Abstract
RNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes RNase H1 and H2 contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 has strict cell cycle requirements, in that it has an essential function in G2/M for both R-loop processing and ribonucleotide excision repair. RNase H1, however, can function independently of the cell cycle to remove R-loops and appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.
Highlights
The RNase H enzymes (RNase H1 and RNase H2) remove RNADNA hybrids through the endonucleolytic cleavage of an RNA moiety that is engaged in the hybrid molecule (Cerritelli and Crouch, 2009)
RNase H1 consists of an N-terminal hybrid binding domain (HBD) and a C-terminal endonuclease motif (Cerritelli and Crouch, 2009), the latter of which must interact with the 20-OH group of four consecutive ribose molecules for optimal activity (Nowotny et al, 2007)
Rnaseh1À/À mice die during embryonic development due to incomplete mitochondrial replication (Cerritelli et al, 2003), a function that is likely not conserved in budding yeast due to the lack of a mitochondrial targeting sequence (MTS) on the yeast protein (Arudchandran et al, 2000)
Summary
The RNase H enzymes (RNase H1 and RNase H2) remove RNADNA hybrids through the endonucleolytic cleavage of an RNA moiety that is engaged in the hybrid molecule (Cerritelli and Crouch, 2009). Defective R-loop removal results in a loss of genome integrity, likely due to DNA replication conflicts (Garcıa-Muse and Aguilera, 2016). RNase H1, coded by the RNH1 gene in yeast, is able to degrade RNA engaged in R-loops, and its overexpression is frequently used as a tool to promote their removal (Wahba et al, 2011; Huertas and Aguilera, 2003). RNase H1 is likely recruited to hybrids via its direct interaction with replication protein A (RPA), a single-stranded binding protein that can coat the displaced DNA strand of an R-loop (Nguyen et al, 2017). Using a chromatin immunoprecipitation (ChIP)-based approach, the Zimmer and Koshland study demonstrated that, Rnh is able to associate with R-loops across the genome, it is only active at a small subset of those loci, at strong R-loop-forming loci (Zimmer and Koshland, 2016).
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