title: 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 asThermococcusrely only upon RNaseH2 to initiate the pathway. BecauseThermococcusDNA 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 archaeonThermococcus kodakarensisbothin vitroandin vivoand 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.
authors.affiliation: From New England Biolabs, Inc., Ipswich, Massachusetts 01938 and.
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Heider
authors.affiliation: the Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80521.
authors.forename: Brett W
authors.initials: BW
authors.lastname: Burkhart
authors.affiliation: the Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80521.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Santangelo
authors.affiliation: From New England Biolabs, Inc., Ipswich, Massachusetts 01938 and gardner@neb.com.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Gardner
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D053904:DNA Breaks, Single-Stranded; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*; D019712:Thermococcus / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D019951:DNA Polymerase beta; D016914:Ribonuclease H; D011088:DNA Ligases
keywords: DNA damage; DNA repair; DNA replication; DNA synthesis; RNaseH2; archaea; enzyme kinetics; pre-steady-state kinetics; ribonucleotide excision repair