The Roles of the RNases H and Chromosomal Sequences in DNA:RNA Hybrid Mediated Genome Instability

DNA:RNA hybrids can lead to DNA damage and genome instability. This damage can be prevented by degradation of the RNA in the hybrid by two evolutionarily conserved enzymes, RNase H1 and RNase H2. Indeed, RNase H deficient cells have increased chromosomal rearrangements. However, the quantitative and spatial contributions of the individual enzymes to hybrid removal has been unclear. Additionally, RNase H2 can remove single ribonucleotides misincorporated into DNA during replication. The relative contribution of DNA:RNA hybrids and misincorporated ribonucleotides to chromosome instability was also uncertain. To address these issues, we studied the rate and location of loss of heterozygosity events on chromosome III in Saccharomyces cerevisiae that were defective for RNase H1, H2 or both. We showed that RNase H2 plays the major role in preventing chromosome III instability through its hybrid-removal activity. Furthermore, RNase H2 acts pervasively at many hybrids along the chromosome. In contrast, RNase H1 acts to prevent LOH within a small region of chromosome III, and this instability is dependent upon two hybrid prone regions. This restriction of RNase H1 activity to a subset of hybrids is not due to its constrained localization as we found it at hybrids genome wide. This result suggests that the genome protection activity of RNase H1 is regulated at a step after hybrid recognition. The global function of RNase H2 and the region specific function of RNase H1 provide insight on why these enzymes with overlapping hybrid-removal activities have been conserved throughout evolution./nAdditionally we developed a novel method to map DNA:RNA hybrids in the yeast genome, termed S1-DRIP-seq. Hybrids were found to form at the same loci in wild- type and RNase H deficient cells and to form at higher levels in RNase H mutants, which corroborates previous observations in the field. This study allowed for the identification of factors that influence hybrid formation and found that high transcription is both strongly correlated with and sufficient for hybrid formation. We then used the information gained from this high-resolution map of hybrids to further probe the factors that influence hybrid formation. We investigated if the increased hybrid levels observed in the RNase H mutants were due to changes in transcript levels. We found that the transcript levels at hybrid-forming loci were not increased in cells lacking the RNases H. We also investigated whether genes that are highly expressed and form hybrids at their endogenous locus can form hybrids at an ectopic locus. We found that hybrid-prone genes did form hybrids when inserted onto a yeast artificial chromosome (YAC), showing that hybrid-prone sequences can form hybrids outside their native chromosomal context. The presence of these hybrid-forming sequences did not cause instability of the YAC, even when overexpressed./nTogether, the work presented in this dissertation identifies several properties of hybrids and the systems in place for their removal from the yeast genome. We found that the RNases H do not act equally to prevent chromosome instability, and they also act differentially at specific hybrids. We found that hybrid-prone sequences can form hybrids outside their native chromosomal context. Finally, our studies of hybrid-related instability on chromosome III and the YAC showed that not all hybrid-forming sequences lead to increased chromosome instability.

Advisor: Koshland, Douglas