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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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THF bypass is associated with increased mutagenesis downstream of the lesion.(A) Distribution of mutations found in the products of TLS through the THF lesion. The THF position is indicated in red. Each vertical line represents a single mutation; the mutation found twice is marked with the asterisk. Mutations in the TLS products within 220 bp from the lesion are in black, other mutations in TLS products are in grey. Blue lines below the horizontal scale bar represent mutations found in the control substrates without the THF. The data are based on DNA sequence analysis of 394 THF bypass products and 456 products of the control plasmid replication. P-value (Fisher’s exact test) indicates the significance of differences in the frequency of mutation in the 220-nucleotide region between TLS products and control plasmids. (B) Types of mutations observed in the THF bypass products and control plasmids. C → T changes are shown for the transcribed strand that is exposed as ssDNA during the plasmid construction. The double asterisk indicates a statistically significant difference (p = 0.0347, Fisher’s exact test).
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pgen.1005110.g002: THF bypass is associated with increased mutagenesis downstream of the lesion.(A) Distribution of mutations found in the products of TLS through the THF lesion. The THF position is indicated in red. Each vertical line represents a single mutation; the mutation found twice is marked with the asterisk. Mutations in the TLS products within 220 bp from the lesion are in black, other mutations in TLS products are in grey. Blue lines below the horizontal scale bar represent mutations found in the control substrates without the THF. The data are based on DNA sequence analysis of 394 THF bypass products and 456 products of the control plasmid replication. P-value (Fisher’s exact test) indicates the significance of differences in the frequency of mutation in the 220-nucleotide region between TLS products and control plasmids. (B) Types of mutations observed in the THF bypass products and control plasmids. C → T changes are shown for the transcribed strand that is exposed as ssDNA during the plasmid construction. The double asterisk indicates a statistically significant difference (p = 0.0347, Fisher’s exact test).

Mentions: A total of 394 THF bypass products and 456 products of the control plasmid replication were analyzed by DNA sequencing. As expected, the majority of TLS events resulted in an A (243/394; 62%) or C (80/394; 20%) incorporation opposite the lesion. T incorporation occurred in 18% of all cases (71/394). A total of 18 mutations were found in the downstream region at distances between 34 and 1529 nucleotides from the THF (Fig. 2A; Table 1). These “hitchhiking” mutations were noticeably concentrated within an approximately 220-nucleotide segment immediately adjacent to the lesion. Although 11 mutations were found among the 456 control plasmids downstream of the lesion site, their distribution was significantly different from that in the TLS products. Mutations in the control plasmids were randomly distributed throughout the sequenced region, with none of the 11 mutations occurring within the first 220 nucleotides, in contrast to ~40% in the THF bypass products (p = 0.0045, Fisher’s exact test). The rate of mutation in the 220-nucleotide region next to the THF site constituted 8.1 x 10-5 per nucleotide (Table 2). This exceeds the genome-wide mutation rate in yeast by approximately 300,000-fold and is close to the rate of errors reported for copying of undamaged DNA by purified Polζ in vitro (5.6 x 10-4, [28]). The frequency of mutations immediately upstream of the lesion site did not differ from that in the control plasmids (Fig. 2A), consistent with the idea that the patch of increased mutagenesis resulted from error-prone DNA synthesis initiated at the lesion site. The frequency of mutations downstream of the lesion was reduced to the background level as the distance from the lesion exceeded 220 nucleotides. Accordingly, the types of mutations in these distant regions were very similar to those in the control plasmids (predominantly C→T transitions and -1 deletions). In contrast, only one C→T transition and no -1 frameshifts were found in the 220-bp region adjacent to the lesion site (Fig. 2B, Table 1). We, therefore, concluded that mutations present in the TLS products outside of the 220-bp region must have resulted from damage of ssDNA during the plasmid construction, and only those observed within the 220-bp region are indicative of error-prone DNA synthesis associated with the THF bypass. We also sequenced the 220-bp region in 47 THF bypass products and 57 control plasmids recovered from msh2Δ strains to determine whether errors made during TLS-associated synthesis are corrected by MMR. We observed no increase in the frequency of untargeted mutations over that in MMR-proficient strains (Table 2), indicating that MMR does not operate in TLS tracts. This is in agreement with a previous report that MMR does not efficiently correct errors made by Polζ [35]. Taken together, these observations suggest that the error-prone synthesis typically continues for approximately 200 nucleotides after the THF bypass is completed. Because of the high level of background mutagenesis in this system, we cannot exclude a possibility that a minor fraction of TLS events could involve more extensive low-fidelity synthesis.


