Available online 29 March 2023, 103489
Author links open overlay panel, , , , , AbstractTranscription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ~15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.
Section snippetsINTRODUCTIONMutations generally have negative consequences but are required for adaptation under stress conditions and for evolutionary change. Most mutations arise during genome duplication and reflect either rare errors of the high-fidelity replicative DNA polymerases or the bypass of DNA damage by error-prone translesion-synthesis DNA polymerases. In addition to replication-associated changes in DNA sequence, mutations also arise in the context of transcription. This may be a particularly important
Strain constructionAll strains used for mutational analyses (Table S1) were derived from SJR282 (MATα ade2–101OChis3Δ200 ura3ΔNco gal80Δ::HIS3) by transformation. As previously described [5], high transcription of CAN1 was achieved by replacing the endogenous promoter with the GAL1 promoter (pGAL) linked to the bacterial kan marker [29]. FCY1, MSH6, MFT1, RNH1, RNH201, TOP1 or UNG1 was deleted by one-step allele replacement using PCR-generated cassettes containing an appropriate selectable marker. For
RESULTSRelative levels of spontaneous DNA damage associated with transcription of a target gene can be inferred by examining the mutagenic consequences. An inherent complication with most base damages is that they or their repair intermediates slow or stall subsequent DNA replication and transcription. This can trigger specialized bypass mechanisms during replication and/or template-specific engagement of nucleotide excision repair during transcription, which complicates subsequent analyses and
DISCUSSIONThe goal of the current study was to determine whether there is a transcription-associated strand bias in the hydrolytic deamination of cytosine to uracil. The CAN1 gene was used as a forward-mutation reporter, with C>T and G>A mutations reflecting NTS and TS deamination, respectively. Under high-txn conditions we observed proportionally fewer missense than nonsense mutations in CAN1, suggesting that increased expression can compensate for reduced protein function. To eliminate
CRediT authorship contribution statementJDW: Conceptualization, methodology, investigation, formal analysis, writing – editing and review, supervision, DZ: Investigation, formal analysis, MGR: Investigation, formal analysis, SS: Investigation, formal analysis, AA: Funding acquisition, writing – editing and review, SJR: Conceptualization, methodology, formal analysis, writing – original draft, visualization, project administration, funding acquisition
AcknowledgementsWe thank Tom Petes and members of the SJR lab for discussions throughout the course of this work and for helpful comments on the manuscript. Work in the SJR was supported by National Institutes of Health grant R35GM118077 and JDW was supported by a Tri-Institutional Molecular Mycology and Pathogenesis Training Program (5T32AI052080) postdoctoral fellowship. Work in the AA lab was supported by the grant PID2019-104270GB-I00/BMC from the Agencia Estatal de Investigación of the Spanish Ministry of
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