Very high levels of misincorporated ribonucleotides increase Topoisomerase1 related genome alterations

Abstract

A combination of high-resolution mapping of genomic rearrangements throughout the genome using microarrays, and measurements of loss of heterozygosity (LOH) on the right arm of chromosome IV were used to examine the effects of misincorporated ribonucleotides (rNMPs) on genome stability in diploid Saccharomyces cerevisiae strains. The effects of three types of mutations were examined in my analysis. Strains with a top1 mutation lack the Topoisomerase 1 enzyme, an enzyme that is involved in relaxing supercoils and in the removal of rNMPs from the genome. Strains with the rnh201 mutation lack RNase H2, an enzyme that removes both R-loops (RNA-DNA hybrids formed during transcription) and misincorporated rNMPs. Lastly, strains with the pol2-M644G mutation have a mutant form of DNA polymerase ε that misincorporates about 10-fold more rNMPs than the wild-type enzyme. My analysis of genetic instability in single mutants and various combinations of double and triple mutants shows that high levels of misincorporated rNMPs elevate mitotic recombination. Since mitotic recombination events are initiated in yeast by double-stranded DNA breaks (DSBs), my results suggest that high levels of misincorporated rNMPs result in elevated levels of DNA breaks.