title: RNase H2-initiated ribonucleotide excision repair.
abstract: Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. Here, we present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. RER is most efficient when the ribonucleotide is incised by RNase H2, and further excised by the flap endonuclease FEN1 with strand displacement synthesis carried out by DNA polymerase δ, the PCNA clamp, its loader RFC, and completed by DNA ligase I. We observed partial redundancy for several of the enzymes in this pathway. Exo1 substitutes for FEN1 and Pol ε for Pol δ with reasonable efficiency. However, RNase H1 fails to substitute for RNase H2 in the incision step of RER.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Justin L
authors.initials: JL
authors.lastname: Sparks
authors.forename: Hyongi
authors.initials: H
authors.lastname: Chon
authors.forename: Susana M
authors.initials: SM
authors.lastname: Cerritelli
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kunkel
authors.forename: Erik
authors.initials: E
authors.lastname: Johansson
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Crouch
authors.forename: Peter M
authors.initials: PM
authors.lastname: Burgers
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D018809:Proliferating Cell Nuclear Antigen; D012265:Ribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D000123:Acetyltransferases; C107118:ELO2 protein, S cerevisiae; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C091139:ribonuclease HII; D016914:Ribonuclease H; C078856:ribonuclease HI; D011088:DNA Ligases; D000072481:DNA Ligase ATP