Capture of Extranuclear DNA at Fission Yeast Double-Strand Breaks

Decottignies, Anabelle

doi:10.1534/genetics.105.046144

Cited by 33 publications

(52 citation statements)

References 74 publications

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“…Typically, 100 ng of linearized or supercoiled DNA were transformed into yeast by using electroporation (31). We chose to use a carrier-free transformation procedure to eliminate possible complications from plasmid recombination reactions with carrier DNA (19,32). Electroporation conditions were 0.75 V, 25 μF, and 200 Ω by using cuvettes with a 0.1-cm gap width (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Yeast Tdp1 regulates the fidelity of nonhomologous end joining

Bahmed

Nitiss

2010

Proc. Natl. Acad. Sci. U.S.A.

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Tyrosyl-DNA-phosphodiesterase 1 (Tdp1) can disjoin peptides covalently bound to DNA. We assessed the role of Tdp1 in nonhomologous end joining (NHEJ) and found that linear DNA molecules with 5′ extensions showed a high frequency of misrepair in Δtdp1 cells. The joining errors in Δtdp1 cells were predominantly 2-4 nucleotide insertions. Ends with 3′ extensions or blunt ends did not show enhanced frequencies of errors, although Δtdp1 cells repaired blunt DNA ends with greater efficiency than WT cells. We found that insertions required Ku80 and DNA ligase IV, as well as polymerase IV. Our results show that yeast Tdp1 is a component of the NHEJ pathway. We suggest that Tdp1p 3′ nucleosidase activity regulates the processing of DNA ends by generating a 3′ phosphate, thereby restricting the ability of polymerases and other enzymes from acting at DNA ends. In support of this model, we found that overexpression of Tpp1, a yeast DNA 3′ phosphatase, also leads to a higher frequency of insertions, suggesting that the generation of a 3′ phosphate is a key step in Tdp1-mediated error prevention during NHEJ.Tpp1 | nucleosidase | repair accuracy | break repair N onhomologous end joining (NHEJ) is a critical pathway for repairing DNA double-strand breaks. In higher eukaryotes, it functions as a primary repair pathway for repairing doublestrand breaks from exogenous DNA damage and is also required for gene rearrangements in the immune system (1). This pathway is conserved in lower eukaryotes and bacteria where it functions mainly as a secondary repair pathway for the repair of doublestrand breaks (2, 3). Because NHEJ does not rely on DNA homology for carrying out repair, it is an intrinsically error-prone pathway. The detailed steps of how accuracy is maintained during NHEJ processes remain poorly understood.NHEJ is carried out by a set of well-conserved proteins including the Ku70/Ku80 proteins that bind to DNA ends, serving as a scaffold for subsequent repair reactions, and DNA ligase IV (4). In mammalian cells, a DNA-dependent protein kinase DNAPKcs also plays critical roles (5). In addition to the core factors that are absolutely required for the NHEJ pathway, other factors such as nucleases and DNA polymerases participate in NHEJ (6, 7).Tdp1p was identified on the basis of its ability to remove peptides covalently bound to DNA (8-10). Yeast Tdp1 can remove phosphotyrosyl-linked peptides from both the 3′ and 5′ ends of DNA (8,11). Tdp1 also has the ability to remove damaged and undamaged nucleosides from the 3′ end of DNA through an activity that is mechanistically identical to the esterase activity that removes peptides (12). Because human Tdp1 is mutated in a DNA repair disorder [spinocerebellar ataxia with axonal neuropathy (13)], we assessed additional roles of this enzyme by using yeast repair mutants. In this paper, we demonstrate that yeast Tdp1 plays an important and unique role in NHEJ and affects the accuracy of the formation of repair junctions. ResultsYeast Tdp1 is Required for Accurate Joining of some NHEJ Subst...

