Authors

Discussion

Stable RNA-DNA hybrids (R-loops) act in several genomic processes, but their roles in DNA replication are unclear. By using DRIP-seq, we show that R-loop distribution within each chromosome parallels the spatial, temporal and functional compartments of the unconventional DNA replication programme in Leishmania. Strikingly, R-loop levels correlate with chromosome size, which in turn correlate with replication timing. MFA-seq analyses shows that DiCre-mediated RNase H1 KO results in origin-independent DNA replication initiation, profoundly changing the DNA replication programme. Such alteration leads to genome-wide, chromosome-size dependent instability, including aneuploidy, SNPs and InDels, as revealed by whole genome sequencing. Therefore, our data reveal a crucial role of RNase H1 in controlling R-loop-mediated DNA replication initiation, favouring conventional origin-directed initiation, and places the hybrids as a pivotal player in Leishmania global genome instability.