rfs-1 and helq-1) suggest that jmjd-5 prevents the formation of stalled postsynaptic recombination intermediates and favors RAD-51 removal. As these phenotypes are all recapitulated by a catalytically inactive jmjd-5 mutant, we propose a novel role for H3K36me2 regulation during late steps of homologous recombination critical to preserve genome integrity. PMID:28207814

  15. Camptothecin enhances the frequency of oligonucleotide-directed gene repair in mammalian cells by inducing DNA damage and activating homologous recombination.

    PubMed

    Ferrara, Luciana; Kmiec, Eric B

    2004-01-01

    Camptothecin (CPT) is an anticancer drug that promotes DNA breakage at replication forks and the formation of lesions that activate the processes of homologous recombination (HR) and nonhomologous end joining. We have taken advantage of the CPT-induced damage response by coupling it to gene repair directed by synthetic oligonucleotides, a process in which a mutant base pair is converted into a wild-type one. Here, we show that pretreating DLD-1 cells with CPT leads to a significant stimulation in the frequency of correction of an integrated mutant enhanced green fluorescent protein gene. The stimulation is dose-dependent and coincident with the formation of double-strand DNA breaks. Caffeine, but not vanillin, blocks the enhancement of gene repair suggesting that, in this system, HR is the pathway most responsible for elevating the frequency of correction. The involvement of HR is further proven by studies in which wortmannin was seen to inhibit gene repair at high concentrations but not at lower levels that are known to inhibit DNA-PK activity. Taken together, our results suggest that DNA damage induced by CPT activates a cellular response that stimulates gene repair in mammalian cells.

  16. Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: predominance of strand transfer.

    PubMed

    Izhar, Lior; Goldsmith, Moshe; Dahan, Ronny; Geacintov, Nicholas; Lloyd, Robert G; Livneh, Zvi

    2008-09-12

    Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichia coli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.

  17. Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination.

    PubMed

    Trojanek, Joanna; Ho, Thu; Del Valle, Luis; Nowicki, Michal; Wang, Jin Ying; Lassak, Adam; Peruzzi, Francesca; Khalili, Kamel; Skorski, Tomasz; Reiss, Krzysztof

    2003-11-01

    The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.

  18. Human genetic variants of homologous recombination repair genes first found to be associated with Epstein-Barr virus antibody titers in healthy Cantonese.

    PubMed

    Shen, Guo-Ping; Pan, Qing-Hua; Hong, Ming-Huang; Qin, Hai-De; Xu, Ya-Fei; Chen, Li-Zhen; Feng, Qi-Sheng; Jorgensen, Timothy J; Shugart, Yin Yao; Zeng, Yi-Xin; Jia, Wei-Hua

    2011-09-15

    Epstein-Barr virus (EBV) infection is a major risk factor for nasopharyngeal carcinoma (NPC). Despite high prevalence of infection among the general population worldwide, only a small proportion of infected individuals presents with seropositivity for EBV-specific IgA antibodies. This seropositive subgroup of EBV carriers has an elevated cumulative risk for NPC during their lifetime. Previous studies reported that the host homologous recombination repair (HRR) system participates in EBV lytic replication, suggesting a potential mechanism to influence EBV reactivation status and thus seropositivity. To investigate whether genetic variants of HRR genes are associated with the serostatus in a healthy population, we investigated the association between seropositivity for anti-VCA-IgA and 156 tagging SNPs in 35 genes connected with HRR in an observational study among 755 healthy Cantonese speakers in southern China. Six variant alleles of MDC1, RAD54L, TP53BP1, RPA1, LIG3 and RFC1 exhibited associations with seropositivity (p(trend) from 0.0085 to 0.00027). Our study provides evidence that genetic variation within the HRR might affect an individual's propensity for EBV seropositive status of anti-VCA IgA antibody.

  19. The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

    PubMed Central

    Amundsen, Susan K.; Taylor, Andrew F.; Smith, Gerald R.

