Journal articles on the topic 'Recombination Repair Pathway'
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1
Kuzminov, Andrei. "Recombinational Repair of DNA Damage inEscherichia coli and Bacteriophage λ." Microbiology and Molecular Biology Reviews 63, no. 4 (December 1, 1999): 751–813. http://dx.doi.org/10.1128/mmbr.63.4.751-813.1999.
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SUMMARY Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.
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Kang, Josephine, and Martin J. Blaser. "Repair and Antirepair DNA Helicases in Helicobacter pylori." Journal of Bacteriology 190, no. 12 (March 28, 2008): 4218–24. http://dx.doi.org/10.1128/jb.01848-07.
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ABSTRACT Orthologs of RecG and RuvABC are highly conserved among prokaryotes; in Escherichia coli, they participate in independent pathways that branch migrate Holliday junctions during recombinational DNA repair. RecG also has been shown to directly convert stalled replication forks into Holliday junctions. The bacterium Helicobacter pylori, with remarkably high levels of recombination, possesses RecG and RuvABC homologs, but in contrast to E. coli, H. pylori RecG limits recombinational repair. We now show that the RuvABC pathway plays the prominent, if not exclusive, repair role. By introducing an E. coli resolvase (RusA) into H. pylori, the repair and recombination phenotypes of the ruvB mutant but not the recG mutant were improved. Our results indicate that RecG and RuvB compete for Holliday junction structures in recombinational repair, but since a classic RecG resolvase is absent from H. pylori, deployment of the RecG pathway is lethal. We propose that evolutionary loss of the H. pylori RecG resolvase provides an “antirepair” pathway allowing for selection of varied strains. Such competition between repair and antirepair provides a novel mechanism to maximize fitness at a bacterial population level.
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Rocha, Pedro P., Yi Fu, JungHyun Kim, and Jane Skok. "The Impact of Nuclear Organization and Homolgous Recombination in Repair of DNA Damage Introduced By Aid during Class Switch Recombination." Blood 124, no. 21 (December 6, 2014): 2738. http://dx.doi.org/10.1182/blood.v124.21.2738.2738.
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Abstract Class Switch Recombination (CSR) involves the introduction of double stranded breaks (DSBs) at the switch regions of the immunoglulin heavy chain (Igh) locus by the enzyme Activation Cytidine Deaminse (AID). AID can also act as a general mutator targeting other loci in the genome which can then either be repaired faithfully or in an error-prone fashion introducing mutations and potentially initiating B cell lymphoma. The factors contributing to the choice of repair pathway are not fully understood. Here we tested the hypothesis that repair pathway choice is influenced by differential accessibility and expression levels of target loci across cell cycle. More specifically in the context of CSR we tested whether differential regulation of gene accessibility across cell cycle is an important determinant for AID binding and subsequent repair pathway choice as different repair pathways predominate at different stages of cell cycle. Using 3D-FISH in conjunction with Immunofluorescence we observed that AID target genes that are faithfully repaired are more accessible (found in euchromatic regions) in the G2 phase of the cell cycle then genes that are frequently mutated. In contrast, those genes which are repaired in an error prone fashion are more accessible in the G1 phase of cell cycle. Since Homologous Recombination mediated repair (HR), which is a faithful repair mechanism, occurs in G2 we speculate that accessibility of these genes at this stage of cell cycle facilitates action by this repair pathway. Conversely, genes that are more accessible during the G1 phase of cell cycle will be repaired by the non-homologous end joining (NHEJ) repair pathway and therefore are more likely to be mutated. Thus, HR could be the pathway by which faithful repair is accomplished and use of the NHEJ pathway on the other hand could contribute to the introduction of dangerous DNA mutations that might lead to B cell transformation and cancer. To connect differences in accessibility with repair pathway usage, we used a mouse model carrying a hypomorphic mutation in BRCA2, a protein involved in HR. This is the first mouse model impaired in HR that eludes embryonic lethality and allows inspection of the role of this pathway in maintaining genomic stability in splenocytes undergoing CSR. Our preliminary investigations indicate that in Brca2 mutant B cells not only is the integrity of fathfully repaired loci compromised, but the Igh locus is also damaged. Taken together these results support our hypothesis and further indicate that the HR pathway is involved in repairing Igh. Given that approximately 95% of lymphomas are of B cell origin and many of these are associated with AID mediated breaks, it is crucial for us to understand the factors that influence targeting and repair. Disclosures No relevant conflicts of interest to declare.
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Mendonca, V. M., and S. W. Matson. "Genetic analysis of delta helD and delta uvrD mutations in combination with other genes in the RecF recombination pathway in Escherichia coli: suppression of a ruvB mutation by a uvrD deletion." Genetics 141, no. 2 (October 1, 1995): 443–52. http://dx.doi.org/10.1093/genetics/141.2.443.
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Abstract Helicase II (uvrD gene product) and helicase IV (helD gene product) have been shown previously to be involved in the RecF pathway of recombination. To better understand the role of these two proteins in homologous recombination in the RecF pathway [recBCsbcB(C) background, we investigated the interactions between helD, uvrD and the following RecF pathway genes: recF, recO, recN and ruvAB. We observed synergistic interactions between uvrD ant the recF, recN, recO and recG genes in both conjugational recombination and the repair of methylmethane sulfonate (MMS)-induced DNA damage. No synergistic interactions were detected between helD and the recF, recO and regN genes when conjugational recombination was analyzed. We did, however, detect synergistic interactions between helD and recF/recO in recombinational repair. Surprisingly, the uvrD deletion completely suppressed the phenotype of a ruvB mutation in a recBCsbcB(C) background. Both conjugational recombination efficiency and MMS-damaged DNA repair proficiency returned to wild-type levels in the deltauvrDruvB9 double mutant. Suppression of the effects of the ruvB mutation by a uvrD deletion was dependent on the recG and recN genes and not dependent on the recF/O/R genes. These data are discussed in the context of two "RecF" homologous recombination pathways operating in a recBCsbcB(C) strain background.