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)

THF bypass is associated with increased mutagenesis downstream of the lesion.(A) Distribution of mutations found in the products of TLS through the THF lesion. The THF position is indicated in red. Each vertical line represents a single mutation; the mutation found twice is marked with the asterisk. Mutations in the TLS products within 220 bp from the lesion are in black, other mutations in TLS products are in grey. Blue lines below the horizontal scale bar represent mutations found in the control substrates without the THF. The data are based on DNA sequence analysis of 394 THF bypass products and 456 products of the control plasmid replication. P-value (Fisher’s exact test) indicates the significance of differences in the frequency of mutation in the 220-nucleotide region between TLS products and control plasmids. (B) Types of mutations observed in the THF bypass products and control plasmids. C → T changes are shown for the transcribed strand that is exposed as ssDNA during the plasmid construction. The double asterisk indicates a statistically significant difference (p = 0.0347, Fisher’s exact test).
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Related In: Results  -  Collection

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pgen.1005110.g002: THF bypass is associated with increased mutagenesis downstream of the lesion.(A) Distribution of mutations found in the products of TLS through the THF lesion. The THF position is indicated in red. Each vertical line represents a single mutation; the mutation found twice is marked with the asterisk. Mutations in the TLS products within 220 bp from the lesion are in black, other mutations in TLS products are in grey. Blue lines below the horizontal scale bar represent mutations found in the control substrates without the THF. The data are based on DNA sequence analysis of 394 THF bypass products and 456 products of the control plasmid replication. P-value (Fisher’s exact test) indicates the significance of differences in the frequency of mutation in the 220-nucleotide region between TLS products and control plasmids. (B) Types of mutations observed in the THF bypass products and control plasmids. C → T changes are shown for the transcribed strand that is exposed as ssDNA during the plasmid construction. The double asterisk indicates a statistically significant difference (p = 0.0347, Fisher’s exact test).
Mentions: A total of 394 THF bypass products and 456 products of the control plasmid replication were analyzed by DNA sequencing. As expected, the majority of TLS events resulted in an A (243/394; 62%) or C (80/394; 20%) incorporation opposite the lesion. T incorporation occurred in 18% of all cases (71/394). A total of 18 mutations were found in the downstream region at distances between 34 and 1529 nucleotides from the THF (Fig. 2A; Table 1). These “hitchhiking” mutations were noticeably concentrated within an approximately 220-nucleotide segment immediately adjacent to the lesion. Although 11 mutations were found among the 456 control plasmids downstream of the lesion site, their distribution was significantly different from that in the TLS products. Mutations in the control plasmids were randomly distributed throughout the sequenced region, with none of the 11 mutations occurring within the first 220 nucleotides, in contrast to ~40% in the THF bypass products (p = 0.0045, Fisher’s exact test). The rate of mutation in the 220-nucleotide region next to the THF site constituted 8.1 x 10-5 per nucleotide (Table 2). This exceeds the genome-wide mutation rate in yeast by approximately 300,000-fold and is close to the rate of errors reported for copying of undamaged DNA by purified Polζ in vitro (5.6 x 10-4, [28]). The frequency of mutations immediately upstream of the lesion site did not differ from that in the control plasmids (Fig. 2A), consistent with the idea that the patch of increased mutagenesis resulted from error-prone DNA synthesis initiated at the lesion site. The frequency of mutations downstream of the lesion was reduced to the background level as the distance from the lesion exceeded 220 nucleotides. Accordingly, the types of mutations in these distant regions were very similar to those in the control plasmids (predominantly C→T transitions and -1 deletions). In contrast, only one C→T transition and no -1 frameshifts were found in the 220-bp region adjacent to the lesion site (Fig. 2B, Table 1). We, therefore, concluded that mutations present in the TLS products outside of the 220-bp region must have resulted from damage of ssDNA during the plasmid construction, and only those observed within the 220-bp region are indicative of error-prone DNA synthesis associated with the THF bypass. We also sequenced the 220-bp region in 47 THF bypass products and 57 control plasmids recovered from msh2Δ strains to determine whether errors made during TLS-associated synthesis are corrected by MMR. We observed no increase in the frequency of untargeted mutations over that in MMR-proficient strains (Table 2), indicating that MMR does not operate in TLS tracts. This is in agreement with a previous report that MMR does not efficiently correct errors made by Polζ [35]. Taken together, these observations suggest that the error-prone synthesis typically continues for approximately 200 nucleotides after the THF bypass is completed. Because of the high level of background mutagenesis in this system, we cannot exclude a possibility that a minor fraction of TLS events could involve more extensive low-fidelity synthesis.

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