    2000-01-01

    The RecBCD enzyme is required for homologous recombination and DNA repair in Escherichia coli. The structure and function of RecBCD enzyme is altered on its interaction with the recombination hotspot Chi (5′-GCTGGTGG-3′). It has been hypothesized that the RecD subunit plays a role in Chi-dependent regulation of enzyme activity [Thaler, D. S., Sampson, E., Siddiqi, I., Rosenberg, S. M., Stahl, F. W. & Stahl, M. (1988) in Mechanisms and Consequences of DNA Damage Processing, eds. Friedberg, E. & Hanawalt, P. (Liss, New York), pp. 413–422; Churchill, J. J., Anderson, D. G. & Kowalczykowski, S. C. (1999) Genes Dev. 13, 901–911]. We tested the hypothesis that the RecD subunit inhibits recombination by deleting recD from the nuclease- and recombination-deficient mutant recBD1080ACD. We report here that the resulting strain, recBD1080AC, was proficient for recombination and DNA repair. Recombination proficiency was accompanied by a change in enzyme activity: RecBD1080AC enzyme loaded RecA protein onto DNA during DNA unwinding whereas RecBD1080ACD enzyme did not. Together, these genetic and biochemical results demonstrate that RecA loading by RecBCD enzyme is required for recombination in E. coli cells and suggest that RecD interferes with the enzyme domain required for its loading. A nuclease-dependent signal appears to be required for a change in RecD that allows RecA loading. Because RecA loading is not observed with wild-type RecBCD enzyme until it acts at a Chi site, our observations support the view that RecD inhibits recombination until the enzyme acts at Chi. PMID:10840065

  20. XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

    SciTech Connect

    Yamada, N; Hinz, J; Kopf, V L; Segalle, K; Thompson, L

    2004-02-25

    Homologous recombinational repair is a major DNA repair pathway that preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 appears to exist in a single complex with Rad51C. To begin to examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a non-conservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of the mutants' dysfunction, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon coexpression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in HRR.

  1. GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis.

    PubMed

    Böhmdorfer, Gudrun; Schleiffer, Alexander; Brunmeir, Reinhard; Ferscha, Stefan; Nizhynska, Viktoria; Kozák, Jaroslav; Angelis, Karel J; Kreil, David P; Schweizer, Dieter

    2011-08-01

    DNA double-strand breaks (DSBs) pose one of the most severe threats to genome integrity, potentially leading to cell death. After detection of a DSB, the DNA damage and repair response is initiated and the DSB is repaired by non-homologous end joining and/or homologous recombination. Many components of these processes are still unknown in Arabidopsis thaliana. In this work, we characterized γ-irradiation and mitomycin C induced 1 (GMI1), a member of the SMC-hinge domain-containing protein family. RT-PCR analysis and promoter-GUS fusion studies showed that γ-irradiation, the radio-mimetic drug bleocin, and the DNA cross-linking agent mitomycin C strongly enhance GMI1 expression particularly in meristematic tissues. The induction of GMI1 by γ-irradiation depends on the signalling kinase Ataxia telangiectasia-mutated (ATM) but not on ATM and Rad3-related (ATR). Epistasis analysis of single and double mutants demonstrated that ATM acts upstream of GMI1 while the atr gmi1-2 double mutant was more sensitive than the respective single mutants. Comet assay revealed a reduced rate of DNA double-strand break repair in gmi1 mutants during the early recovery phase after exposure to bleocin. Moreover, the rate of homologous recombination of a reporter construct was strongly reduced in gmi1 mutant plants upon exposure to bleocin or mitomycin C. GMI1 is the first member of its protein family known to be involved in DNA repair.

  2. Tet(C) Gene Transfer between Chlamydia suis Strains Occurs by Homologous Recombination after Co-infection: Implications for Spread of Tetracycline-Resistance among Chlamydiaceae

    PubMed Central

    Marti, Hanna; Kim, Hoyon; Joseph, Sandeep J.; Dojiri, Stacey; Read, Timothy D.; Dean, Deborah