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Wang, Xin, Carolyn A. Peterson, Huyong Zheng, Rodney S. Nairn, Randy J. Legerski, and Lei Li. "Involvement of Nucleotide Excision Repair in a Recombination-Independent and Error-Prone Pathway of DNA Interstrand Cross-Link Repair." Molecular and Cellular Biology 21, no. 3 (February 1, 2001): 713–20. http://dx.doi.org/10.1128/mcb.21.3.713-720.2001.
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ABSTRACT DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.
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Shcherbakov, Victor P. "Mismatch repair in recombination of bacteriophage T4." BioMolecular Concepts 3, no. 6 (December 1, 2012): 523–34. http://dx.doi.org/10.1515/bmc-2012-0021.
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AbstractThe review focuses on the mechanism of mismatch repair in bacteriophage T4. It was first observed in T4 as an extra recombination mechanism, which contributed to the general recombination only when particular rII mutations were used as genetic markers (high-recombination markers), whereas it was inactive toward other rII mutations (low-recombination markers). This marker-dependent recombination pathway was identified as a repair of mismatches in recombinational heteroduplexes. Comparison of the structure of markers enabled us to make several specific conclusions on the nature of the marker discrimination by the mismatch repair system operating during T4 crosses. First, heteroduplexes with one mismatched base pair (either of transition or of transversion type) as well as single-nucleotide mismatches of indel type are not efficiently repaired. Second, among the repairable mismatches, those with two or more contiguous mismatched nucleotides are the most effectively repaired, whereas insertion of one correct pair between two mismatched ones reduces the repairability. Third, heteroduplexes containing insertion mutations are repaired asymmetrically, the longer strand being preferentially removed. Fourth, the sequence environment is an important factor. Inspection of the sequences flanking mismatches shows that runs of A:T pairs directly neighboring the mismatches greatly promote repair. The mismatch is recognized by T4 endonuclease VII and nicked on the 3′ side. The nonpaired 3′ terminus is attacked by the proofreading 3′→5′ exonuclease of T4 DNA polymerase that removes the mismatched nucleotides along with several (~25) complementary nucleotides (the repair tract) and then switches to polymerization. The residual nick is ligated by DNA ligase (gp30). Most probably, the T4 system repairs replication and other mismatches as well; however, it might not discriminate old and new DNA strands and so does not seem to be aimed at repair of replication errors, in contrast to the most commonly studied examples of mismatch repair.
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Berardini, Mark, Patricia L. Foster, and Edward L. Loechler. "DNA Polymerase II (polB) Is Involved in a New DNA Repair Pathway for DNA Interstrand Cross-Links inEscherichia coli." Journal of Bacteriology 181, no. 9 (May 1, 1999): 2878–82. http://dx.doi.org/10.1128/jb.181.9.2878-2882.1999.
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ABSTRACT DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be ∼0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in apolB + (∼0.6) than in a ΔpolB(∼0.1) strain. Complementation with apolB +-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type <polB < recA < polB recA ∼ uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing,E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.
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Symington, Lorraine S. "Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair." Microbiology and Molecular Biology Reviews 66, no. 4 (December 2002): 630–70. http://dx.doi.org/10.1128/mmbr.66.4.630-670.2002.
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SUMMARY The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.
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Tamburini, Beth A., and Jessica K. Tyler. "Localized Histone Acetylation and Deacetylation Triggered by the Homologous Recombination Pathway of Double-Strand DNA Repair." Molecular and Cellular Biology 25, no. 12 (June 15, 2005): 4903–13. http://dx.doi.org/10.1128/mcb.25.12.4903-4913.2005.
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ABSTRACT Many recent studies have demonstrated recruitment of chromatin-modifying enzymes to double-strand breaks. Instead, we wanted to examine chromatin modifications during the repair of these double-strand breaks. We show that homologous recombination triggers the acetylation of N-terminal lysines on histones H3 and H4 flanking a double-strand break, followed by deacetylation of H3 and H4. Consistent with a requirement for acetylation and deacetylation during homologous recombination, Saccharomyces cerevisiae with substitutions of the acetylatable lysines of histone H4, deleted for the N-terminal tail of histone H3 or H4, deleted for the histone acetyltransferase GCN5 gene or the histone deacetylase RPD3 gene, shows inviability following induction of an HO lesion that is repaired primarily by homologous recombination. Furthermore, the histone acetyltransferases Gcn5 and Esa1 and the histone deacetylases Rpd3, Sir2, and Hst1 are recruited to the HO lesion during homologous recombinational repair. We have also observed a distinct pattern of histone deacetylation at the donor locus during homologous recombination. Our results demonstrate that dynamic changes in histone acetylation accompany homologous recombination and that the ability to modulate histone acetylation is essential for viability following homologous recombination.
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Zahradka, Ksenija, Sanela Šimić, Maja Buljubašić, Mirjana Petranović, Damir Đermić, and Davor Zahradka. "sbcB15 and ΔsbcB Mutations Activate Two Types of RecF Recombination Pathways in Escherichia coli." Journal of Bacteriology 188, no. 21 (August 25, 2006): 7562–71. http://dx.doi.org/10.1128/jb.00613-06.
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ABSTRACT Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and ΔsbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and ΔsbcB mutations on DNA repair after UV and γ irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than ΔsbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as ΔsbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC ΔsbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.
