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

Genes ◽  
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.

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 Conservative Repair of a Chromosomal Double-Strand Break by Single-Strand DNA through Two Steps of Annealing

2006 ◽  
Vol 26 (20) ◽  
pp. 7645-7657 ◽  
Author(s):  
Francesca Storici ◽  
Joyce R. Snipe ◽  
Godwin K. Chan ◽  
Dmitry A. Gordenin ◽  
Michael A. Resnick
Keyword(s):  
Normal Cell ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Repair Mechanisms ◽  
Strand Invasion ◽  
Strand Annealing

ABSTRACT The repair of chromosomal double-strand breaks (DSBs) is essential to normal cell growth, and homologous recombination is a universal process for DSB repair. We explored DSB repair mechanisms in the yeast Saccharomyces cerevisiae using single-strand oligonucleotides with homology to both sides of a DSB. Oligonucleotide-directed repair occurred exclusively via Rad52- and Rad59-mediated single-strand annealing (SSA). Even the SSA domain of human Rad52 provided partial complementation for a null rad52 mutation. The repair did not involve Rad51-driven strand invasion, and moreover the suppression of strand invasion increased repair with oligonucleotides. A DSB was shown to activate targeting by oligonucleotides homologous to only one side of the break at large distances (at least 20 kb) from the break in a strand-biased manner, suggesting extensive 5′ to 3′ resection, followed by the restoration of resected DNA to the double-strand state. We conclude that long resected chromosomal DSB ends are repaired by a single-strand DNA oligonucleotide through two rounds of annealing. The repair by single-strand DNA can be conservative and may allow for accurate restoration of chromosomal DNAs with closely spaced DSBs.

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

Multi-Pathway DNA Double-Strand Break Repair Reporters Reveal Extensive Cross-Talk Between End-Joining, Single Strand Annealing, and Homologous Recombination

2021 ◽  
Author(s):  
Bert van de Kooij ◽  
Alex Kruswick ◽  
Haico van Attikum ◽  
Michael B. Yaffe
Keyword(s):  
Homologous Recombination ◽  
Cross Talk ◽  
Single Strand ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Alu Elements ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
End Resection

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to extensive mutagenesis and genomic loss. Repair pathway cross-talk and compensation within the DSB-repair network is incompletely understood, despite its importance for genomic stability, oncogenesis, and the outcome of genome editing by CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple distinct pathways to repair of a DSB. These reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibition of the essential c-NHEJ factor DNA-PKcs not only increases repair by HR, but also results in a substantial increase in mutagenic repair by SSA. We show that SSA-mediated repair of Cas9-generated DSBs can occur between Alu elements at endogenous genomic loci, and is enhanced by inhibition of DNA-PKcs. Finally, we demonstrate that the short-range end-resection factors CtIP and Mre11 promote both SSA and HR, whereas the long-range end-resection factors DNA2 and Exo1 promote SSA, but reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the rapid and comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.

scholarly journals BCR-ABL promotes the frequency of mutagenic single-strand annealing DNA repair

Blood ◽  
2009 ◽  
Vol 114 (9) ◽  
pp. 1813-1819 ◽  
Author(s):  
Margret S. Fernandes ◽  
Mamatha M. Reddy ◽  
Jeffrey R. Gonneville ◽  
Scott C. DeRoo ◽  
Klaus Podar ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Growth Factor Receptor ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Model Systems ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Strand Annealing

Intracellular oxidative stress in cells transformed by the BCR-ABL oncogene is associated with increased DNA double-strand breaks. Imprecise repair of these breaks can result in the accumulation of mutations, leading to therapy-related drug resistance and disease progression. Using several BCR-ABL model systems, we found that BCR-ABL specifically promotes the repair of double-strand breaks through single-strand annealing (SSA), a mutagenic pathway that involves sequence repeats. Moreover, our results suggest that mutagenic SSA repair can be regulated through the interplay between BCR-ABL and extrinsic growth factors. Increased SSA activity required Y177 in BCR-ABL, as well as a functional PI3K and Ras pathway downstream of this site. Furthermore, our data hint at a common pathway for DSB repair whereby BCR-ABL, Tel-ABL, Tel-PDGFR, FLT3-ITD, and Jak2V617F all increase mutagenic repair. This increase in SSA may not be sufficiently suppressed by tyrosine kinase inhibitors in the stromal microenvironment. Therefore, drugs that target growth factor receptor signaling represent potential therapeutic agents to combat tyrosine kinase-induced genomic instability.

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