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DNA polymerase ζ-dependent lesion bypass in Saccharomyces cerevisiae is accompanied by error-prone copying of long stretches of adjacent DNA.

Kochenova OV, Daee DL, Mertz TM, Shcherbakova PV - PLoS Genet. (2015)

Bottom Line: The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro.Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells.These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

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Frequency of UV-induced reversion of the ura3-G764A allele in the wild-type and Polζ-deficient (rev3Δ) strains.The data are mean frequencies for at least six determinations. Error bars are shown unless they are smaller than the plot symbol and represent standard errors.
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pgen.1005110.g004: Frequency of UV-induced reversion of the ura3-G764A allele in the wild-type and Polζ-deficient (rev3Δ) strains.The data are mean frequencies for at least six determinations. Error bars are shown unless they are smaller than the plot symbol and represent standard errors.

Mentions: As described previously, sensitivity of the plasmid TLS assay is limited by the high background likely resulting from spontaneous damage during the plasmid construction. To overcome this limitation and to ascertain that the extended stretches of error-prone DNA synthesis is not a peculiar feature of the plasmid assay, we next developed an approach to study mutagenesis associated with the Polζ-dependent bypass of a chromosomal DNA lesion. Because creating a unique site-specific abasic site in a yeast chromosome is technically challenging, we chose to use a lesion induced by UV irradiation at a specific dipyrimidine sequence in the chromosomal URA3 gene. Cis-syn cyclobutane pyrimidine dimers and (6–4) photoproducts are major types of DNA lesion induced by UV irradiation and can be generated at any of the four pyrimidine doublets, TT, CT, TC, and CC [36]. Error-prone bypass of UV lesions in vivo, like that of abasic sites, occurs via a Polζ/Rev1-dependent pathway [37–39]. We introduced a single nucleotide substitution at position 764 of the chromosomal URA3 gene (the ura3-G764A mutation) that leads to a Ura- phenotype and creates a dipyrimidine sequence (TC), a possible site for UV lesion formation (Fig. 3). The ura3-G764A strains can revert to the Ura+ phenotype via several base substitutions at the 3' C or 5' T of the dinucleotide (Fig. 3A). The occurrence of either substitution upon UV irradiation of yeast cells indicates that the lesion formation and the mutagenic TLS have taken place at this site. The frequency of the ura3-G764A reversion increased in a dose-dependent manner in wild-type strains, but not in rev3Δ mutants lacking Polζ (Fig. 4). This indicated that UV irradiation readily induces lesions that are bypassed via Polζ-dependent synthesis to produce the Ura+ revertants. DNA sequence analysis of the URA3 locus of 165 independent revertants obtained after irradiation with 60 J/m2 UV light showed that all of the revertants contained base substitutions at 3' C, 5' T or both positions of the dipyrimidine at the site of ura3-G764A mutation (S1 Table). Thus, the system is highly efficient in the identification of products of mutagenic TLS through a site-specific chromosomal lesion. To determine the extent of error-prone synthesis associated with the bypass of this lesion, total DNA was isolated from the 165 Ura+ revertants, and 2.5-kb regions upstream and downstream from the reversion site in respect to the direction of TLS were amplified by PCR and sequenced (Fig. 3B).


DNA polymerase ζ-dependent lesion bypass in Saccharomyces cerevisiae is accompanied by error-prone copying of long stretches of adjacent DNA.

Kochenova OV, Daee DL, Mertz TM, Shcherbakova PV - PLoS Genet. (2015)

Frequency of UV-induced reversion of the ura3-G764A allele in the wild-type and Polζ-deficient (rev3Δ) strains.The data are mean frequencies for at least six determinations. Error bars are shown unless they are smaller than the plot symbol and represent standard errors.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380420&req=5

pgen.1005110.g004: Frequency of UV-induced reversion of the ura3-G764A allele in the wild-type and Polζ-deficient (rev3Δ) strains.The data are mean frequencies for at least six determinations. Error bars are shown unless they are smaller than the plot symbol and represent standard errors.
Mentions: As described previously, sensitivity of the plasmid TLS assay is limited by the high background likely resulting from spontaneous damage during the plasmid construction. To overcome this limitation and to ascertain that the extended stretches of error-prone DNA synthesis is not a peculiar feature of the plasmid assay, we next developed an approach to study mutagenesis associated with the Polζ-dependent bypass of a chromosomal DNA lesion. Because creating a unique site-specific abasic site in a yeast chromosome is technically challenging, we chose to use a lesion induced by UV irradiation at a specific dipyrimidine sequence in the chromosomal URA3 gene. Cis-syn cyclobutane pyrimidine dimers and (6–4) photoproducts are major types of DNA lesion induced by UV irradiation and can be generated at any of the four pyrimidine doublets, TT, CT, TC, and CC [36]. Error-prone bypass of UV lesions in vivo, like that of abasic sites, occurs via a Polζ/Rev1-dependent pathway [37–39]. We introduced a single nucleotide substitution at position 764 of the chromosomal URA3 gene (the ura3-G764A mutation) that leads to a Ura- phenotype and creates a dipyrimidine sequence (TC), a possible site for UV lesion formation (Fig. 3). The ura3-G764A strains can revert to the Ura+ phenotype via several base substitutions at the 3' C or 5' T of the dinucleotide (Fig. 3A). The occurrence of either substitution upon UV irradiation of yeast cells indicates that the lesion formation and the mutagenic TLS have taken place at this site. The frequency of the ura3-G764A reversion increased in a dose-dependent manner in wild-type strains, but not in rev3Δ mutants lacking Polζ (Fig. 4). This indicated that UV irradiation readily induces lesions that are bypassed via Polζ-dependent synthesis to produce the Ura+ revertants. DNA sequence analysis of the URA3 locus of 165 independent revertants obtained after irradiation with 60 J/m2 UV light showed that all of the revertants contained base substitutions at 3' C, 5' T or both positions of the dipyrimidine at the site of ura3-G764A mutation (S1 Table). Thus, the system is highly efficient in the identification of products of mutagenic TLS through a site-specific chromosomal lesion. To determine the extent of error-prone synthesis associated with the bypass of this lesion, total DNA was isolated from the 165 Ura+ revertants, and 2.5-kb regions upstream and downstream from the reversion site in respect to the direction of TLS were amplified by PCR and sequenced (Fig. 3B).

Bottom Line: The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro.Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells.These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

Show MeSH
Related in: MedlinePlus