UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA... more UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA polymerase η (Polη), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Polη result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Here, we test Polη for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than a cis-syn TT dimer. Opposite the 3′ T of a (6-4) TT photoproduct, both yeast and human Polη preferentially insert a G residue, but they are unable to extend from the inserted nucleotide. DNA Polζ, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3′ T of the (6-4) TT lesion by Polη, and Polζ inserts the correct nucleotide A opposite the 5′ T of the lesion. Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Polη and Polζ, wherein Polη inserts a nuc...
Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bas... more Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1(REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1Δ apn2Δ strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes. Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase ζ mediates the mutagenic bypass of AP sites.
The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replicat... more The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases and and postreplicational repair (PRR) of discontinuities that form in the newly synthesized DNA opposite from DNA lesions, mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Rad5 is an SWI/SNF family ATPase, and additionally, it functions as a ubiquitin ligase in the ubiquitin conjugation reaction. To decipher the roles of these Rad5 activities in lesion bypass, here we examine the effects of mutations in the Rad5 ATPase and ubiquitin ligase domains on the PRR of UV-damaged DNA and on UV-induced mutagenesis. Even though the ATPase-defective mutation confers only a modest degree of UV sensitivity whereas the ubiquitin ligase mutation causes a high degree of UV sensitivity, we find that both of these mutations produce the same high level of PRR defect as that conferred by the highly UV-sensitive rad5⌬ mutation. From these studies, we infer a requirement of the Rad5 ATPase and ubiquitin ligase activities in PRR, and based upon the effects of different rad5 mutations on UV mutagenesis, we suggest a role for Rad5 in affecting the efficiency of lesion bypass by the TLS polymerases. In contrast to the role of Rad5 in PRR, however, where its function is coupled with that of Mms2-Ubc13, Rad5 function in TLS would be largely independent of this ubiquitin-conjugating enzyme complex.
Proceedings of The National Academy of Sciences, 2008
Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a ... more Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA polymerases (Pols). Here we determine the mutational specificity and the genetic control of translesion synthesis (TLS) opposite an AP site in yeast by using a double-stranded plasmid system that we have devised in which
UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA... more UV light-induced DNA lesions block the normal replication machinery. Eukaryotic cells possess DNA polymerase η (Polη), which has the ability to replicate past a cis-syn thymine-thymine (TT) dimer efficiently and accurately, and mutations in human Polη result in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Here, we test Polη for its ability to bypass a (6-4) TT lesion which distorts the DNA helix to a much greater extent than a cis-syn TT dimer. Opposite the 3′ T of a (6-4) TT photoproduct, both yeast and human Polη preferentially insert a G residue, but they are unable to extend from the inserted nucleotide. DNA Polζ, essential for UV induced mutagenesis, efficiently extends from the G residue inserted opposite the 3′ T of the (6-4) TT lesion by Polη, and Polζ inserts the correct nucleotide A opposite the 5′ T of the lesion. Thus, the efficient bypass of the (6-4) TT photoproduct is achieved by the combined action of Polη and Polζ, wherein Polη inserts a nuc...
Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bas... more Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1(REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1Δ apn2Δ strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes. Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase ζ mediates the mutagenic bypass of AP sites.
The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replicat... more The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases and and postreplicational repair (PRR) of discontinuities that form in the newly synthesized DNA opposite from DNA lesions, mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Rad5 is an SWI/SNF family ATPase, and additionally, it functions as a ubiquitin ligase in the ubiquitin conjugation reaction. To decipher the roles of these Rad5 activities in lesion bypass, here we examine the effects of mutations in the Rad5 ATPase and ubiquitin ligase domains on the PRR of UV-damaged DNA and on UV-induced mutagenesis. Even though the ATPase-defective mutation confers only a modest degree of UV sensitivity whereas the ubiquitin ligase mutation causes a high degree of UV sensitivity, we find that both of these mutations produce the same high level of PRR defect as that conferred by the highly UV-sensitive rad5⌬ mutation. From these studies, we infer a requirement of the Rad5 ATPase and ubiquitin ligase activities in PRR, and based upon the effects of different rad5 mutations on UV mutagenesis, we suggest a role for Rad5 in affecting the efficiency of lesion bypass by the TLS polymerases. In contrast to the role of Rad5 in PRR, however, where its function is coupled with that of Mms2-Ubc13, Rad5 function in TLS would be largely independent of this ubiquitin-conjugating enzyme complex.
Proceedings of The National Academy of Sciences, 2008
Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a ... more Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA polymerases (Pols). Here we determine the mutational specificity and the genetic control of translesion synthesis (TLS) opposite an AP site in yeast by using a double-stranded plasmid system that we have devised in which
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Papers by Robert Johnson