Genome-wide ribonucleotide detection in Archaea

Genome integrity is constantly challenged by the incorporation of ribonucleotides ribonucleoside monophosphates (rNMPs) during DNA synthesis. Covalently linked single and several consecutive rNMPs occur in the genome of a number of organisms. They are mainly introduced by DNA polymerases during DNA replication and repair. In general, cells evolved ribonucleases H (RNases H) specialized in the removal of rNMPs from DNA to avoid any detrimental consequences on genome stability. Here, we describe the involvement of types 1 and/or 2 RNases H in processing embedded rNMPs in the genome of two archaeal species Haloferax volcanii and Thermococcus barophilus. Genome-wide, nucleotide-resolution maps of embedded rNMPs reveal oriC-centered strand-switching profiles in H. volcanii ΔrnhB, indicating origin firing in native cells, while their absence in T. barophilus reflects low origin usage. The data also define archaeal sequence-context rules for rNMP embedment, confirm the predominant role of RNase HII in rNMP removal with evidence of compensatory repair pathways, and link incorporation patterns to measured rNTP/dNTP pools. Together, these findings uncover archaeal-specific mechanisms of rNMP incorporation and repair with implications for replication and genome stability.