Defects in RNase H2 Stimulate DNA Break Repair by RNA Reverse Transcribed into cDNA

Eukaryotic ribonucleases (RNase) H1 and H2 are endonucleases that cleave RNA in a double- stranded RNA-DNA molecule. RNase H2 can also cleave a single ribonucleotide embedded in DNA duplex. While the activity of RNase H1 and H2 has been extensively characterized in vitro, still much is unclear about the specific targets of these enzymes in vivo. We recently demonstrated that yeast cells can repair a double-strand break (DSB) in DNA by homologous recombination (HR) using antisense (non-coding) RNA, either directly, or indirectly after converting RNA into cDNA. In wildtype RNase H1 and/or H2 cells, repair by cDNA dominates, whereas in the absence of RNase H1 and H2 functions cDNA and, in particular, direct transcript-RNA repair mechanisms are markedly stimulated. Here we found that null alleles of any of the three RNase H2 subunits stimulate DSB repair by cDNA significantly more than a null allele of RNase H1. These results show that RNase H2 is the preferred RNase H enzyme to target cDNA in yeast. Targeting of cDNA by RNase H2 does not require RNase H2 interaction with the DNA clamp proliferating cell nuclear antigen (PCNA). Moreover, yeast RNase H2 orthologous mutants of two common RNase H2 defects associated with Aicardi Goutières syndrome (AGS) in humans, displayed elevated cDNA-driven repair of a DSB when combined with each other or with RNase H1 null mutation. Our findings support the hypothesis that defective RNase H2 alleles have higher level of cDNA derived from either coding or non-coding RNA in the form of RNA-cDNA hybrids. Keywords: Aicardi Goutières syndrome, cDNA, double-strand break repair, RNA-DNA hybrids, RNase H, Rnh203, transcript RNA, Ty retrotransposon.