DEFECTS IN BASE EXCISION-REPAIR COMBINED WITH ELEVATED INTRACELLULAR DCTP LEVELS DRAMATICALLY REDUCE MUTATION-INDUCTION IN YEAST BY ETHYL METHANESULFONATE AND N-METHYL-N'-NITRO-N-NITROSOGUANIDINE

Previously, we determined that elimination of deoxycytidylate (dCMP) d eaminase (DCD1) in the yeast Saccharomyces cerevisiae increases the in tracellular dCTP:dTTP ratio and reduces the induction of G.C --> A.T t ransitions in the SUP4-o gene by ethyl methanesulfonate (EMS) and N-me thyl-N'-nitro-N-nitrosoguanidine (MNNG). Simultaneously, the G.C --> C .G transversion frequency rises substantially. We attributed the first response to dCTP outcompeting dTTP for incorporation opposite O-6-alk ylguanine, and the second outcome to the increased dCTP pool causing e rror-prone repair of apurinic (AP) sites resulting from the removal or lability of N-7-alkylguanine. To test the latter hypothesis, we used isogenic dcd1 strains deleted for either of two genes (MAG1: 3-methyla denine glycosylase; APN1: apurinic endonuclease) involved in the repai r of N-7-alkylguanine. In these backgrounds, EMS or MNNG induction of total SUP4-o mutations, G.C --> A.T transitions and G.C --> C.G transv ersions were reduced by >98%, >97%, and >80%, respectively. Mutation f requencies in the dcd1 apn1 strain were close to those for spontaneous mutagenesis in the wild-type parent. These findings argue that misinc orporation of dCTP during repair of alkylation-induced AP sites is res ponsible for the increased G.C --> C.G transversion frequency in the d cd1 strain treated with EMS or MNNG. The data also demonstrate that de fective repair of AP sites coupled with an elevated dCTP:dTTP ratio el iminates most EMS and MNNG mutagenesis. In addition, the results point to a role for AP sites in the production of some EMS- and MNNG-induce d G.C --> A.T transitions as well as other substitutions in the dcd1 s train. (C) 1998 Wiley-Liss, Inc.