Article

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

University of Michigan, Ann Arbor, Michigan, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2003; 278(33):31434-43. DOI: 10.1074/jbc.M304586200
Source: PubMed

ABSTRACT Tpp1 is a DNA 3'-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3'-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3'-phosphodiesterase function. This species difference was due to the absence of delta-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, beta-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3' alpha,beta-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3'-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3'-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3' blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3'-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3'-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast delta-lyase, the activity of the 3'-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.

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    • "The 3′-phosphate blocks polymerases (and other DNA metabolic enzymes) until the phosphate is removed. Strong evidence for this model comes from the observation that over-expression of Tpp1, a phosphatase that is specific for 3′-PO4 ends (37,38) leads to the same types of errors seen when Tdp1 activity is absent (23). Generation of additions in the absence of Tdp1 occurs through the canonical NHEJ pathway since additions are not seen when yKu80 or DNA ligase IV is absent. "
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    ABSTRACT: Non-homologous end-joining (NHEJ) is a critical error-prone pathway of double strand break repair. We recently showed that tyrosyl DNA phosphodiesterase 1 (Tdp1) regulates the accuracy of NHEJ repair junction formation in yeast. We assessed the role of other enzymes in the accuracy of junction formation using a plasmid repair assay. We found that exonuclease 1 (Exo1) is important in assuring accurate junction formation during NHEJ. Like tdp1Δ mutants, exo1Δ yeast cells repairing plasmids with 5'-extensions can produce repair junctions with templated insertions. We also found that exo1Δ mutants have a reduced median size of deletions when joining DNA with blunt ends. Surprisingly, exo1Δ pol4Δ mutants repair blunt ends with a very low frequency of deletions. This result suggests that there are multiple pathways that process blunt ends prior to end-joining. We propose that Exo1 acts at a late stage in end-processing during NHEJ. Exo1 can reverse nucleotide additions occurring due to polymerization, and may also be important for processing ends to expose microhomologies needed for NHEJ. We propose that accurate joining is controlled at two steps, a first step that blocks modification of DNA ends, which requires Tdp1, and a second step that occurs after synapsis that requires Exo1.
    Nucleic Acids Research 10/2010; 39(3):970-8. DOI:10.1093/nar/gkq886 · 9.11 Impact Factor
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    • "The breaks are likely to have arisen by enzymatic incision of AP sites since the formation of breaks was prevented by a mag1 deletion mutant that abolishes AP formation. Based on the synthetic lethality between the double mutation apn1 apn2 and either rad1 or rad10, it has been suggested that the 3′-endonuclease activity of Rad1-Rad10 can release 3′-blocked termini thereby providing an alternative branch of BER (1,5,8). The apn1 apn2 double mutant carries wild-type RAD1 and RAD10 and still showed accumulation of breaks, rather than repair. "
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    ABSTRACT: Base excision repair (BER) provides relief from many DNA lesions. While BER enzymes have been characterized biochemically, BER functions within cells are much less understood, in part because replication bypass and double-strand break (DSB) repair can also impact resistance to base damage. To investigate BER in vivo, we examined the repair of methyl methanesulfonate (MMS) induced DNA damage in haploid G1 yeast cells, so that replication bypass and recombinational DSB repair cannot occur. Based on the heat-lability of MMS-induced base damage, an assay was developed that monitors secondary breaks in full-length yeast chromosomes where closely spaced breaks yield DSBs that are observed by pulsed-field gel electrophoresis. The assay detects damaged bases and abasic (AP) sites as heat-dependent breaks as well as intermediate heat-independent breaks that arise during BER. Using a circular chromosome, lesion frequency and repair kinetics could be easily determined. Monitoring BER in single and multiple glycosylase and AP-endonuclease mutants confirmed that Mag1 is the major enzyme that removes MMS-damaged bases. This approach provided direct physical evidence that Apn1 and Apn2 not only repair cellular base damage but also prevent break accumulation that can result from AP sites being channeled into other BER pathway(s).
    Nucleic Acids Research 05/2008; 36(6):1836-46. DOI:10.1093/nar/gkm1148 · 9.11 Impact Factor
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