    2017-01-01

    Chlamydia suis is a swine pathogen that has also recently been found to cause zoonotic infections of the human eye, pharynx, and gastrointestinal tract. Many strains contain a tetracycline class C gene [tet(C)] cassette that confers tetracycline resistance. The cassette was likely originally acquired by horizontal gene transfer from a Gram-negative donor after the introduction of tetracycline into animal feed in the 1950s. Various research groups have described the capacity for different Chlamydia species to exchange DNA by homologous recombination. Since over 90% of C. suis strains are tetracycline resistant, they represent a potential source for antibiotic-resistance spread within and between Chlamydiaceae species. Here, we examined the genetics of tet(C)-transfer among C. suis strains. Tetracycline-sensitive C. suis strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant C. suis strains in McCoy cells. Potential recombinants were clonally purified by a harvest assay derived from the classic plaque assay. C. suis strain Rogers132, lacking transposases IS200 and IS605, was the most efficient donor, producing two unique recombinants detected in three of the 56 (5.4%) clones screened. Recombinants were found to have a minimal inhibitory concentration (MIC) of 8-16 μg/mL for tetracycline. Resistance remained stable over 10 passages as long as recombinants were initially grown in tetracycline at twice the MIC of S45 (0.032 μg/mL). Genomic analysis revealed that tet(C) had integrated into the S45 genome by homologous recombination at two unique sites depending on the recombinant: a 55 kb exchange between nrqF and pckG, and a 175 kb exchange between kdsA and cysQ. Neither site was associated with inverted repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS200/IS605 transposases, is stable if initially grown in tetracycline, and results in

  3. Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae.

    PubMed

    Lee, Yoonna; Kim, Kangil; Kang, Kyu-Tae; Lee, Jong-Soo; Yang, Sang Sik; Chung, Woo-Hyun

    2014-10-15

    Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death.

  4. Phenotypically silent Cre recombination within the postnatal ventricular conduction system

    PubMed Central

    Bhattacharyya, Samadrita; Bhakta, Minoti

    2017-01-01

    The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3’ untranslated region (3’UTR). Here we show that Cntn23’UTR-IRES-Cre-EGFP/+ mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that Cntn23’UTR-IRES-Cre-EGFP/+ mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function. PMID:28358866

  5. Mouse homolog of Saccharomyces cerevisiae spo11 is induced in normal mu(+)B-cells by stimuli that cause germline C(H) transcription and subsequent class switch recombination.

    PubMed

    Tokuyama, H; Tokuyama, Y

    2000-05-25

    The first step of Ig heavy chain class switch recombination (CSR) is considered to be DNA double strand break (DSB) formation in the two switch (S) regions (S(mu) and downstream S(H)), although the underlying mechanism is unknown. Recently, it has been demonstrated that at least Spo11, a homolog of the novel type II topoisomerase (topo VI) that catalyzes DSB formation, is involved in the initiation of meiotic recombination of Saccaromyces cerevisiae. In the present study, we examined whether the mouse homolog of Spo11 is induced in normal mouse mu(+)B-cells by stimuli that cause an early step of CSR, germline C(H) transcription, and subsequent CSR. Two CSR systems were used: IgA CSR induced by all-trans retinoic acid, IL-5, and LPS, and IgG1 CSR induced by IL-4 and LPS. Germline transcript and mouse Spo11 expression were analyzed by RT-PCR. In both systems, first germline transcripts were clearly detected on day 2 and then Spo11 was detected on day 3, increasing thereafter with time. The time course of changes in Spo11 expression coincided with that of CSR. Spo11 seems to be induced by CSR-inducing stimuli, regardless of the direction of CSR. These results suggested that mouse Spo11 might participate in the initiation step of CSR.

  6. Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids

    PubMed Central

    Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E.; Zickler, Denise; Espagne, Eric

    2014-01-01

    Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid–Sad1p, UNC-84–domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid “twin” nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly. PMID:25210014

  7. Absence of SUN-domain protein Slp1 blocks karyogamy and switches meiotic recombination and synapsis from homologs to sister chromatids.

    PubMed

    Vasnier, Christelle; de Muyt, Arnaud; Zhang, Liangran; Tessé, Sophie; Kleckner, Nancy E; Zickler, Denise; Espagne, Eric