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Pike, Brietta L., and Jörg Heierhorst. "Mdt1 Facilitates Efficient Repair of Blocked DNA Double-Strand Breaks and Recombinational Maintenance of Telomeres." Molecular and Cellular Biology 27, no. 18 (July 16, 2007): 6532–45. http://dx.doi.org/10.1128/mcb.00471-07.
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ABSTRACT DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3′-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Δ also hypersensitizes partially recombination-defective cells to camptothecin-induced 3′-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Δ cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair “clean” endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Δ leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Δ causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance.
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Ivančić-Baće, Ivana, Erika Salaj-Šmic, and Krunoslav Brčić-Kostić. "Effects of recJ, recQ, and recFOR Mutations on Recombination in Nuclease-Deficient recB recD Double Mutants of Escherichia coli." Journal of Bacteriology 187, no. 4 (February 15, 2005): 1350–56. http://dx.doi.org/10.1128/jb.187.4.1350-1356.2005.
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ABSTRACT The two main recombination pathways in Escherichia coli (RecBCD and RecF) have different recombination machineries that act independently in the initiation of recombination. Three essential enzymatic activities are required for early recombinational processing of double-stranded DNA ends and breaks: a helicase, a 5′→3′ exonuclease, and loading of RecA protein onto single-stranded DNA tails. The RecBCD enzyme performs all of these activities, whereas the recombination machinery of the RecF pathway consists of RecQ (helicase), RecJ (5′→3′ exonuclease), and RecFOR (RecA-single-stranded DNA filament formation). The recombination pathway operating in recB (nuclease-deficient) mutants is a hybrid because it includes elements of both the RecBCD and RecF recombination machineries. In this study, genetic analysis of recombination in a recB (nuclease-deficient) recD double mutant was performed. We show that conjugational recombination and DNA repair after UV and gamma irradiation in this mutant are highly dependent on recJ, partially dependent on recFOR, and independent of recQ. These results suggest that the recombination pathway operating in a nuclease-deficient recB recD double mutant is also a hybrid. We propose that the helicase and RecA loading activities belong to the RecBCD recombination machinery, while the RecJ-mediated 5′→3′ exonuclease is an element of the RecF recombination machinery.
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Cano, David A., M. Graciela Pucciarelli, Francisco García-del Portillo, and Josep Casadesús. "Role of the RecBCD Recombination Pathway in Salmonella Virulence." Journal of Bacteriology 184, no. 2 (January 15, 2002): 592–95. http://dx.doi.org/10.1128/jb.184.2.592-595.2002.
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ABSTRACT Mutants of Salmonella enterica lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N. A. Buchmeier, C. J. Lipps, M. Y. H. So, and F. Heffron, Mol. Microbiol. 7:933–936, 1993). The virulence-related defects of RecBC− mutants are not suppressed by sbcB and sbcCD mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD. Functions of the RecF pathway such as RecJ and RecF are not required for virulence. Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by S. enterica may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes. Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during Salmonella infection.
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Saparbaev, Murat, Louise Prakash, and Satya Prakash. "Requirement of Mismatch Repair Genes MSH2 and MSH3 in the RAD1-RAD10 Pathway of Mitotic Recombination in Saccharomyces cerevisiae." Genetics 142, no. 3 (March 1, 1996): 727–36. http://dx.doi.org/10.1093/genetics/142.3.727.
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Abstract The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3Δ mutation has an effect on recombination similar to that of the rad1Δ and rad10Δ mutations. The msh2Δ mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.
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Shcherbakov, V. P., and L. A. Plugina. "Marker-dependent recombination in T4 bacteriophage. III. Structural prerequisites for marker discrimination." Genetics 128, no. 4 (August 1, 1991): 673–85. http://dx.doi.org/10.1093/genetics/128.4.673.
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Abstract Distance- as well as marker-dependence of genetic recombination of bacteriophage T4 was studied in crosses between rIIB mutants with known base sequences. The notion of a "basic recombination," which is the recombination within distances shorter than hybrid DNA length in the absence of mismatch repair and any marker effects, was substantiated. The basic recombination frequency per base pair can serve as an objective parameter (natural constant) of general recombination reflecting its intensity. Comparative studies of the recombination properties of rIIB mutants with various sequence changes in the mutated sites showed that the main factor determining the probability of mismatch repair in recombination heteroduplexes is the length of a continuous heterologous region. A run of A:T pairs immediately adjoining the mismatch appears to stimulate its repair. In the case of mismatches with DNA strands of unequal length, formed by frameshift mutations, the repair is asymmetric, the longer strand (bulge) being preferentially removed. A pathway for mismatch repair including sequential action of endonuclease VII (gp49)----3'----5' exonuclease (gp43)----DNA polymerase (gp43)----DNA ligase (gp30) was proposed. A possible identity of the recombinational mismatch repair mechanism to that operating to produce mutations via sequence conversion is discussed.
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Bjorkman, Andrea, Likun Du, Annika Lindblom, and Qiang Pan-Hammarstrom. "Altered class switch recombination junctions in patients with deficiency in Mlh1 and Brca1 (109.6)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 109.6. http://dx.doi.org/10.4049/jimmunol.188.supp.109.6.
