Mutagenic specificity of endogenously generated abasic sites in Saccharomyces cerevisiae chromosomal DNA.
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.
Auerbach
Paul
P
Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
Bennett
Richard A O
RA
Bailey
Elisabeth A
EA
Krokan
Hans E
HE
Demple
Bruce
B
D015825:Chromosomes, Fungal / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism; D000099270:Y-Family DNA Polymerases
D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase; D009713:Nucleotidyltransferases; D029701:Saccharomyces cerevisiae Proteins; D051981:Uracil-DNA Glycosidase; C425929:DNA polymerase zeta; C100951:REV1 protein, S cerevisiae; D000099270:Y-Family DNA Polymerases