The Role of Yeast DNA 3′-Phosphatase Tpp1 and Rad1/Rad10 Endonuclease in Processing Spontaneous and Induced Base Lesions

Karumbati, Anandi S.; Deshpande, Rajashree A.; Jilani, Arshad; Vance, John R.; Ramotar, Dindial; Wilson, Thomas E.

doi:10.1074/jbc.m304586200

Cited by 38 publications

(40 citation statements)

References 44 publications

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“…6). The residual repair of these DSBs in this strain likely represents removal of the 3Ј phosphate by Rad1/Rad10, which has been shown to remove nonhomologous flaps in SSA (11) and to participate in the processing of 3Ј phosphates (16,33). We could not test this directly, because rad1⌬ apn1⌬ apn2⌬ tpp1⌬ yeast cells are inviable.…”

Section: Discussionmentioning

confidence: 95%

Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

Daley

Wilson²

2005

Molecular and Cellular Biology

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The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of singlestranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

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Section: Discussionmentioning

confidence: 95%

Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

Daley

Wilson²

2005

Molecular and Cellular Biology

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“…Wilson and colleagues showed that Tpp1, a yeast phosphatase homolog of polynucleotide kinase, is involved in removal of the 3′ PO 4 following the repair of Top1-mediated DNA damage. Tpp1 has 3′ DNA phosphatase activity and lacks 5′ DNA phosphatase, 5′ polynucleotide kinase, and 3′ exonuclease activities; i.e., its only known activity is 3′ DNA phosphatase (24,25). We overexpressed either WT Tpp1 or a mutant Tpp1 with a change at a key catalytic residue (Asp35 mutated to Ala).…”

Section: Resultsmentioning

confidence: 99%

“…The number of identical sequences recovered is indicated for each sequence. (24) or pTW375 D35A (as pTW375, but with a Tpp1 mutation changing Asp 35 to Ala) (24) were transfected with YCplac111 that had been linearized with HindIII. Accuracy of repair of the linearized DNA was determined as described in the legend for Fig.…”

Section: Resultsmentioning

confidence: 99%

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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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“…In this case the NER protein plays a NER-independent role. Similarly, flap endonuclease activity of the budding yeast Rad1p-Rad10p complex is required for removal of 3′ blocked ends from DNA strand breaks induced by reactive oxygen species (Guillet and Boiteux, 2002;Karumbati et al, 2003;Guzder et al, 2004). The S. pombe NER genes rad16, swi10 and rhp14 (XPA homolog) also have a function in msh2/pms1-independent mismatch repair (Fleck et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Fission yeast homologs of human XPC and CSB, rhp41 and rhp26, are involved in transcription-coupled repair of methyl methanesulfonate-induced DNA damage

Kanamitsu

Ikeda

2011

Genes Genet. Syst.

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Methyl methanesulfonate (MMS) methylates nitrogen atoms in purines, and predominantly produces 7-methylguanine and 3-methyladenine (3-meA). Previously, we showed that base excision repair (BER) and nucleotide excision repair (NER) synergistically function to repair MMS-induced DNA damage in the fission yeast Schizosaccharomyces pombe. Here, we studied the roles of NER components in repair of 3-meA and BER intermediates such as the AP site and single strand breaks. Mutants of rhp41 (XPC homolog) and rhp26 (CSB homolog) exhibited moderate sensitivity to MMS. Transcription of the fbp1 gene, which is induced by glucose starvation, was strongly inhibited by MMS damage in rhp41Δ and rhp26Δ strains but not in wild type and 3-meA DNA glycosylase-deficient cells. The results indicate that Rhp41p and Rhp26p are involved in transcriptioncoupled repair (TCR) of MMS-induced DNA damage. In the BER pathway of S. pombe, AP lyase activity of Nth1p mainly incises the AP site to generate a 3′-blocked end, which is in turn converted to 3′-OH by Apn2p. Deletion of rad16 or rhp26 in the nth1Δ strain greatly enhanced MMS sensitivity, suggesting that the AP site could also be corrected by TCR. Double mutant apn2Δ/rad16Δ exhibited hypersensitivity to MMS, implying that Rad16p provides a backup pathway for removal of the 3′-blocked end. Moreover, an rhp51Δ strain was extremely sensitive to MMS and double mutants of nth1Δ/rhp51Δ and apn2Δ/rhp51Δ increased the sensitivity, suggesting that homologous recombination is necessary for repair of three different types of lesions, 3-meA, AP sites and 3′-blocked ends.

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The Role of Yeast DNA 3′-Phosphatase Tpp1 and Rad1/Rad10 Endonuclease in Processing Spontaneous and Induced Base Lesions

Cited by 38 publications

References 44 publications

Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

Yeast Tdp1 regulates the fidelity of nonhomologous end joining

Fission yeast homologs of human XPC and CSB, rhp41 and rhp26, are involved in transcription-coupled repair of methyl methanesulfonate-induced DNA damage

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