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Section: Methodsmentioning

confidence: 99%

Yeast Tdp1 regulates the fidelity of nonhomologous end joining

Bahmed

Nitiss

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Two micrograms of phenol/chloroform-purified DNA were used for each yeast transformation. Yeast transformations were performed using a protocol adapted from the lithium acetate method (Okazaki et al 1990) described in Decottignies (2005). The number of Ura 1 colonies was scored after 4 days of incubation at 32°.…”

Section: Methodsmentioning

confidence: 99%

“…Strikingly, this Ku-independent MMEJ pathway was highly reminiscent of a major DSB repair mechanism operating in mammalian cells since short stretches of homologous nucleotides (1-4 bp) are often involved in end-joining events (Roth and Wilson 1986;Thacker et al 1992;King et al 1993;Sasaki et al 2003) although it is not clearly established whether these events are the result of NHEJ or MMEJ. Soon after the experiments were performed in yku70D budding yeast cells, evidence for the conservation of MMEJ throughout evolution was provided by other studies in NHEJ-deficient cells from mammals (Kabotyanski et al 1998;Feldmann et al 2000;Zhong et al 2002;Bentley et al 2004;Guirouilh-Barbat et al 2004;Tsujiet al 2004), Arabidopsis (Heacock et al 2004), S. cerevisiae (Ma et al 2003;Yu and Gabriel 2003), and S. pombe (Manolis et al 2001;Decottignies 2005). Moreover, biochemical purification approaches confirmed that enzymatic requirements for MMEJ are clearly distinct from NHEJ requirements in Xenopus laevis egg extracts (Gottlich et al 1998).…”

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confidence: 86%

“…Hence, when this work was initiated, the nature of MMEJ was still unclear. To clarify the mechanism(s) underlying MMEJ, this study relied on the highly flexible PCR-based EC DSB repair assay established previously (Decottignies 2005). Although the chromatinization state of EC DNA may be different from the one encountered in a chromosomal break, data from budding yeast suggest that the ratio of HR/NHEJ events is highly similar in both types of DSB repair assays and that plasmid assays mirror chromosomal assays for other aspects of NHEJ and HR (SSA) regulation (Karathanasis and Wilson 2002).…”

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confidence: 99%

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Microhomology-Mediated End Joining in Fission Yeast Is Repressed by Pku70 and Relies on Genes Involved in Homologous Recombination

Decottignies

2007

Genetics

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115

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Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joining (MMEJ) mechanism in fission yeast. MMEJ was found to require at least five homologous nucleotides and its efficiency was decreased by the presence of nonhomologous nucleotides either within the overlapping sequences or at DSB ends. Exo1 exonuclease and Rad22, a Rad52 homolog, were required for repair, suggesting that MMEJ is related to the single-strand-annealing (SSA) pathway of HR. In addition, MMEJ-dependent repair of DSBs with discontinuous microhomologies was strictly dependent on Pol4, a PolX DNA polymerase. Although not strictly required, Msh2 and Pms1 mismatch repair proteins affected the pattern of MMEJ repair. Strikingly, Pku70 inhibited MMEJ and increased the minimal homology length required for efficient MMEJ. Overall, this study strongly suggests that MMEJ does not define a distinct DSB repair mechanism but reflects ''micro-SSA.''

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“…b Insertions of 100-300 bp that might come from the carrier DNA (Decottignies 2005). c Boldface type indicates bases stemming from the 39 overhangs; underlining indicates possible base pairing involved in the junction.…”

Section: Notementioning

confidence: 99%

Mismatch Tolerance by DNA Polymerase Pol4 in the Course of Nonhomologous End Joining in Saccharomyces cerevisiae

Pardo

Marcand

2006

Genetics

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In yeast, the nonhomologous end joining pathway (NHEJ) mobilizes the DNA polymerase Pol4 to repair DNA double-strand breaks when gap filling is required prior to ligation. Using telomere-telomere fusions caused by loss of the telomeric protein Rap1 and double-strand break repair on transformed DNA as assays for NHEJ between fully uncohesive ends, we show that Pol4 is able to extend a 39-end whose last bases are mismatched, i.e., mispaired or unpaired, to the template strand.

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Capture of Extranuclear DNA at Fission Yeast Double-Strand Breaks

Cited by 33 publications

References 74 publications

Yeast Tdp1 regulates the fidelity of nonhomologous end joining

Yeast Tdp1 regulates the fidelity of nonhomologous end joining

Microhomology-Mediated End Joining in Fission Yeast Is Repressed by Pku70 and Relies on Genes Involved in Homologous Recombination

Mismatch Tolerance by DNA Polymerase Pol4 in the Course of Nonhomologous End Joining in Saccharomyces cerevisiae

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