    2014-09-23

    Karyogamy, the process of nuclear fusion is required for two haploid gamete nuclei to form a zygote. Also, in haplobiontic organisms, karyogamy is required to produce the diploid nucleus/cell that then enters meiosis. We identify sun like protein 1 (Slp1), member of the mid-Sad1p, UNC-84-domain ubiquitous family, as essential for karyogamy in the filamentous fungus Sordaria macrospora, thus uncovering a new function for this protein family. Slp1 is required at the last step, nuclear fusion, not for earlier events including nuclear movements, recognition, and juxtaposition. Correspondingly, like other family members, Slp1 localizes to the endoplasmic reticulum and also to its extensions comprising the nuclear envelope. Remarkably, despite the absence of nuclear fusion in the slp1 null mutant, meiosis proceeds efficiently in the two haploid "twin" nuclei, by the same program and timing as in diploid nuclei with a single dramatic exception: the normal prophase program of recombination and synapsis between homologous chromosomes, including loading of recombination and synaptonemal complex proteins, occurs instead between sister chromatids. Moreover, the numbers of recombination-initiating double-strand breaks (DSBs) and ensuing recombinational interactions, including foci of the essential crossover factor Homo sapiens enhancer of invasion 10 (Hei10), occur at half the diploid level in each haploid nucleus, implying per-chromosome specification of DSB formation. Further, the distribution of Hei10 foci shows interference like in diploid meiosis. Centromere and spindle dynamics, however, still occur in the diploid mode during the two meiotic divisions. These observations imply that the prophase program senses absence of karyogamy and/or absence of a homolog partner and adjusts the interchromosomal interaction program accordingly.

  8. Direct evaluation of a kinetic model for RecA-mediated DNA-strand exchange: the importance of nucleic acid dynamics and entropy during homologous genetic recombination.

    PubMed

    Xiao, Jie; Lee, Andrew M; Singleton, Scott F

    2006-08-01

    The Escherichia coli RecA protein is the prototype of a class of proteins that play central roles in genomic repair and recombination in all organisms. The unresolved mechanistic strategy by which RecA aligns a single strand of DNA with a duplex DNA and mediates a DNA strand switch is central to understanding homologous recombination. We explored the mechanism of RecA-mediated DNA-strand exchange using oligonucleotide substrates with the intrinsic fluorophore 6-methylisoxanthopterin. Pre-steady-state spectrofluorometric analysis elucidated the earliest transient intermediates formed during recombination and delineated the mechanistic strategy by which RecA facilitates this process. The structural features of the first detectable intermediate and the energetic characteristics of its formation were consistent with interactions between a few bases of the single-stranded DNA and the minor groove of a locally melted or stretched duplex DNA. Further analysis revealed RecA to be an unusual enzyme in that entropic rather than enthalpic contributions dominate its catalytic function, and no unambiguously active role for the protein was detected in the earliest molecular events of recombination. The data best support the conclusion that the mechanistic strategy of RecA likely relies on intrinsic DNA dynamics.

  9. Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis.

    PubMed

    Schröpfer, Susan; Kobbe, Daniela; Hartung, Frank; Knoll, Alexander; Puchta, Holger

    2014-02-01

    RecQ helicases are critical for the maintenance of genomic stability. The Arabidopsis RecQ helicase RECQ4A is the functional counterpart of human BLM, which is mutated in the genetic disorder Bloom's syndrome. RECQ4A performs critical roles in regulation of homologous recombination (HR) and DNA repair. Loss of RECQ4A leads to elevated HR frequencies and hypersensitivity to genotoxic agents. Through complementation studies, we were now able to demonstrate that the N-terminal region and the helicase activity of RECQ4A are both essential for the cellular response to replicative stress induced by methyl methanesulfonate and cisplatin. In contrast, loss of helicase activity or deletion of the N-terminus only partially complemented the mutant hyper-recombination phenotype. Furthermore, the helicase-deficient protein lacking its N-terminus did not complement the hyper-recombination phenotype at all. Therefore, RECQ4A seems to possess at least two different and independent sub-functions involved in the suppression of HR. By in vitro analysis, we showed that the helicase core was able to regress an artificial replication fork. Swapping of the terminal regions of RECQ4A with the closely related but functionally distinct helicase RECQ4B indicated that in contrast to the C-terminus, the N-terminus of RECQ4A was required for its specific functions in DNA repair and recombination.

  10. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  11. Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

    PubMed

    Muñoz-Galván, Sandra; López-Saavedra, Ana; Jackson, Stephen P; Huertas, Pablo; Cortés-Ledesma, Felipe; Aguilera, Andrés

    2013-02-01

    While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs.

  12. A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.

    PubMed Central

    Chiang, C C; Kennell, J C; Wanner, L A; Lambowitz, A M

    1994-01-01

    The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery. Images PMID:7523850

  13. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

    PubMed

    Bao, Zehua; Xiao, Han; Liang, Jing; Zhang, Lu; Xiong, Xiong; Sun, Ning; Si, Tong; Zhao, Huimin

    2015-05-15

    One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

  14. Alcohol homologation

    DOEpatents

    Wegman, R.W.; Moloy, K.G.