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Abstract Class switch recombination (CSR) is a B-cell specific process that results in the change of immunoglobulin isotype from IgM to IgA, IgG or IgE. It involves the creation of DNA double strand breaks (DSBs) in switch regions, which are sensed and repaired by DNA damage response proteins and the non-homologous end-joining (NHEJ) pathway. Here we have studied CSR in patients with heterozygous mutations in both the mismatch repair (MMR) gene Mlh1 and the breast cancer susceptibility gene Brca1 and in patients with mutations in either Mlh1 or Brca1 alone. Brca1 is involved in several processes ranging from DNA DSB repair, particularly through homologous recombination, and cell cycle checkpoint control to ubiquitination and chromatin remodeling. Its function in NHEJ as well as in CSR is unknown. The MMR pathway normally repairs mismatches arising in the DNA, while during CSR it seems to be involved in the creation of DSBs by processing of mismatches in the switch regions. To study CSR in the patients we have analyzed the in vivo generated recombination switch junctions, formed after the repair of two switch regions during CSR. We have found that the switch recombination junctions are altered in all patients, suggesting the importance of these proteins during CSR in human B cells.
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Ozenberger, B. A., and G. S. Roeder. "A unique pathway of double-strand break repair operates in tandemly repeated genes." Molecular and Cellular Biology 11, no. 3 (March 1991): 1222–31. http://dx.doi.org/10.1128/mcb.11.3.1222-1231.1991.
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The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.
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Ozenberger, B. A., and G. S. Roeder. "A unique pathway of double-strand break repair operates in tandemly repeated genes." Molecular and Cellular Biology 11, no. 3 (March 1991): 1222–31. http://dx.doi.org/10.1128/mcb.11.3.1222.
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The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.
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Yokochi, T., K. Kusano, and I. Kobayashi. "Evidence for conservative (two-progeny) DNA double-strand break repair." Genetics 139, no. 1 (January 1, 1995): 5–17. http://dx.doi.org/10.1093/genetics/139.1.5.
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Abstract The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage lambda Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.
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Summers, K. C., F. Shen, E. A. Sierra Potchanant, E. A. Phipps, R. J. Hickey, and L. H. Malkas. "Phosphorylation: The Molecular Switch of Double-Strand Break Repair." International Journal of Proteomics 2011 (May 18, 2011): 1–8. http://dx.doi.org/10.1155/2011/373816.
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Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.
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Rattray, Alison J., Brenda K. Shafer, and David J. Garfinkel. "The Saccharomyces cerevisiae DNA Recombination and Repair Functions of the RAD52 Epistasis Group Inhibit Ty1 Transposition." Genetics 154, no. 2 (February 1, 2000): 543–56. http://dx.doi.org/10.1093/genetics/154.2.543.
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Abstract RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or δ) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.
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Shcherbakov, V. P., L. A. Plugina, and E. A. Kudryashova. "Marker-dependent recombination in T4 bacteriophage. IV. Recombinational effects of antimutator T4 DNA polymerase." Genetics 140, no. 1 (May 1, 1995): 13–25. http://dx.doi.org/10.1093/genetics/140.1.13.
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Abstract Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3'-->5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49)-->3'-->5' exonuclease (gp43)-->DNA polymerase (gp43)-->DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.
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Kang, Josephine, and Martin J. Blaser. "UvrD Helicase Suppresses Recombination and DNA Damage-Induced Deletions." Journal of Bacteriology 188, no. 15 (August 1, 2006): 5450–59. http://dx.doi.org/10.1128/jb.00275-06.
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ABSTRACT UvrD, a highly conserved helicase involved in mismatch repair, nucleotide excision repair (NER), and recombinational repair, plays a critical role in maintaining genomic stability and facilitating DNA lesion repair in many prokaryotic species. In this report, we focus on the UvrD homolog in Helicobacter pylori, a genetically diverse organism that lacks many known DNA repair proteins, including those involved in mismatch repair and recombinational repair, and that is noted for high levels of inter- and intragenomic recombination and mutation. H. pylori contains numerous DNA repeats in its compact genome and inhabits an environment rich in DNA-damaging agents that can lead to increased rearrangements between such repeats. We find that H. pylori UvrD functions to repair DNA damage and limit homologous recombination and DNA damage-induced genomic rearrangements between DNA repeats. Our results suggest that UvrD and other NER pathway proteins play a prominent role in maintaining genome integrity, especially after DNA damage; thus, NER may be especially critical in organisms such as H. pylori that face high-level genotoxic stress in vivo.
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Nussbaum, A., M. Shalit, and A. Cohen. "Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli." Genetics 130, no. 1 (January 1, 1992): 37–49. http://dx.doi.org/10.1093/genetics/130.1.37.
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Abstract 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.
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Weinstock, David M., and Maria Jasin. "Alternative Pathways for the Repair of RAG-Induced DNA Breaks." Molecular and Cellular Biology 26, no. 1 (January 1, 2006): 131–39. http://dx.doi.org/10.1128/mcb.26.1.131-139.2006.
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ABSTRACT RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be ≥40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70 − / − cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.
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Spell, Rachelle Miller, and Sue Jinks-Robertson. "Role of Mismatch Repair in the Fidelity ofRAD51- andRAD59-Dependent Recombination inSaccharomyces cerevisiae." Genetics 165, no. 4 (December 1, 2003): 1733–44. http://dx.doi.org/10.1093/genetics/165.4.1733.
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AbstractTo prevent genome instability, recombination between sequences that contain mismatches (homeologous recombination) is suppressed by the mismatch repair (MMR) pathway. To understand the interactions necessary for this regulation, the genetic requirements for the inhibition of homeologous recombination were examined using mutants in the RAD52 epistasis group of Saccharomyces cerevisiae. The use of a chromosomal inverted-repeat recombination assay to measure spontaneous recombination between 91 and 100% identical sequences demonstrated differences in the fidelity of recombination in pathways defined by their dependence on RAD51 and RAD59. In addition, the regulation of homeologous recombination in rad51 and rad59 mutants displayed distinct patterns of inhibition by different members of the MMR pathway. Whereas the requirements for the MutS homolog, MSH2, and the MutL homolog, MLH1, in the suppression of homeologous recombination were similar in rad51 strains, the loss of MSH2 caused a greater loss in homeologous recombination suppression than did the loss of MLH1 in a rad59 strain. The nonequivalence of the regulatory patterns in the wild-type and mutant strains suggests an overlap between the roles of the RAD51 and RAD59 gene products in potential cooperative recombination mechanisms used in wild-type cells.
