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) deaminase (DCD1) in the yeast Saccharomyces cerevisiae increases the intracellular dCTP:dTTP ratio and reduces the induction of G x C --> A x T transitions in the SUP4-o gene by ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Simultaneously, the G x C --> C x G transversion frequency rises substantially. We attributed the first response to dCTP outcompeting dTTP for incorporation opposite O6-alkylguanine, and the second outcome to the increased dCTP pool causing error-prone repair of apurinic (AP) sites resulting from the removal or lability of N7-alkylguanine. To test the latter hypothesis, we used isogenic dcd1 strains deleted for either of two genes (MAG1: 3-methyladenine glycosylase; APN1: apurinic endonuclease) involved in the repair of N7-alkylguanine. In these backgrounds, EMS or MNNG induction of total SUP4-o mutations, G x C --> A x T transitions and G x C --> C x G transversions were reduced by >98%, >97%, and >80%, respectively. Mutation frequencies in the dcd1 apn1 strain were close to those for spontaneous mutagenesis in the wild-type parent. These findings argue that misincorporation of dCTP during repair of alkylation-induced AP sites is responsible for the increased G x C --> C x G transversion frequency in the dcd1 strain treated with EMS or MNNG. The data also demonstrate that defective repair of AP sites coupled with an elevated dCTP:dTTP ratio eliminates most EMS and MNNG mutagenesis. In addition, the results point to a role for AP sites in the production of some EMS- and MNNG-induced G x C --> A x T transitions as well as other substitutions in the dcd1 strain.
School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria, Australia. bkunz@deakin.edu.au
B A
BA
Kunz
E S
ES
Henson
R
R
Karthikeyan
T
T
Kuschak
S A
SA
McQueen
C A
CA
Scott
W
W
Xiao
D000477:Alkylating Agents / Q000633:toxicity*; D000478:Alkylation; D019757:Carbon-Oxygen Lyases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases; D011088:DNA Ligases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D004271:DNA, Fungal / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D005020:Ethyl Methanesulfonate / Q000037:antagonists & inhibitors* / Q000633:toxicity; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D016153:Genes, Suppressor / Q000187:drug effects; D007424:Intracellular Fluid; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D016296:Mutagenesis / Q000187:drug effects*; D009699:N-Glycosyl Hydrolases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects*
D000477:Alkylating Agents; D018736:DNA Adducts; D004271:DNA, Fungal; D003845:Deoxycytosine Nucleotides; D005656:Fungal Proteins; D008769:Methylnitronitrosoguanidine; C024107:2'-deoxycytidine 5'-triphosphate; D005020:Ethyl Methanesulfonate; D043223:Deoxyribonuclease IV (Phage T4-Induced); C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D011088:DNA Ligases