    1988-02-23

    A process is described for the homologation of an alkanol by reaction with synthesis gas in contact with a system containing rhodium atom, ruthenium atom, iodine atom and a bis(diorganophosphino) alkane to selectivity produce the next higher homologue.

  15. Alcohol homologation

    DOEpatents

    Wegman, Richard W.; Moloy, Kenneth G.

    1988-01-01

    A process for the homologation of an alkanol by reaction with synthesis gas in contact with a system containing rhodium atom, ruthenium atom, iodine atom and a bis(diorganophosphino) alkane to selectivity produce the next higher homologue.

  16. Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair

    PubMed Central

    Shinohara, Takeshi; Ikawa, Shukuko; Iwasaki, Wakana; Hiraki, Toshiki; Hikima, Takaaki; Mikawa, Tsutomu; Arai, Naoto; Kamiya, Nobuo; Shibata, Takehiko

    2015-01-01

    In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. PMID:25561575

  17. The extent of error-prone replication restart by homologous recombination is controlled by Exo1 and checkpoint proteins.

    PubMed

    Tsang, Ellen; Miyabe, Izumi; Iraqui, Ismail; Zheng, Jiping; Lambert, Sarah A E; Carr, Antony M

    2014-07-01

    Genetic instability, a hallmark of cancer, can occur when the replication machinery encounters a barrier. The intra-S-phase checkpoint maintains stalled replication forks in a replication-competent configuration by phosphorylating replisome components and DNA repair proteins to prevent forks from catastrophically collapsing. Here, we report a novel function of the core Schizosaccharomyces pombe checkpoint sensor kinase, Rad3 (an ATR orthologue), that is independent of Chk1 and Cds1 (a CHK2 orthologue); Rad3(ATR) regulates the association of recombination factors with collapsed forks, thus limiting their genetic instability. We further reveal antagonistic roles for Rad3(ATR) and the 9-1-1 clamp - Rad3(ATR) restrains MRN- and Exo1-dependent resection, whereas the 9-1-1 complex promotes Exo1 activity. Interestingly, the MRN complex, but not its nuclease activity, promotes resection and the subsequent association of recombination factors at collapsed forks. The biological significance of this regulation is revealed by the observation that Rad3(ATR) prevents Exo1-dependent genome instability upstream of a collapsed fork without affecting the efficiency of recombination-mediated replication restart. We propose that the interplay between Rad3(ATR) and the 9-1-1 clamp functions to fine-tune the balance between the need for the recovery of replication through recombination and the risk of increased genome instability.

  18. Phosphorylation of cohesin Rec11/SA3 by casein kinase 1 promotes homologous recombination by assembling the meiotic chromosome axis.

    PubMed

    Sakuno, Takeshi; Watanabe, Yoshinori

    2015-01-26

    In meiosis, cohesin is required for sister chromatid cohesion, as well as meiotic chromosome axis assembly and recombination. However, mechanisms underlying the multifunctional nature of cohesin remain elusive. Here, we show that fission yeast casein kinase 1 (CK1) plays a crucial role in assembling the meiotic chromosome axis (so-called linear element: LinE) and promoting recombination. An in vitro phosphorylation screening assay identified meiotic cohesin subunit Rec11/SA3 as an excellent substrate of CK1. The phosphorylation of Rec11 by CK1 mediates the interaction with the Rec10/Red1/SCP2 axis component, a key step in meiotic chromosome axis assembly, and is dispensable for sister chromatid cohesion. Crucially, the expression of Rec11-Rec10 fusion protein nearly completely bypasses the requirement for CK1 or cohesin phosphorylation for LinE assembly and recombination. This study uncovers a central mechanism of the cohesin-dependent assembly of the meiotic chromosome axis and recombination apparatus that acts independently of sister chromatid cohesion.

