Mitotic cells can repair DNA double-strand breaks via a homology-directed pathway

Abstract The choice of repair pathways of DNA double-strand breaks (DSBs) is dependent upon the cell cycle phases. While homologous recombination repair (HRR) is active between the S and G2 phases, its involvement in mitotic DSB repair has not been examined in detail. In the present study, we developed a new reporter assay system to detect homology-directed repair (HDR), a major pathway used for HRR, in combination with an inducible DSB-generation system. As expected, the maximal HDR activity was observed in the late S phase, along with minimal activity in the G1 phase and at the G1/S boundary. Surprisingly, significant HDR activity was observed in M phase, and the repair efficiency was similar to that observed in late S phase. HDR was also confirmed in metaphase cells collected with continuous colcemid exposure. ChIP assays revealed the recruitment of RAD51 to the vicinity of DSBs in M phase. In addition, the ChIP assay for gamma-H2AX and phosphorylated DNA-PKcs indicated that a part of M-phase cells with DSBs could proceed into the next G1 phase. These results provide evidence showing that a portion of mitotic cell DSBs are undoubtedly repaired through action of the HDR repair pathway.

Download Full-text

DNA damage triggers increased mobility of chromosomes in G1-phase cells

Molecular Biology of the Cell ◽

10.1091/mbc.e19-08-0469 ◽

2019 ◽

Vol 30 (21) ◽

pp. 2620-2625 ◽

Cited By ~ 5

Author(s):

Michael J. Smith ◽

Eric E. Bryant ◽

Fraulin J. Joseph ◽

Rodney Rothstein

Keyword(s):

Dna Damage ◽

S Phase ◽

Double Strand Breaks ◽

Homology Search ◽

G1 Phase ◽

Dna Damage Checkpoint ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Global Mobility ◽

Break Site

During S phase in Saccharomyces cerevisiae, chromosomal loci become mobile in response to DNA double-strand breaks both at the break site (local mobility) and throughout the nucleus (global mobility). Increased nuclear exploration is regulated by the recombination machinery and the DNA damage checkpoint and is likely an important aspect of homology search. While mobility in response to DNA damage has been studied extensively in S phase, the response in interphase has not, and the question of whether homologous recombination proceeds to completion in G1 phase remains controversial. Here, we find that global mobility is triggered in G1 phase. As in S phase, global mobility in G1 phase is controlled by the DNA damage checkpoint and the Rad51 recombinase. Interestingly, despite the restriction of Rad52 mediator foci to S phase, Rad51 foci form at high levels in G1 phase. Together, these observations indicate that the recombination and checkpoint machineries promote global mobility in G1 phase, supporting the notion that recombination can occur in interphase diploids.

Download Full-text

Poly(ADP-ribose) glycohydrolase promotes formation and homology-directed repair of meiotic DNA double-strand breaks independent of its catalytic activity

10.1101/2020.03.12.988840 ◽

2020 ◽

Cited By ~ 2

Author(s):

Eva Janisiw ◽

Marilina Raices ◽

Fabiola Balmir ◽

Luis Paulin Paz ◽

Antoine Baudrimont ◽

...

Keyword(s):

Catalytic Activity ◽

Synaptonemal Complex ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Post Translational Modification ◽

Dna Breaks ◽

Strand Breaks ◽

Homology Directed Repair ◽

Damage Repair ◽

Somatic Tissues

SummaryPoly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in promoting the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.

Download Full-text

Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells

Nucleic Acids Research ◽

10.1093/nar/gkh703 ◽

2004 ◽

Vol 32 (12) ◽

pp. 3683-3688 ◽

Cited By ~ 155

Author(s):

N. Saleh-Gohari

Keyword(s):

Cell Cycle ◽

Homologous Recombination ◽

S Phase ◽

Human Cells ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks

Download Full-text

Protein phosphatase PP6 is required for homology-directed repair of DNA double-strand breaks

Cell Cycle ◽

10.4161/cc.10.9.15479 ◽

2011 ◽

Vol 10 (9) ◽

pp. 1411-1419 ◽

Cited By ~ 26

Author(s):

Jianing Zhong ◽

Ji Liao ◽

Xin Liu ◽

Pei Wang ◽

Jinping Liu ◽

...

Keyword(s):

Protein Phosphatase ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Homology Directed Repair

Download Full-text

Abstract 688: Acetylation of poly-ADP-ribose polymerase and Ku70 by histone deacetylase inhibitors promote abnormal binding to DNA double strand breaks and decrease repair efficiency in leukemia cells

10.1158/1538-7445.am10-688 ◽

2010 ◽

Author(s):

Carine Robert ◽

Ivana Gojo ◽

Feyruz Rassool

Keyword(s):

Histone Deacetylase ◽

Histone Deacetylase Inhibitors ◽

Double Strand Breaks ◽

Leukemia Cells ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Repair Efficiency

Download Full-text

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507833112 ◽

2015 ◽

Vol 112 (50) ◽

pp. E6907-E6916 ◽

Cited By ~ 16

Author(s):

Damon Meyer ◽

Becky Xu Hua Fu ◽

Wolf-Dietrich Heyer

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Polymerase ◽

Genome Stability ◽

Double Strand Breaks ◽

Dna Polymerase Delta ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Repair Efficiency ◽

Repair Pathways

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.

Download Full-text

Evaluation of DNA Double Strand Breaks Repair Efficiency in Head and Neck Cancer

DNA and Cell Biology ◽

10.1089/dna.2011.1325 ◽

2012 ◽

Vol 31 (3) ◽

pp. 298-305 ◽

Cited By ~ 10

Author(s):

Anna Walczak ◽

Pawel Rusin ◽

Lukasz Dziki ◽

Hanna Zielinska-Blizniewska ◽

Jurek Olszewski ◽

...

Keyword(s):

Head And Neck Cancer ◽

Head And Neck ◽

Neck Cancer ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Repair Efficiency

Download Full-text

The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks

Genes ◽

10.3390/genes12091430 ◽

2021 ◽

Vol 12 (9) ◽

pp. 1430

Author(s):

Ian Yannuzzi ◽

Margaret A. Butler ◽

Joel Fernandez ◽

Jeannine R. LaRocque

Keyword(s):

Direct Repeat ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Strand Breaks ◽

Homology Directed Repair ◽

Dna End Resection ◽

The Impact ◽

End Resection

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.

Download Full-text