Surtees, J. A. | Search bioRxiv

bioRxiv2021

Complex Mutation Profiles in Mismatch Repair and Ribonucleotide Reductase Mutations Reveal Novel Repair Substrate Specificity of MutS Homolog (MSH) Complexes

Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate (dNTP) pools by ribonucleotide reductase (RNR) and repair of replication errors by the mismatch repair (MMR) system. We identified genetic interactions between rnr1 alleles that elevate dNTP levels and MMR. We then utilized a targeted deep-sequencing approach to determine mutational signatures associated with MMR pathway defects. By combining rnr1 and msh mutations to increase dNTP levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct repair of G/C single base deletions in GC runs, while Msh2-Msh6 specifically directs repair of substitutions at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when MMR is defective.

genetics↗

bioRxiv2020

Targeted Next Generation Sequencing in Saccharomyces cerevisiae Reveals Complex Mutation Profiles in the Presence of Mismatch Repair and Ribonucleotide Reductase Mutations that Compromise the Fidelity of DNA Replication

Distinct mutation signatures arise from environmental exposures and/or from defects in metabolic pathways that promote genome stability. The presence of a particular mutation signature in a cell or a tumor can therefore predict the underlying mechanism of mutagenesis, which, in practice, may be clinically important. These insults to the genome often alter dNTP pools, which itself impacts replication fidelity. Therefore, the impact of altered dNTP pools should be considered when making mechanistic predictions based on mutation signatures. We developed a targeted deep-sequencing approach on the CAN1 gene in Saccharomyces cerevisiae to define information-rich mutational profiles associated with distinct rnr1 backgrounds that alter replication fidelity by elevating dNTP levels.. The mutation spectra of rnr1Y285F and rnr1Y285A alleles were characterized previously; our analysis was consistent with this prior work but the sequencing depth achieved in our study allowed a significantly more robust and nuanced computational analysis of the variants observed, generating profiles that integrated information about mutation spectra, position effects, and sequence context. This approach revealed novel, genotype-specific mutation profiles in the presence of even modest changes in dNTP pools. Furthermore, we identified broader sequence contexts and specific nucleotide motifs that influenced variant profiles in different rnr1 backgrounds, which allowed us to make specific mechanistic predictions about the impact of altered dNTP pools on replication fidelity.

genetics↗