  19. The Cre/loxP recombination system for production of infectious mouse polyomavirus.

    PubMed

    Hron, Tomáš; Spanielová, Hana; Suchanová, Jiřina; Forstová, Jitka

    2013-09-01

    Murine polyomavirus mutants are frequently produced for experimental as well as therapy purposes. Commonly used methods for preparation of mutant viral genomes from recombinant vectors are laborious and give variable yields and quality. We describe an efficient and reproducible Cre/loxP-mediated recombination system that generates polyomavirus genomes from recombinant plasmid in vivo. We designed and constructed two variants of recombinant vectors containing the wild-type polyomavirus genome flanked by loxP homologous sites. The loxP sites were introduced either into the intronic region of early genes or between the two poly(A) signal sites of convergent transcriptional units. After cotransfection of the recombinant plasmids with the Cre-expressing vector into mouse 3T6 cells, we obtained infectious virus from the genome variant containing loxP site in the intronic region, but we failed to isolate any infectious virus from the viral genome containing loxP site between poly(A) signals. We show that the Cre/loxP-based method of polyomavirus production is simple, expedient, and reproducible and works with satisfactory efficiency.

  20. Polymorphisms of Homologous Recombination RAD51, RAD51B, XRCC2, and XRCC3 Genes and the Risk of Prostate Cancer

    PubMed Central

    Nowacka-Zawisza, Maria; Wiśnik, Ewelina; Wasilewski, Andrzej; Skowrońska, Milena; Forma, Ewa; Bryś, Magdalena; Różański, Waldemar; Krajewska, Wanda M.

    2015-01-01

    Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland. PMID:26339569

  1. Polymorphisms of homologous recombination RAD51, RAD51B, XRCC2, and XRCC3 genes and the risk of prostate cancer.

    PubMed

    Nowacka-Zawisza, Maria; Wiśnik, Ewelina; Wasilewski, Andrzej; Skowrońska, Milena; Forma, Ewa; Bryś, Magdalena; Różański, Waldemar; Krajewska, Wanda M

    2015-01-01

    Genetic polymorphisms in DNA repair genes may induce individual variations in DNA repair capacity, which may in turn contribute to the risk of cancer developing. Homologous recombination repair (HRR) plays a critical role in maintaining chromosomal integrity and protecting against carcinogenic factors. The aim of the present study was to evaluate the relationship between prostate cancer risk and the presence of single nucleotide polymorphisms (SNPs) in the genes involved in HRR, that is, RAD51 (rs1801320 and rs1801321), RAD51B (rs10483813 and rs3784099), XRCC2 (rs3218536), and XRCC3 (rs861539). Polymorphisms were analyzed by PCR-RFLP and Real-Time PCR in 101 patients with prostate adenocarcinoma and 216 age- and sex-matched controls. A significant relationship was detected between the RAD51 gene rs1801320 polymorphism and increased prostate cancer risk. Our results indicate that the RAD51 gene rs1801320 polymorphism may contribute to prostate cancer susceptibility in Poland.

  2. Inhibition of homologous recombination repair with Pentoxifylline targets G2 cells generated by radiotherapy and induces major enhancements of the toxicity of cisplatin and melphalan given after irradiation

    PubMed Central

    Bohm, Lothar

    2006-01-01

    The presentation reviews the modus operandi of the dose modifying drug Pentoxifylline and the dose enhancement factors which can be achieved in different cell types. Preclinical and clinical data show that Pentoxifylline improves the oxygenation of hypoxic tumours and enhances tumour control by irradiation. In vitro experiments demonstrate that Pentoxifylline also operates when oxygen is not limiting and produces dose modifying factors in the region of 1.2 – 2.0. This oxygen independent effect is poorly understood. In p53 mutant cells irradiation induces a G2 block which is abrogated by Pentoxifylline. The enhancement of cell kill observed when Pentoxifylline and irradiation are given together could arise from rapid entry of damaged tumour cells into mitosis and propagation of DNA lesions as the result of curtailment of repair time. Recovery ratios and repair experiments using CFGE after high dose irradiation demonstrate that Pentoxifylline inhibits repair directly and that curtailment of repair time is not the explanation. Use of the repair defective xrs1 and the parental repair competent CHO-K1 cell line shows that Pentoxifylline inhibits homologous recombination repair which operates predominantly in the G2 phase of the cell cycle. When irradiated cells residing in G2 phase are exposed to very low doses of cisplatin at a toxic dose of 5 %. (TC: 0.05) massive toxicity enhancements up to a factor of 80 are observed in melanoma, squamous carcinoma and prostate tumour cell lines. Enhancements of radiotoxicity seen when Pentoxifylline and radiation are applied together are small and do not exceed a factor of 2.0. The capacity of Pentoxifyline to inhibit homologous recombination repair has not as yet been clinically utilized. A suitable application could be in the treatment of cervical carcinoma where irradiation and cisplatin are standard modality. In vitro data also strongly suggest that regimes where irradiation is used in combination with alkylating drugs may

  3. Inhibition of homologous recombination repair with Pentoxifylline targets G2 cells generated by radiotherapy and induces major enhancements of the toxicity of cisplatin and melphalan given after irradiation.

