scholarly journals ATRX and RECQ5 define distinct homologous recombination subpathways

2021 ◽  
Vol 118 (3) ◽  
pp. e2010370118
Author(s):  
Amira Elbakry ◽  
Szilvia Juhász ◽  
Ki Choi Chan ◽  
Markus Löbrich
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Strand Break ◽  
Sister Chromatid Exchanges ◽  
Holliday Junctions ◽  
Nuclear Antigen ◽  
Sequence Information ◽  
Dna Double Strand Break ◽  
Break Site ◽  
Strand Annealing

Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

scholarly journals Repairing a double–strand chromosome break by homologous recombination: revisiting Robin Holliday's model

2004 ◽  
Vol 359 (1441) ◽  
pp. 79-86 ◽  
Author(s):  
James E. Haber ◽  
Gregorz Ira ◽  
Anna Malkova ◽  
Neal Sugawara
Keyword(s):  
Homologous Recombination ◽  
Repair Process ◽  
Strand Break ◽  
Holliday Junctions ◽  
Chromosome Break ◽  
Invasion Mechanism ◽  
Strand Invasion ◽  
Strand Annealing ◽  
Break Induced Replication ◽  
Mutant Cells

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

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 The Structure-Specific Endonuclease Ercc1-Xpf Is Required To Resolve DNA Interstrand Cross-Link-Induced Double-Strand Breaks

2004 ◽  
Vol 24 (13) ◽  
pp. 5776-5787 ◽  
Author(s):  
Laura J. Niedernhofer ◽  
Hanny Odijk ◽  
Magda Budzowska ◽  
Ellen van Drunen ◽  
Alex Maas ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Strand Break ◽  
Sister Chromatid Exchanges ◽  
Wild Type ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Normal Metabolism ◽  
Cross Links

ABSTRACT Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (γ-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced γ-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, γ-H2AX foci were also induced in Ercc1 −/− cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1 −/− cells, MMC-induced γ-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1 −/− and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

scholarly journals Mechanistic view and genetic control of DNA recombination during meiosis

2017 ◽  
Author(s):  
Marie-Claude Marsolier-Kergoat ◽  
Md Muntaz Khan ◽  
Jonathan Schott ◽  
Xuan Zhu ◽  
Bertrand Llorente
Keyword(s):  
Meiotic Recombination ◽  
Dna Recombination ◽  
Strand Break ◽  
Holliday Junctions ◽  
Switching Frequency ◽  
Template Switching ◽  
Genome Wide ◽  
Dna Double Strand Break ◽  
Strand Annealing ◽  
Mechanistic View

ABSTRACTMeiotic recombination is essential for fertility and allelic shuffling. Canonical recombination models fail to capture the observed complexity of meiotic recombinants. Here we revisit these models by analyzing meiotic heteroduplex DNA tracts genome-wide in combination with meiotic DNA double-strand break (DSB) locations. We provide unprecedented support to the synthesis-dependent strand annealing model and establish estimates of its associated template switching frequency and polymerase processivity. We show that resolution of double Holliday junctions (dHJs) is biased toward cleavage of the pair of strands containing newly synthesized DNA near the junctions. The suspected dHJ resolvase Mlh1-3 as well as Mlh1-2, Exo1 and Sgs1 promote asymmetric positioning of crossover intermediates relative to the initiating DSB and bidirectional conversions. Finally, we show that crossover-biased dHJ resolution depends on Mlh1-3, Exo1, Msh5 and to a lesser extent on Sgs1. These properties are likely conserved in eukaryotes containing the ZMM proteins, which includes mammals.

scholarly journals Break-Induced Loss of Heterozygosity in Fission Yeast: Dual Roles for Homologous Recombination in Promoting Translocations and Preventing De Novo Telomere Addition

2007 ◽  
Vol 27 (21) ◽  
pp. 7745-7757 ◽  
Author(s):  
Jason K. Cullen ◽  
Sharon P. Hussey ◽  
Carol Walker ◽  
John Prudden ◽  
Boon-Yu Wee ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Loss Of Heterozygosity ◽  
De Novo ◽  
Strand Break ◽  
Dual Roles ◽  
Dna Double Strand Break ◽  
Break Site ◽  
Break Induced Replication ◽  
End Resection ◽  
Causal Event

ABSTRACT Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss ∼25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32 +, rad50 +, nbs1 +, rhp51 +, rad22 +, rhp55 +, rhp54 +, and mus81 +. Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Δ and rhp55Δ backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Δ rhp55Δ strains, were reduced in exo1Δ rhp55Δ and an rhp55Δ strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.

