“…Cells harbouring nuclease dead dna2 -1 were more sensitive than dna2Δ pif1 -m2 mutants to Phleomycin, an agent that causes DNA double strand breaks, but less sensitive to hydroxyurea (HU), an agent inducing replication stress (Fig. 1A, [26, 27]). While sensitivities to various genotoxic stressors have been previously reported with dna2 mutants, there has been little work explaining why dna2 -1 mutants show greater sensitivity to DSB causing agents compared to cells harbouring the deletion of DNA2 or its binding partner, SGS1 (Figs.…”
Section: Resultsmentioning
confidence: 99%
“…However, there is a gap in understanding the regulation of Dna2 at DSBs as nuclease deficient dna2 -1 (P504S) shows greater sensitivity to DSB-inducing agents compared to dna2Δ pif1 -m2 (Fig. 1A) [26, 27].…”
A DNA double strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination (HR) or non-homologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of HR, is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harbouring nuclease dead dna2-1 compared to dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harbouring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater defect in resection compared to cells where DNA2 was deleted. Both the decreased level of resection and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change, however Nej1 increased as did the frequency of alt-EJ/ MMEJ repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.
“…Cells harbouring nuclease dead dna2 -1 were more sensitive than dna2Δ pif1 -m2 mutants to Phleomycin, an agent that causes DNA double strand breaks, but less sensitive to hydroxyurea (HU), an agent inducing replication stress (Fig. 1A, [26, 27]). While sensitivities to various genotoxic stressors have been previously reported with dna2 mutants, there has been little work explaining why dna2 -1 mutants show greater sensitivity to DSB causing agents compared to cells harbouring the deletion of DNA2 or its binding partner, SGS1 (Figs.…”
Section: Resultsmentioning
confidence: 99%
“…However, there is a gap in understanding the regulation of Dna2 at DSBs as nuclease deficient dna2 -1 (P504S) shows greater sensitivity to DSB-inducing agents compared to dna2Δ pif1 -m2 (Fig. 1A) [26, 27].…”
A DNA double strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination (HR) or non-homologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of HR, is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harbouring nuclease dead dna2-1 compared to dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harbouring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater defect in resection compared to cells where DNA2 was deleted. Both the decreased level of resection and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change, however Nej1 increased as did the frequency of alt-EJ/ MMEJ repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.
“…The generated gap is filled by the DNA polymerase ο (Pol ο) or Pol χ holoenzyme (37,41,42). When EXO1 is not available, a 5’ strand break created by the activated MutLα endonuclease is utilized by the DNA polymerase ο holoenzyme to remove the mismatch in a strand-displacement DNA synthesis reaction (38,42) enhanced by the nuclease activity of DNA2 (39).…”
Section: Introductionmentioning
confidence: 99%
“…Eukaryotic MMR proceeds via a series of coordinated events that include mismatch recognition, incision of the daughter strand near the mismatch, and mismatch removal (9,26,36). Biochemical studies have resulted in the reconstitution of MutLa-dependent and independent MMR pathways from purified components (37)(38)(39). A critical step in MutLa-dependent MMR pathways is a mismatch-, MutSa-, PCNA-, RFC-, and ATP-dependent incision of the discontinuous strand by the endonuclease activity of MutLa (23).…”
The DNA mismatch repair (MMR) system promotes genome stability and protects humans from certain types of cancer. Its primary function is the correction of DNA polymerase errors. MutLα is an important eukaryotic MMR factor. We have examined the contributions of MutLα to maintaining genome stability. We show here that loss of MutLα in yeast increases the genome-wide mutation rate by ~130-fold and generates a genome-wide mutation spectrum that consists of small indels and base substitutions. We also show that loss of yeast MutLα leads to error-prone MMR that produces T>C base substitutions in 5'-ATA-3' sequences. In agreement with this finding, our examination of human whole genome DNA sequencing data has revealed that loss of MutLα in induced pluripotent stem cells triggers error-prone MMR that leads to the formation of T>C mutations in 5'-NTN-3' sequences. Our further analysis has shown that MutLα-independent MMR plays a role in suppressing base substitutions in N3homopolymeric runs. In addition, we describe that MutLα preferentially defends noncoding DNA from mutations. Our study defines the contributions of MutLα-dependent and independent mechanisms to genome-wide MMR.
“…However, there is a gap in understanding the regulation of Dna2 at DSBs as nuclease-deficient dna2 -1 (P504S) shows greater sensitivity to DSB-inducing agents compared with dna2 Δ pif1 -m2 ( Fig. 1 A ) ( 26 , 27 ). Figure 1 Nuclease-deficient dna2 -1 leads to compromised resection at DSB.…”
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