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Dang, Tuyen T., and Julio C. Morales. "XRN2 Links RNA:DNA Hybrid Resolution to Double Strand Break Repair Pathway Choice." Cancers 12, no. 7 (July 7, 2020): 1821. http://dx.doi.org/10.3390/cancers12071821.
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It was recently shown that the 5’ to 3’ exoribonuclease XRN2 is involved in the DNA damage response. Importantly, loss of XRN2 abrogates DNA double stranded break repair via the non-homologous end-joining pathway. However, the mechanistic details of how XRN2 functions in the non-homologous end-joining repair process are unknown. In this study, we elucidated that XRN2-mediated RNA:DNA hybrid resolution is required to allow Ku70 binding to DNA ends. These data suggest that XRN2 is required for the initiation of non-homologous end-joining repair. Interestingly, we uncovered a role for XRN2 in the homologous recombination repair pathway. Loss of XRN2 lead to a decrease in the repair of double strand breaks by homologous recombination. Strikingly, when we removed RNA:DNA hybrids by RNaseH1 over-expression, homologous recombination was not restored. We found RNA:DNA hybrid formation at and downstream of the DSB site, suggesting that unregulated transcription inhibits homologous recombination repair. In summary, our results indicate a relation between RNA:DNA hybrid resolution and double strand break repair pathway choice.
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Che, Shiyou, Yujing Chen, Yakun Liang, Qionglin Zhang, and Mark Bartlam. "Crystal structure of RecR, a member of the RecFOR DNA-repair pathway, fromPseudomonas aeruginosaPAO1." Acta Crystallographica Section F Structural Biology Communications 74, no. 4 (March 22, 2018): 222–30. http://dx.doi.org/10.1107/s2053230x18003503.
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DNA damage is usually lethal to all organisms. Homologous recombination plays an important role in the DNA damage-repair process in prokaryotic organisms. Two pathways are responsible for homologous recombination inPseudomonas aeruginosa: the RecBCD pathway and the RecFOR pathway. RecR is an important regulator in the RecFOR homologous recombination pathway inP. aeruginosa. It forms complexes with RecF and RecO that can facilitate the loading of RecA onto ssDNA in the RecFOR pathway. Here, the crystal structure of RecR fromP. aeruginosaPAO1 (PaRecR) is reported.PaRecR crystallizes in space groupP6122, with two monomers per asymmetric unit. Analytical ultracentrifugation data show thatPaRecR forms a stable dimer, but can exist as a tetramer in solution. The crystal structure shows that dimericPaRecR forms a ring-like tetramer architectureviacrystal symmetry. The presence of a ligand in the Walker B motif of one RecR subunit suggests a putative nucleotide-binding site.
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Bashkirov, Vladimir I., Jeff S. King, Elena V. Bashkirova, Jacqueline Schmuckli-Maurer, and Wolf-Dietrich Heyer. "DNA Repair Protein Rad55 Is a Terminal Substrate of the DNA Damage Checkpoints." Molecular and Cellular Biology 20, no. 12 (June 15, 2000): 4393–404. http://dx.doi.org/10.1128/mcb.20.12.4393-4404.2000.
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ABSTRACT Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.
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Zhao, Lei, Chengyu Bao, Yuxuan Shang, Xinye He, Chiyuan Ma, Xiaohua Lei, Dong Mi, and Yeqing Sun. "The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks." BioMed Research International 2020 (August 25, 2020): 1–12. http://dx.doi.org/10.1155/2020/4834965.
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Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.
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Jalan, Manisha, Juber Patel, Kyrie S. Olsen, Sana Ahmed-Seghir, Daniel S. Higginson, Jorge S. Reis-Filho, Nadeem Riaz, and Simon N. Powell. "Abstract 5688: RNA-mediated DNA repair: A novel repair pathway in homologous recombination-deficient cancers." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5688. http://dx.doi.org/10.1158/1538-7445.am2022-5688.
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Abstract Genome instability has long been considered the primary driver of most cancer types. A double strand break (DSB) in DNA can have deleterious consequences for a cell, which if not repaired faithfully, can lead to mutations and chromosomal rearrangements, or even cell death. DSBs can be processed by several DNA repair pathways, of which homologous recombination (HR) is the preferred method due to its error-free nature. HR uses an intact homologous DNA sequence as a template for recovering the information lost at the break site. A significant proportion of all cancers, especially triple-negative breast, ovarian pancreatic and prostate cancers, have loss of function alterations affecting genes involved in HR-mediated DNA repair. Alternate repair pathways operate when HR is defective in tumors, but the pathways operative in this context remain a matter of contention. Previous work in vivo in yeast and in vitro systems has established a new role of RNA in DNA repair. Owing to its abundance in the cell and its sequence similarity to parental DNA, we sought to define whether RNA can act as a template for the repair of DSBs in human cells. We developed a novel high throughput assay to test if DNA breaks can be repaired using RNA as an alternative template in mammalian cells. Human cells were transfected with a guide RNA cloned in a Cas9 expression vector to generate a site-specific DSB at the AAVS1 locus, a safe harbour, in the human genome. Furthermore, a donor template in the form of DNA or RNA (homologous to the DSB locus) containing a unique mutational signature was provided at the time of transfection. The unique mutational signature enables us to determine if the donor has been utilized as a template for DNA repair. Using this assay, we demonstrate that cells can use a spliced RNA transcript as a functional template to repair a DSB. We have identified that Rev3L, a key component of the translesion synthesis polymerase Pol Zeta (ζ), has a novel reverse-transcriptase activity in human cells and can help repair the DSB using RNA as a template. Further characterization of this repair pathway and its associated mutational scar will provide new insights into the mutational signatures seen in HR-defective cancers, enabling a better understanding of the DNA repair pathways upregulated in these tumours. The proposed studies could help prioritize novel therapeutic approaches by exploiting synthetic lethality in HR-deficient cancers as well as HR-proficient cancers when used in combinatorial cancer therapy. Citation Format: Manisha Jalan, Juber Patel, Kyrie S Olsen, Sana Ahmed-Seghir, Daniel S Higginson, Jorge S Reis-Filho, Nadeem Riaz, Simon N Powell. RNA-mediated DNA repair: A novel repair pathway in homologous recombination-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5688.
