Faculty Opinions recommendation of Phosphorylation of Slx4 by Mec1 and Tel1 regulates the single-strand annealing mode of DNA repair in budding yeast.

ABSTRACT Budding yeast (Saccharomyces cerevisiae) Slx4 is essential for cell viability in the absence of the Sgs1 helicase and for recovery from DNA damage. Here we report that cells lacking Slx4 have difficulties in completing DNA synthesis during recovery from replisome stalling induced by the DNA alkylating agent methyl methanesulfonate (MMS). Although DNA synthesis restarts during recovery, cells are left with unreplicated gaps in the genome despite an increase in translesion synthesis. In this light, epistasis experiments show that SLX4 interacts with genes involved in error-free bypass of DNA lesions. Slx4 associates physically, in a mutually exclusive manner, with two structure-specific endonucleases, Rad1 and Slx1, but neither of these enzymes is required for Slx4 to promote resistance to MMS. However, Rad1-dependent DNA repair by single-strand annealing (SSA) requires Slx4. Strikingly, phosphorylation of Slx4 by the Mec1 and Tel1 kinases appears to be essential for SSA but not for cell viability in the absence of Sgs1 or for cellular resistance to MMS. These results indicate that Slx4 has multiple functions in responding to DNA damage and that a subset of these are regulated by Mec1/Tel1-dependent phosphorylation.

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

Journal of Bacteriology ◽

10.1128/jb.00290-15 ◽

2015 ◽

Vol 197 (19) ◽

pp. 3121-3132 ◽

Cited By ~ 11

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.

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Strand asymmetry influences mismatch repair during single-strand annealing

10.1101/847160 ◽

2019 ◽

Cited By ~ 1

Author(s):

Victoria O. Pokusaeva ◽

Aránzazu Rosado Diez ◽

Lorena Espinar ◽

Guillaume J. Filion

Keyword(s):

Dna Repair ◽

Mismatch Repair ◽

Tandem Repeats ◽

Embryonic Stem ◽

Intrinsic Property ◽

Strand Break ◽

Single Strand ◽

A Genome ◽

Single Strand Annealing ◽

Strand Annealing

ABSTRACTBiases of DNA repair can shape the nucleotide landscape of genomes at evolutionary timescales. However, such biases have not yet been measured in chromatin for lack of technologies. Here we develop a genome-wide assay whereby the same DNA lesion is repaired in different chromatin contexts. We insert thousands of barcoded transposons carrying a reporter of DNA mismatch repair in the genome of mouse embryonic stem cells. Upon inducing a double-strand break between tandem repeats, a mismatch is generated when the single strand annealing repair pathway is used. Surprisingly, the mismatch repair machinery favors the same strand 60-80% of the time. The location of the lesion in the genome and the type of mismatch have little influence on the repair bias in this context. Using machine learning, we further show that both the repair bias and the efficiency of the repair are independent of known chromatin features. These results suggest that some intrinsic property of the lesion can have a large influence on the outcome of DNA repair, irrespective of the surrounding chromatin context.

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Single-Strand Annealing in Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms22042167 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2167

Author(s):

Janusz Blasiak

Keyword(s):

Dna Repair ◽

Synthetic Lethality ◽

Single Strand ◽

Repair System ◽

Dna Double Strand Breaks ◽

End Joining ◽

Cancer Pathogenesis ◽

Single Strand Annealing ◽

Non Homologous End Joining ◽

Strand Annealing

DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.

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

Genetics ◽

10.1093/genetics/159.2.515 ◽

2001 ◽

Vol 159 (2) ◽

pp. 515-525 ◽

Cited By ~ 2

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.

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