RNA–DNA hybrids on protein coding genes are stabilized by loss of RNase H and are associated with DNA damages during S-phase in fission yeast

RNA–DNA hybrid is a part of the R-loop which is an important non-standard nucleic acid structure. RNA–DNA hybrid/R-loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA–DNA hybrids and map their genomic locations in fission yeast cells. The RNA–DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild-type. The majority of RNA–DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S-phase and about 20% of the RNA–DNA hybrids overlap with Rad52 loci. During S-phase, more robust association of Rad52 with RNA–DNA hybrids was observed in the protein coding region than in M-phase. These results suggest that persistent RNA–DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S-phase, potentially through collision with DNA replication forks.