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Matthews, Allysia J., Jee Eun Choi, and Bao Q. Vuong. "AID phosphorylation is required for base excision repair during class switch recombination." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 195.19. http://dx.doi.org/10.4049/jimmunol.198.supp.195.19.
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Abstract Activation-induced cytidine deaminase (AID) generates dU:dG mismatches in the immunoglobulin heavy chain locus during class switch recombination (CSR). The resolution of these mismatches occurs primarily through the base excision repair (BER) pathway and additionally through the mismatch repair (MMR) pathway. The disruption of either pathway results in a reduction in class switch recombination, whereas ablation of both pathways completely blocks CSR. The phosphorylation of AID at Ser38 (pS38-AID) is required for wild-type levels of CSR, in part through its interaction with APE1, an essential component of the BER pathway. B cells carrying a homozygous knock-in mutation of the AID phosphorylation site (AIDS38A/S38A) have intermediate levels of CSR as compared to wild-type and AID−/− B cells. CSR in AIDS38A/S38AUNG−/− and AIDS38A/S38AMSH2−/− B cells is completely blocked. This data parallels the CSR defect observed in UNG−/−MSH2−/− B cells as compared to UNG−/− or MSH2−/− B cells and genetically confirms the critical role of pS38-AID in the BER repair pathway during CSR.
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Sargent, R. G., M. A. Brenneman, and J. H. Wilson. "Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination." Molecular and Cellular Biology 17, no. 1 (January 1997): 267–77. http://dx.doi.org/10.1128/mcb.17.1.267.
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In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.
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Hill, Stuart A., Tracy Woodward, Andrew Reger, Rachel Baker, and Theresa Dinse. "Role for the RecBCD Recombination Pathway for pilE Gene Variation in Repair-Proficient Neisseria gonorrhoeae." Journal of Bacteriology 189, no. 22 (September 14, 2007): 7983–90. http://dx.doi.org/10.1128/jb.00980-07.
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ABSTRACT The role of the RecBCD recombination pathway in PilE antigenic variation in Neisseria gonorrhoeae is contentious and appears to be strain dependent. In this study, N. gonorrhoeae strain MS11 recB mutants were assessed for recombination/repair. MS11 recB mutants were found to be highly susceptible to DNA treatments that caused double-chain breaks and were severely impaired for growth; recB growth suppressor mutants arose at high frequencies. When the recombination/repair capacity of strain MS11 was compared to that of strains FA1090 and P9, innate differences were observed between the strains, with FA1090 and P9 rec + bacteria presenting pronounced recombination/repair defects. Consequently, MS11 recB mutants present a more robust phenotype than the other strains that were tested. In addition, MS11 recB mutants are also shown to be defective for pilE/pilS recombination. Moreover, pilE/pilS recombination is shown to proceed with gonococci that carry inverted pilE loci. Consequently, a novel RecBCD-mediated double-chain-break repair model for PilE antigenic variation is proposed.
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Jenkins, Chelsea, Jenny Kan, and Maureen E. Hoatlin. "Targeting the Fanconi Anemia Pathway to Identify Tailored Anticancer Therapeutics." Anemia 2012 (2012): 1–7. http://dx.doi.org/10.1155/2012/481583.
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The Fanconi Anemia (FA) pathway consists of proteins involved in repairing DNA damage, including interstrand cross-links (ICLs). The pathway contains an upstream multiprotein core complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and a downstream pathway that converges with a larger network of proteins with roles in homologous recombination and other DNA repair pathways. Selective killing of cancer cells with an intact FA pathway but deficient in certain other DNA repair pathways is an emerging approach to tailored cancer therapy. Inhibiting the FA pathway becomes selectively lethal when certain repair genes are defective, such as the checkpoint kinase ATM. Inhibiting the FA pathway in ATM deficient cells can be achieved with small molecule inhibitors, suggesting that new cancer therapeutics could be developed by identifying FA pathway inhibitors to treat cancers that contain defects that are synthetic lethal with FA.
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West, Stephen C. "Links Between Defective DNA Strand Break Repair and Genome Instability in Fanconi Anemia." Blood 120, no. 21 (November 16, 2012): SCI—2—SCI—2. http://dx.doi.org/10.1182/blood.v120.21.sci-2.sci-2.
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Abstract Abstract SCI-2 All living organisms feature DNA repair pathways that safeguard the integrity of the genome, and mutations in proteins that mediate key events in DNA repair have been linked to genome instability and tumorigenesis. Homologous recombination provides an important DNA repair pathway that is needed for the restoration and restart of broken replication forks, for the rejoining of chromosome/chromatid breaks, and for the processing of DNA cross-links. Mutations in genes that encode a variety of recombination proteins are linked to breast cancers and to heritable diseases such as Bloom syndrome (BS) and Fanconi anemia (FA). In recent work, we purified the BLM protein (defective in BS), the BRCA2 (FANCND1) and PALB2 (FANCN) tumor suppressors (mutated in some cases of FA), and the newly discovered FANCP protein, also known as SLX4, and have initiated structure-function analyses to elucidate their molecular functions. How these proteins process DNA, and how they are regulated and controlled to direct the outcome of recombinational repair is now revealing unexpected insights that extend our understanding of efficient DNA repair and tumor avoidance. Disclosures: No relevant conflicts of interest to declare.
