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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 of 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 ([DELTA]apn1) 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 dTMP 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 nontranscribed strands of URA3. The basis for this strand discrimination requires further exploration.

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase [Beta] (Pol[Beta]) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein-protein contact between Pol[Beta] and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Pol[Beta] into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Pol[Beta] exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5[prime]-terminal deoxyribose 5-phosphate by Pol[Beta]. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.