scholarly journals Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast

PLoS Genetics ◽  
2017 ◽  
Vol 13 (9) ◽  
pp. e1007013 ◽  
Author(s):  
Arancha Sanchez ◽  
Mariana C. Gadaleta ◽  
Oliver Limbo ◽  
Paul Russell
Keyword(s):  
Fission Yeast ◽  
Single Strand ◽  
Replication Forks ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Single Strand Breaks ◽  
Polynucleotide Kinase

scholarly journals Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in polynucleotide kinase/phosphatase mutant of fission yeast

2017 ◽  
Author(s):  
Arancha Sanchez ◽  
Mariana C. Gadaleta ◽  
Oliver Limbo ◽  
Paul Russell
Keyword(s):  
Dna Repair ◽  
Sister Chromatid ◽  
Neurological Disease ◽  
Single Strand ◽  
Replication Forks ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Single Strand Breaks ◽  
Polynucleotide Kinase ◽  
Sister Chromatid Recombination

ABSTRACTThe DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) protects genome integrity by restoring ligatable 5’-phosphate and 3’-hydroxyl termini at single-strand breaks (SSBs). In humans, PNKP mutations underlie the neurological disease known as MCSZ, but these individuals are not predisposed for cancer, implying effective alternative repair pathways in dividing cells. Homology-directed repair (HDR) of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells, but the critical HDR protein Rad51 is not required in PNKP-null (pnk1Δ) cells of Schizosaccharomyces pombe. Here, we report that pnk1Δ cells have enhanced requirements for Rad3 (ATR/Mec1) and Chk1 checkpoint kinases, and the multi-BRCT domain protein Brc1 that binds phospho-histone H2A (γH2A) at damaged replication forks. The viability of pnk1Δ cells depends on Mre11 and Ctp1 (CtIP/Sae2) double-strand break (DSB) resection proteins, Rad52 DNA strand annealing protein, Mus81-Eme1 Holliday junction resolvase, and Rqh1 (BLM/WRN/Sgs1) DNA helicase. Eliminating Pnk1 strongly sensitizes mre11Δ pku80Δ cells to DNA damaging agents that collapse replication forks, indicating a requirement for Mre11-Rad50-Nbs1 (MRN) protein complex that cannot be efficiently replaced by Exo1 5’-3’ exonuclease. Coupled with increased sister chromatid recombination and Rad52 repair foci in pnk1Δ cells, these findings indicate that lingering SSBs in pnk1Δ cells trigger Rad51-independent homology-directed repair of collapsed replication forks.AUTHOR SUMMARYDNA is constantly damaged by normal cellular metabolism, for example production of reactive oxygen species, or from exposure to external DNA damaging sources, such as radiation from the sun or chemicals in the environment. These genotoxic agents create thousands of single-strand breaks/cell/day in the human body. An essential DNA repair protein known as polynucleotide kinase/phosphatase (PNKP) makes sure the single-strand breaks have 5’ phosphate and 3’ hydroxyl ends suitable for healing by DNA ligase. Mutations that reduce PNKP activity cause a devastating neurological disease but surprisingly not cancer, suggesting that other DNA repair mechanisms step into the breach in dividing PNKP-deficient cells. One popular candidate was homology-directed repair (HDR) of replication forks that collapse at single-strand breaks, but the crucial HDR protein Rad51 was found to be non-essential in PNKP-deficient cells of fission yeast. In this study, Sanchez and Russell revive the HDR model by showing that SSBs in PNKP-deficient cells are repaired by a variant HDR mechanism that bypasses the requirement for Rad51. Notably, Mus81 endonuclease that resolves sister chromatid recombination structures formed during HDR of collapsed replication forks was found to be essential in PNKP-deficient cells.

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

2010 ◽  
Vol 402 (1) ◽  
pp. 70-82 ◽  
Author(s):  
Petra Groth ◽  
Simon Ausländer ◽  
Muntasir Mamun Majumder ◽  
Niklas Schultz ◽  
Fredrik Johansson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Strand ◽  
Replication Forks ◽  
Strand Breaks ◽  
Methylated Dna ◽  
Single Strand Breaks

scholarly journals Spontaneous Homologous Recombination Is Induced by Collapsed Replication Forks That Are Caused by Endogenous DNA Single-Strand Breaks

2005 ◽  
Vol 25 (16) ◽  
pp. 7158-7169 ◽  
Author(s):  
Nasrollah Saleh-Gohari ◽  
Helen E. Bryant ◽  
Niklas Schultz ◽  
Kayan M. Parker ◽  
Tobias N. Cassel ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Line ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Fibroblast Cell ◽  
Single Strand ◽  
Replication Forks ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Recombination Repair

ABSTRACT Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous γH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that γH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.

Mechanism of chromosome rearrangement arising from single-strand breaks

2021 ◽  
Author(s):  
Palina Kot ◽  
Takaaki Yasuhara ◽  
Atsushi Shibata ◽  
Miyako Hirakawa ◽  
Yu Abe ◽  
...  
Keyword(s):  
Chromosome Rearrangement ◽  
Single Strand ◽  
Strand Breaks ◽  
Single Strand Breaks
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