Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

Abstract

Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity. Here, we show that ribonucleotides are incorporated by the hyperthermophilic archaeon Thermococcus kodakarensis both in vitro and in vivo and a robust ribonucleotide excision repair pathway is critical to keeping incorporation levels low in wild-type cells. Using pre-steady-state and steady-state kinetics experiments, we also show that archaeal RNaseH2 rapidly cleaves at embedded ribonucleotides (200-450 s−1), but exhibits an ∼1,000-fold slower turnover rate (0.06–0.17 s−1), suggesting a potential role for RNaseH2 in protecting or marking nicked sites for further processing. We found that following RNaseH2 cleavage, the combined activities of polymerase B (PolB), flap endonuclease (Fen1), and DNA ligase are required to complete ribonucleotide processing. PolB formed a ribonucleotide-containing flap by strand displacement synthesis that was cleaved by Fen1, and DNA ligase sealed the nick for complete repair. Our study reveals conservation of the overall mechanism of ribonucleotide excision repair across domains of life. The lack of redundancies in ribonucleotide repair in archaea perhaps suggests a more ancestral form of ribonucleotide excision repair compared with the eukaryotic pathway.

Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2

“…Increased body of evidence supports the notion that incorporation of rNMPs in DNA is a frequent phenomenon, having profound detrimental effects on genome stability of both prokaryotes and eukaryotes ( 1 , 2 , 63 , 64 ). In humans, as well as in yeast and bacteria including Archaea, the main processing pathway responsible for repairing of these lesions is the RER pathway, which involves the RNase H2 enzyme ( 65 , 66 ). RNase H2 importance in higher organisms is testified by its essentiality for embryonic development in mouse ( 67 ).…”

Genome Replication in Thermococcus kodakarensis Independent of Cdc6 and an Origin of Replication

“…In E. coli , long-lived R-loops accumulate in strains lacking RNase H, and these R-loops can facilitate initiator protein (DnaA) independent constitutive stable DNA replication, but their growth is very slow (Masai et al, 1994 ; Masai and Arai, 1996 ; Maduike et al, 2014 ). The T. kodakarensis strains investigated here all express TK0805, the gene that encodes RNase HII (Heider et al, 2017 ) and thus R-loop accumulation is unlikely to be responsible for origin-independent genome replication in T. kodakarensis .…”

Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair

“…The steady-state kinetic rate was reduced 6-fold. RNAse H2 shows burst kinetics, indicative of a rate-limiting step following cleavage (38,101). The amplitude of the burst (Fig.…”