scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

scholarly journals Effects of DNA Structure and Homology Length on Vaccinia Virus Recombination

2001 ◽  
Vol 75 (15) ◽  
pp. 6923-6932 ◽  
Author(s):  
Xiao-Dan Yao ◽  
David H. Evans
Keyword(s):  
Vaccinia Virus ◽  
Dna Sequences ◽  
Accurate Method ◽  
Single Strand ◽  
Recombination Mechanism ◽  
Virus Recombination ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACT Replicating poxviruses catalyze high-frequency recombination reactions by a process that is not well understood. Using transfected DNA substrates we show that these viruses probably use a single-strand annealing recombination mechanism. Plasmids carrying overlapping portions of a luciferase gene expression cassette and luciferase assays were first shown to provide an accurate method of assaying recombinant frequencies. We then transfected pairs of DNAs into virus-infected cells and monitored the efficiencies of linear-by-linear, linear-by-circle, and circle-by-circle recombination. These experiments showed that vaccinia virus recombination systems preferentially catalyze linear-by-linear reactions much more efficiently than circle-by-circle reactions and catalyze circle-by-circle reactions more efficiently than linear-by-circle reactions. Reactions involving linear substrates required surprisingly little sequence identity, with only 16-bp overlaps still permitting ∼4% recombinant production. Masking the homologies by adding unrelated DNA sequences to the ends of linear substrates inhibited recombination in a manner dependent upon the number of added sequences. Circular molecules were also recombined by replicating viruses but at frequencies 15- to 50-fold lower than are linear substrates. These results are consistent with mechanisms in which exonuclease or helicase processing of DNA ends permits the forming of recombinants through annealing of complementary single strands. Our data are not consistent with a model involving strand invasion reactions, because such reactions should favor mixtures of linear and circular substrates. We also noted that many of the reaction features seen in vivo were reproduced in a simple in vitro reaction requiring only purified vaccinia virus DNA polymerase, single-strand DNA binding protein, and pairs of linear substrates. The 3′-to-5′ exonuclease activity of poxviral DNA polymerases potentially catalyzes recombination in vivo.

scholarly journals Rejoining of DNA double-strand breaks in vitro by single-strand annealing

1998 ◽  
Vol 258 (2) ◽  
pp. 387-395 ◽  
Author(s):  
Bernd Gottlich ◽  
Susanne Reichenberger ◽  
Elke Feldmann ◽  
Petra Pfeiffer
Keyword(s):  
Double Strand Breaks ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Strand Annealing

scholarly journals Expanding Diversity of Firmicutes Single-Strand Annealing Proteins: A Putative Role of Bacteriophage-Host Arms Race

2021 ◽  
Vol 12 ◽  
Author(s):  
Kamil Steczkiewicz ◽  
Eric Prestel ◽  
Elena Bidnenko ◽  
Agnieszka K. Szczepankowska
Keyword(s):  
Dna Sequences ◽  
Sequence Similarity ◽  
Reca Protein ◽  
Arms Race ◽  
Single Strand ◽  
Bacterial Phyla ◽  
Single Strand Annealing ◽  
Strand Annealing

Bacteriophage-encoded single strand annealing proteins (SSAPs) are recombinases which can substitute the classical, bacterial RecA and manage the DNA metabolism at different steps of phage propagation. SSAPs have been shown to efficiently promote recombination between short and rather divergent DNA sequences and were exploited for in vivo genetic engineering mainly in Gram-negative bacteria. In opposition to the conserved and almost universal bacterial RecA protein, SSAPs display great sequence diversity. The importance for SSAPs in phage biology and phage-bacteria evolution is underlined by their role as key players in events of horizontal gene transfer (HGT). All of the above provoke a constant interest for the identification and study of new phage recombinase proteins in vivo, in vitro as well as in silico. Despite this, a huge body of putative ssap genes escapes conventional classification, as they are not properly annotated. In this work, we performed a wide-scale identification, classification and analysis of SSAPs encoded by the Firmicutes bacteria and their phages. By using sequence similarity network and gene context analyses, we created a new high quality dataset of phage-related SSAPs, substantially increasing the number of annotated SSAPs. We classified the identified SSAPs into seven distinct families, namely RecA, Gp2.5, RecT/Redβ, Erf, Rad52/22, Sak3, and Sak4, organized into three superfamilies. Analysis of the relationships between the revealed protein clusters led us to recognize Sak3-like proteins as a new distinct SSAP family. Our analysis showed an irregular phylogenetic distribution of ssap genes among different bacterial phyla and specific phages, which can be explained by the high rates of ssap HGT. We propose that the evolution of phage recombinases could be tightly linked to the dissemination of bacterial phage-resistance mechanisms (e.g., abortive infection and CRISPR/Cas systems) targeting ssap genes and be a part of the constant phage-bacteria arms race.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

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

2015 ◽  
Vol 197 (19) ◽  
pp. 3121-3132 ◽  
Author(s):  
Richa Gupta ◽  
Stewart Shuman ◽  
Michael S. Glickman
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Single Strand ◽  
Central Position ◽  
End Joining ◽  
Dna Double Strand Break ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACTMycobacteria 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 ofadnABorrecOindividually causes partial impairment of HR, but loss ofadnABandrecOin combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNAin 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 ofrecFandrecRin 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.IMPORTANCEThis 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.

scholarly journals RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair

2018 ◽  
Vol 115 (35) ◽  
pp. E8286-E8295 ◽  
Author(s):  
Liwei An ◽  
Chao Dong ◽  
Junshi Li ◽  
Jie Chen ◽  
Jingsong Yuan ◽  
...  
Keyword(s):  
Single Strand ◽  
Fine Tuning ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Molecular Rheostat ◽  
Dna End Resection ◽  
Strand Annealing ◽  
End Resection

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

scholarly journals Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB

2016 ◽  
Vol 113 (16) ◽  
pp. 4308-4313 ◽  
Author(s):  
Seiji N. Sugiman-Marangos ◽  
Yoni M. Weiss ◽  
Murray S. Junop
Keyword(s):  
Deinococcus Radiodurans ◽  
Double Strand Breaks ◽  
Single Strand ◽  
High Fidelity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Single Strand Annealing ◽  
Dna Strands ◽  
Strand Annealing

Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.

scholarly journals FKBP25 participates in DNA double-strand break repair

2020 ◽  
Vol 98 (1) ◽  
pp. 42-49 ◽  
Author(s):  
David Dilworth ◽  
Fade Gong ◽  
Kyle Miller ◽  
Christopher J. Nelson
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Peptide Bonds ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Fk506 Binding Proteins

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis–trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25’s catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

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