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Yasmin, Tahirah, Benura Azeroglu, Charlotte A. Cockram, and David R. F. Leach. "Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair." PLOS Genetics 17, no. 8 (August 25, 2021): e1009717. http://dx.doi.org/10.1371/journal.pgen.1009717.
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Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.
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Lewis, L. Kevin, James W. Westmoreland, and Michael A. Resnick. "Repair of Endonuclease-Induced Double-Strand Breaks in Saccharomyces cerevisiae: Essential Role for Genes Associated with Nonhomologous End-Joining." Genetics 152, no. 4 (August 1, 1999): 1513–29. http://dx.doi.org/10.1093/genetics/152.4.1513.
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Abstract Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development.
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Kooistra, R., K. Vreeken, J. B. Zonneveld, A. de Jong, J. C. Eeken, C. J. Osgood, J. M. Buerstedde, P. H. Lohman, and A. Pastink. "The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination." Molecular and Cellular Biology 17, no. 10 (October 1997): 6097–104. http://dx.doi.org/10.1128/mcb.17.10.6097.
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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.
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Galitski, Timothy, and John R. Roth. "Pathways for Homologous Recombination Between Chromosomal Direct Repeats in Salmonella typhimurium." Genetics 146, no. 3 (July 1, 1997): 751–67. http://dx.doi.org/10.1093/genetics/146.3.751.
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Homologous recombination pathways probably evolved primarily to accomplish chromosomal repair and the formation and resolution of duplications by sister-chromosome exchanges. Various DNA lesions initiate these events. Classical recombination assays, involving bacterial sex, focus attention on double-strand ends of DNA. Sexual exchanges, initiated at these ends, depend on the RecBCD pathway. In the absence of RecBCD function, mutation of the sbcB and sbcC genes activates the apparently cryptic RecF pathway. To provide a more general view of recombination, we describe an assay in which endogenous DNA damage initiates recombination between chromosomal direct repeats. The repeats flank markers conferring lactose utilization (Lac+) and ampicillin resistance (ApR); recombination generates Lac-ApS segregants. In this assay, the RecF pathway is not cryptic; it plays a major role without sbcBC mutations. Others have proposed that single-strand gaps are the natural substrate for RecF-dependent recombination. Supporting this view, recombination stimulated by a double-strand break (DSB) in a chromosomal repeat depended on RecB function, not RecF function. Without RecBCD function, sbcBC mutations modified the RecF pathway and allowed it to catalyze DSB-stimulated recombination. Sexual recombination assays overestimate the importance of RecBCD and DSBs, and underestimate the importance of the RecF pathway.
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Lovett, S. T., C. Luisi-DeLuca, and R. D. Kolodner. "The genetic dependence of recombination in recD mutants of Escherichia coli." Genetics 120, no. 1 (September 1, 1988): 37–45. http://dx.doi.org/10.1093/genetics/120.1.37.
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Abstract RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.
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Jiang, Yuning. "Contribution of Microhomology to Genome Instability: Connection between DNA Repair and Replication Stress." International Journal of Molecular Sciences 23, no. 21 (October 26, 2022): 12937. http://dx.doi.org/10.3390/ijms232112937.
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Microhomology-mediated end joining (MMEJ) is a highly mutagenic pathway to repair double-strand breaks (DSBs). MMEJ was thought to be a backup pathway of homologous recombination (HR) and canonical nonhomologous end joining (C-NHEJ). However, it attracts more attention in cancer research due to its special function of microhomology in many different aspects of cancer. In particular, it is initiated with DNA end resection and upregulated in homologous recombination-deficient cancers. In this review, I summarize the following: (1) the recent findings and contributions of MMEJ to genome instability, including phenotypes relevant to MMEJ; (2) the interaction between MMEJ and other DNA repair pathways; (3) the proposed mechanistic model of MMEJ in DNA DSB repair and a new connection with microhomology-mediated break-induced replication (MMBIR); and (4) the potential clinical application by targeting MMEJ based on synthetic lethality for cancer therapy.
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Keung, Man, Yanyuan Wu, and Jaydutt Vadgama. "PARP Inhibitors as a Therapeutic Agent for Homologous Recombination Deficiency in Breast Cancers." Journal of Clinical Medicine 8, no. 4 (March 30, 2019): 435. http://dx.doi.org/10.3390/jcm8040435.
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Poly (ADP-ribose) polymerases (PARPs) play an important role in various cellular processes, such as replication, recombination, chromatin remodeling, and DNA repair. Emphasizing PARP’s role in facilitating DNA repair, the PARP pathway has been a target for cancer researchers in developing compounds which selectively target cancer cells and increase sensitivity of cancer cells to other anticancer agents, but which also leave normal cells unaffected. Since certain tumors (BRCA1/2 mutants) have deficient homologous recombination repair pathways, they depend on PARP-mediated base excision repair for survival. Thus, inhibition of PARP is a promising strategy to selectively kill cancer cells by inactivating complementary DNA repair pathways. Although PARP inhibitor therapy has predominantly targeted BRCA-mutated cancers, this review also highlights the growing conversation around PARP inhibitor treatment for non-BRCA-mutant tumors, those which exhibit BRCAness and homologous recombination deficiency. We provide an update on the field’s progress by considering PARP inhibitor mechanisms, predictive biomarkers, and clinical trials of PARP inhibitors in development. Bringing light to these findings would provide a basis for expanding the use of PARP inhibitors beyond BRCA-mutant breast tumors.