    PubMed

    Bohm, Lothar

    2006-05-03

    The presentation reviews the modus operandi of the dose modifying drug Pentoxifylline and the dose enhancement factors which can be achieved in different cell types. Preclinical and clinical data show that Pentoxifylline improves the oxygenation of hypoxic tumours and enhances tumour control by irradiation. In vitro experiments demonstrate that Pentoxifylline also operates when oxygen is not limiting and produces dose modifying factors in the region of 1.2-2.0. This oxygen independent effect is poorly understood. In p53 mutant cells irradiation induces a G2 block which is abrogated by Pentoxifylline. The enhancement of cell kill observed when Pentoxifylline and irradiation are given together could arise from rapid entry of damaged tumour cells into mitosis and propagation of DNA lesions as the result of curtailment of repair time. Recovery ratios and repair experiments using CFGE after high dose irradiation demonstrate that Pentoxifylline inhibits repair directly and that curtailment of repair time is not the explanation. Use of the repair defective xrs1 and the parental repair competent CHO-K1 cell line shows that Pentoxifylline inhibits homologous recombination repair which operates predominantly in the G2 phase of the cell cycle. When irradiated cells residing in G2 phase are exposed to very low doses of cisplatin at a toxic dose of 5%. (TC: 0.05) massive toxicity enhancements up to a factor of 80 are observed in melanoma, squamous carcinoma and prostate tumour cell lines. Enhancements of radiotoxicity seen when Pentoxifylline and radiation are applied together are small and do not exceed a factor of 2.0. The capacity of Pentoxifyline to inhibit homologous recombination repair has not as yet been clinically utilized. A suitable application could be in the treatment of cervical carcinoma where irradiation and cisplatin are standard modality. In vitro data also strongly suggest that regimes where irradiation is used in combination with alkylating drugs may also

  4. A 590 kb deletion caused by non-allelic homologous recombination between two LINE-1 elements in a patient with mesomelia-synostosis syndrome.

    PubMed

    Kohmoto, Tomohiro; Naruto, Takuya; Watanabe, Miki; Fujita, Yuji; Ujiro, Sae; Okamoto, Nana; Horikawa, Hideaki; Masuda, Kiyoshi; Imoto, Issei

    2017-04-01

    Mesomelia-synostoses syndrome (MSS) is a rare, autosomal-dominant, syndromal osteochondrodysplasia characterized by mesomelic limb shortening, acral synostoses, and multiple congenital malformations due to a non-recurrent deletion at 8q13 that always encompasses two coding-genes, SULF1 and SLCO5A1. To date, five unrelated patients have been reported worldwide, and MMS was previously proposed to not be a genomic disorder associated with deletions recurring from non-allelic homologous recombination (NAHR) in at least two analyzed cases. We conducted targeted gene panel sequencing and subsequent array-based copy number analysis in an 11-year-old undiagnosed Japanese female patient with multiple congenital anomalies that included mesomelic limb shortening and detected a novel 590 Kb deletion at 8q13 encompassing the same gene set as reported previously, resulting in the diagnosis of MSS. Breakpoint sequences of the deleted region in our case demonstrated the first LINE-1s (L1s)-mediated unequal NAHR event utilizing two distant L1 elements as homology substrates in this disease, which may represent a novel causative mechanism of the 8q13 deletion, expanding the range of mechanisms involved in the chromosomal rearrangements responsible for MSS.

  5. Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

    PubMed Central

    Ma, Jian Jun; Xu, Peng; Zhang, Wei; Li, Yu Mei; Fu, Qiang; Zhu, Guang Feng; Xue, Wei; Lei, Yong Hua; Gao, Jing Yu; Wang, Juan Ying; Shao, Chen; Yi, Cheng Gang; Wang, He

    2013-01-01

    Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment. PMID:23950876

  6. Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants.

    PubMed

    Asaad, N A; Zeng, Z C; Guan, J; Thacker, J; Iliakis, G

    2000-11-23

    The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected