Genome-wide ribonucleotide detection in <i>Archaea</i>

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.