Mutagenic specificity of endogenously generated abasic sites in Saccharomyces cerevisiae chromosomal DNA
Paul Auerbach1, Richard A O Bennett, Elisabeth A Bailey
1Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
Abstract:
Abasic [apurinic/apyrimidinic (AP)] sites are common, noncoding DNA lesions. Despite extensive investigation, the mutational pattern they provoke in eukaryotic cells remains unresolved. We constructed Saccharomyces cerevisiae strains in which chromosomal AP sites were generated during normal cell growth by altered human uracil-DNA glycosylases that remove undamaged cytosines or thymines. The mutation target was the URA3 gene inserted near the ARS309 origin to allow defined replication polarity. Expression of the altered glycosylases caused a 7- to 18-fold mutator effect in AP endonuclease-deficient (deltaapn1) yeast, which depended highly on the known translesion synthesis enzymes Rev1 and DNA polymerase zeta. For the C-glycosylase, GC>CG transversions were the predominant mutations, followed by GC>AT transitions. AT>CG transversions predominated for the T-glycosylase. These results support a major role for Rev1-dependent dCMP insertion across from AP sites and a lesser role for dAMP insertion. Unexpectedly, there was also a significant proportion of dTMP insertions that suggest another mutational pathway at AP sites. Although replication polarity did not strongly influence mutagenesis at AP sites, for certain mutation types, there was a surprisingly strong difference between the transcribed and non-transcribed strands of URA3. The basis for this strand discrimination requires further exploration.
Insights
Abasic sites, common DNA lesions, cause mutations primarily through Rev1 and DNA polymerase zeta in yeast. Unexpected pathways and strand-specific differences in mutagenesis were also observed.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- Abasic (apurinic/apyrimidinic) sites are frequent DNA lesions.
- The precise mutational outcomes of abasic sites in eukaryotes are not fully understood.
Purpose of the Study:
- To investigate the mutational patterns induced by abasic sites in Saccharomyces cerevisiae.
- To elucidate the roles of translesion synthesis enzymes in abasic site mutagenesis.
Main Methods:
- Generating chromosomal abasic sites using altered human uracil-DNA glycosylases in yeast.
- Utilizing the URA3 gene as a mutation target near a replication origin.
- Assessing mutagenesis in AP endonuclease-deficient strains expressing specific glycosylases.
Main Results:
- A significant mutator effect (7- to 18-fold) was observed in AP endonuclease-deficient yeast.
- Mutagenesis was highly dependent on translesion synthesis enzymes Rev1 and DNA polymerase zeta.
- Specific mutation types (GC>CG, GC>AT, AT>CG) were predominant depending on the glycosylase activity.
- Evidence for Rev1-dependent dCMP and dAMP insertion, along with unexpected dTMP insertion, was found.
- Strand-specific differences in mutagenesis were noted within the URA3 gene.
Conclusions:
- Rev1-dependent nucleotide insertion plays a key role in abasic site mutagenesis.
- Multiple pathways, including unexpected ones, contribute to the mutational spectrum at abasic sites.
- Further research is needed to understand the observed strand discrimination in mutagenesis.
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