scholarly journals Formation of Large Palindromic DNA by Homologous Recombination of Short Inverted Repeat Sequences in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (3) ◽  
pp. 1065-1075
Author(s):  
David K Butler ◽  
David Gillespie ◽  
Brandi Steele
Keyword(s):  
Homologous Recombination ◽  
Inverted Repeat ◽  
Excision Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Repeat Sequence ◽  
Inverted Repeat Sequence ◽  
Repeat Sequences ◽  
Dna Double Strand Break ◽  
Intermolecular Reaction

Abstract Large DNA palindromes form sporadically in many eukaryotic and prokaryotic genomes and are often associated with amplified genes. The presence of a short inverted repeat sequence near a DNA double-strand break has been implicated in the formation of large palindromes in a variety of organisms. Previously we have established that in Saccharomyces cerevisae a linear DNA palindrome is efficiently formed from a single-copy circular plasmid when a DNA double-strand break is introduced next to a short inverted repeat sequence. In this study we address whether the linear palindromes form by an intermolecular reaction (that is, a reaction between two identical fragments in a head-to-head arrangement) or by an unusual intramolecular reaction, as it apparently does in other examples of palindrome formation. Our evidence supports a model in which palindromes are primarily formed by an intermolecular reaction involving homologous recombination of short inverted repeat sequences. We have also extended our investigation into the requirement for DNA double-strand break repair genes in palindrome formation. We have found that a deletion of the RAD52 gene significantly reduces palindrome formation by intermolecular recombination and that deletions of two other genes in the RAD52-epistasis group (RAD51 and MRE11) have little or no effect on palindrome formation. In addition, palindrome formation is dramatically reduced by a deletion of the nucleotide excision repair gene RAD1.

scholarly journals RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

2014 ◽  
Vol 35 (2) ◽  
pp. 406-416 ◽  
Author(s):  
Su Chen ◽  
Chen Wang ◽  
Luxi Sun ◽  
Da-Liang Wang ◽  
Lu Chen ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Open Chromatin ◽  
Dsb Repair ◽  
Heterochromatin Protein ◽  
Dna Double Strand Break ◽  
Recombination Repair ◽  
Ubiquitin Conjugating Enzyme

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.

Structural insights into DNA double-strand break signaling

2021 ◽  
Vol 478 (1) ◽  
pp. 135-156
Author(s):  
Rashmi Panigrahi ◽  
J. N. Mark Glover
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Signaling Proteins ◽  
Protein Signaling ◽  
Strand Breaks ◽  
Large Size ◽  
Dna Double Strand Break ◽  
Break Site ◽  
Anti Cancer ◽  
Structural Principles

Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein–protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

1994 ◽  
Vol 14 (1) ◽  
pp. 400-406
Author(s):  
W P Deng ◽  
J A Nickoloff
Keyword(s):  
Dna Replication ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Strand Break ◽  
Wild Type ◽  
Heteroduplex Dna ◽  
Frameshift Mutations ◽  
Single Strand Annealing ◽  
Extrachromosomal Recombination ◽  
Strand Annealing

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.

scholarly journals Schizosaccharomyces pombe KAT5 contributes to resection and repair of a DNA double strand break

Genetics ◽  
2021 ◽  
Author(s):  
Tingting Li ◽  
Ruben C Petreaca ◽  
Susan L Forsburg
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Chromatin Remodeling ◽  
Dna Damage Response ◽  
Histone Acetyltransferase ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Damage Checkpoint ◽  
Dna Double Strand Break ◽  
Damage Response

Abstract Chromatin remodeling is essential for effective repair of a DNA double strand break. KAT5 (S. pombe Mst1, human TIP60) is a MYST family histone acetyltransferase conserved from yeast to humans that coordinates various DNA damage response activities at a DNA double strand break (DSB), including histone remodeling and activation of the DNA damage checkpoint. In S. pombe, mutations in mst1+ causes sensitivity to DNA damaging drugs. Here we show that Mst1 is recruited to DSBs. Mutation of mst1+ disrupts recruitment of repair proteins and delays resection. These defects are partially rescued by deletion of pku70, which has been previously shown to antagonize repair by homologous recombination. These phenotypes of mst1 are similar to pht1-4KR, a non-acetylatable form of histone variant H2A.Z, which has been proposed to affect resection. Our data suggest that Mst1 functions to direct repair of DSBs towards homologous recombination pathways by modulating resection at the double strand break.

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