Mechanisms of mutagenesis in vivo due to imbalanced dNTP pools.
The mechanisms by which imbalanced dNTPs induce mutations have been well characterized within a test tube, but not in vivo. We have examined mechanisms by which dNTP imbalances induce genome instability in strains of Saccharomyces cerevisiae with different amino acid substitutions in Rnr1, the large subunit of ribonucleotide reductase. These strains have different dNTP imbalances that correlate with elevated CAN1 mutation rates, with both substitution and insertion-deletion rates increasing by 10- to 300-fold. The locations of the mutations in a strain with elevated dTTP and dCTP are completely different from those in a strain with elevated dATP and dGTP. Thus, imbalanced dNTPs reduce genome stability in a manner that is highly dependent on the nature and degree of the imbalance. Mutagenesis is enhanced despite the availability of proofreading and mismatch repair. The mutations can be explained by imbalanced dNTP-induced increases in misinsertion, strand misalignment and mismatch extension at the expense of proofreading. This implies that the relative dNTP concentrations measured in extracts are truly available to a replication fork in vivo. An interesting mutational strand bias is observed in one rnr1 strain, suggesting that the S-phase checkpoint selectively prevents replication errors during leading strand replication.
Kumar
Dinesh
D
Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Abdulovic
Amy L
AL
Viberg
Jörgen
J
Nilsson
Anna Karin
AK
Kunkel
Thomas A
TA
Chabes
Andrei
A
D019943:Amino Acid Substitution; D026923:Amino Acid Transport Systems, Basic / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D054643:INDEL Mutation; D016296:Mutagenesis; D009154:Mutation; D012264:Ribonucleotide Reductases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D013942:Thymine Nucleotides / Q000378:metabolism
D026923:Amino Acid Transport Systems, Basic; C502212:CAN1 protein, S cerevisiae; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C029603:deoxyguanosine triphosphate; D012264:Ribonucleotide Reductases; C551024:Rnr1 protein, S cerevisiae; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate