Physiological, Biochemical, and Molecular-Genetic Events Governing the Recognition and Resolution of RNA/DNA Hybrids

The catalytic mechanisms of RNase H1 and RNaseH2 are very similar, but their overall structures are very different. In vitro, both types hydrolyze the RNA of RNA/DNA hybrids. However, RNase H1 requires a minimum length of four rNMPs in an RNA/DNA hybrid, whereas RNase H2 recognizes and incises at a single rNMP embedded in DNA. Moreover, each enzyme has a unique dominant function leading to different symptoms in patients with RNase H deficiencies: RNase H1 defects lead to mitochondrial myopathy in mice and human patients, whereas RNase H2 mutations lead to a type I interferonopathy. Interestingly, our mouse model with one of the mutations found in patients (G37S-RNASEH2A), which in humans causes interferonopathy, results in perinatal lethality in mice.

Separation of function mutations for RNase H1

Our results obtained in the past indicate that the biochemical properties of mice and human RNases H1 are quite similar. Both enzymes have two isoforms, translated from a single mRNA, that produce mitochondrial and nuclear forms. We found that absence of RNase H1 during development caused defects in mitochondrial DNA replication, resulting in embryonic arrests and, possibly, obscuring any role of the nuclear RNase H1. In 2025, we generated a mouse model that expresses only the mitochondrial RNase H1 in the hope of uncovering functions of the more abundant nuclear RNase H1. The mice are viable and fertile, suggesting that RNase H1 has no essential function in the nucleus, at least under physiological conditions.

Separation of function mutations for RNase H2

RNase H2 has two activities: hydrolysis of RNA in RNA/DNA hybrids and incision of single ribonucleotides (rNMPs) embedded in DNA, otherwise known as ribonucleotide excision repair (RER). We generated a mutant of RNase H2 that retained the ability to hydrolyze RNA in RNA/DNA hybrids but failed to incise at rNMPs in DNA (ribonucleotide excision defective: RED). Mice homozygous for the RED mutation were embryonic-lethal at E9–E10, the same as RNase H2–null mice. We also generated a mutation that retained significant RER activity but less than a few percent of hybrid activity (hybrid defective: HD). HD mice are viable and fertile.

Double mutant of RNase H1 and RNase H2 defective for R-loop processing mice are viable and fertile.

The simple conclusion from these studies is that RNase H1 is needed for mitochondrial DNA replication but has little or no nuclear function, whereas the major role of RNase H2 is removal of single rNMPs in DNA.

We crossed homozygous mice that only express mitochondrial RNase H1 form with homozygous mice that have RNase H2 defective in processing RNA/DNA hybrids. These mice are viable. We are studying the R-loop distribution in these mutants to determine the role of RNase H1 and RNase H2 in R-loop resolution.

The RNA exosome and RNases H cooperate to suppress R-loop–mediated genome instability.

In eukaryotes, the RNA exosome is a major 3′-5′ RNA degradation, multi-subunit complex, which degrades cryptic and defective transcripts, preventing their engagement with DNA and the formation of R-loops. In collaboration with Aziz El Hage and David Tollervey, we are studying the interactions between the RNA exosome and RNases H in suppressing R-loop–mediated genome instability.

Using the yeast S. cerevisiae, we found that cells defective in both RNase H and RNA exosome activities are hypersensitive to the drug hydroxyurea (HU), which induces replicative stress by depleting the cellular dNTP supply. RNase H2–RED variant and over-expression of RNase H1 partially suppressed the growth defects in presence of HU, suggesting that some of the problems in these cells are caused by harmful R-loops.

We determined that R-loop–mediated genome instability and hyper-recombination are increased in mutants defective for RNase H and the RNA exosome, concluding that both pathways converge to prevent R-loop accumulation. We are in the process of identifying the RNA/DNA hybrids that are presents in these cells.