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Bärtsch, Stephan, Leslie E. Kang, and Lorraine S. Symington. "RAD51 Is Required for the Repair of Plasmid Double-Stranded DNA Gaps from Either Plasmid or Chromosomal Templates." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1194–205. http://dx.doi.org/10.1128/mcb.20.4.1194-1205.2000.
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ABSTRACT DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52epistasis group was tested in this system. RAD51,RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered fromrad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, orRAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not onRAD51. The residual repair events in rad51mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms forRAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically inrad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.
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Kennedy, Richard D., and Alan D. D'Andrea. "DNA Repair Pathways in Clinical Practice: Lessons From Pediatric Cancer Susceptibility Syndromes." Journal of Clinical Oncology 24, no. 23 (August 10, 2006): 3799–808. http://dx.doi.org/10.1200/jco.2005.05.4171.
Full textAbstract:
Human cancers exhibit genomic instability and an increased mutation rate due to underlying defects in DNA repair. Cancer cells are often defective in one of six major DNA repair pathways, namely: mismatch repair, base excision repair, nucleotide excision repair, homologous recombination, nonhomologous endjoining and translesion synthesis. The specific DNA repair pathway affected is predictive of the kinds of mutations, the tumor drug sensitivity, and the treatment outcome. The study of rare inherited DNA repair disorders, such as Fanconi anemia, has yielded new insights to drug sensitivity and treatment of sporadic cancers, such as breast or ovarian epithelial tumors, in the general population. The Fanconi anemia pathway is an example of how DNA repair pathways can be deregulated in cancer cells and how biomarkers of the integrity of these pathways could be useful as a guide to cancer management and may be used in the development of novel therapeutic agents.
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Milne, G. T., S. Jin, K. B. Shannon, and D. T. Weaver. "Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae." Molecular and Cellular Biology 16, no. 8 (August 1996): 4189–98. http://dx.doi.org/10.1128/mcb.16.8.4189.
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DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.
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Rijkers, Tonnie, Jody Van Den Ouweland, Bruno Morolli, Anton G. Rolink, Willy M. Baarends, Petra P. H. Van Sloun, Paul H. M. Lohman, and Albert Pastink. "Targeted Inactivation of Mouse RAD52Reduces Homologous Recombination but Not Resistance to Ionizing Radiation." Molecular and Cellular Biology 18, no. 11 (November 1, 1998): 6423–29. http://dx.doi.org/10.1128/mcb.18.11.6423.
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ABSTRACT The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant,MmRAD52 −/− ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.
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Eskes, Robert, Lu Liu, Hongwen Ma, Michael Y. Chao, Lorna Dickson, Alan M. Lambowitz, and Philip S. Perlman. "Multiple Homing Pathways Used by Yeast Mitochondrial Group II Introns." Molecular and Cellular Biology 20, no. 22 (November 15, 2000): 8432–46. http://dx.doi.org/10.1128/mcb.20.22.8432-8446.2000.
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ABSTRACT The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.
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Li, Jinbao, Huize Sun, Yulin Huang, Yali Wang, Yuyan Liu, and Xuefeng Chen. "Pathways and assays for DNA double-strand break repair by homologous recombination." Acta Biochimica et Biophysica Sinica 51, no. 9 (July 10, 2019): 879–89. http://dx.doi.org/10.1093/abbs/gmz076.
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AbstractDouble strand breaks (DSBs) are the most detrimental type of DNA damage that must be repaired to ensure genome integrity and cell survival. Unrepaired or improperly repaired DSBs can potentially cause tumorigenesis or cell death. DSBs are primarily repaired by non-homologous end joining or homologous recombination (HR). The HR pathway is initiated by processing of the 5′-end of DSBs to generate 3′-end single-strand DNA (ssDNA). Furthermore, the intermediate is channeled to one of the HR sub-pathways, including: (i) double Holliday junction (dHJ) pathway, (ii) synthesis-dependent strand annealing (SDSA), (iii) break-induced replication (BIR), and (iv) single-strand annealing (SSA). In the dHJ sub-pathway, the 3′-ssDNA coated with Rad51 recombinase performs homology search and strand invasion, forming a displacement loop (D-loop). Capture of the second end by the D-loop generates a dHJ intermediate that is subsequently dissolved by DNA helicase or resolved by nucleases, producing non-crossover or crossover products. In SDSA, the newly synthesized strand is displaced from the D-loop and anneals to the end on the other side of the DSBs, producing non-crossovers. In contrast, BIR repairs one-end DSBs by copying the sequence up to the end of the template chromosome, resulting in translocation or loss of heterozygosity. SSA takes place when resection reveals flanking homologous repeats that can anneal, leading to deletion of the intervening sequences. A variety of reporter assays have been developed to monitor distinct HR sub-pathways in both Saccharomyces cerevisiae and mammals. Here, we summarize the principles and representative assays for different HR sub-pathways with an emphasis on the studies in the budding yeast.
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Costantino, Lorenzo, Sotirios K. Sotiriou, Juha K. Rantala, Simon Magin, Emil Mladenov, Thomas Helleday, James E. Haber, George Iliakis, Olli P. Kallioniemi, and Thanos D. Halazonetis. "Break-Induced Replication Repair of Damaged Forks Induces Genomic Duplications in Human Cells." Science 343, no. 6166 (December 5, 2013): 88–91. http://dx.doi.org/10.1126/science.1243211.
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In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.