title: RecJ-like protein from Pyrococcus furiosus has 3'-5' exonuclease activity on RNA: implications for proofreading of 3'-mismatched RNA primers in DNA replication.
abstract: Replicative DNA polymerases require an RNA primer for leading and lagging strand DNA synthesis, and primase is responsible for the de novo synthesis of this RNA primer. However, the archaeal primase from Pyrococcus furiosus (Pfu) frequently incorporates mismatched nucleoside monophosphate, which stops RNA synthesis. Pfu DNA polymerase (PolB) cannot elongate the resulting 3'-mismatched RNA primer because it cannot remove the 3'-mismatched ribonucleotide. This study demonstrates the potential role of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading 3'-mismatched ribonucleotides. PfRecJ hydrolyzes single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3'-5' direction, and the kinetic parameters (Km and Kcat) of PfRecJ during RNA strand digestion are consistent with a role in proofreading 3'-mismatched RNA primers. Replication protein A, the single-stranded DNA-binding protein, stimulates the removal of 3'-mismatched ribonucleotides of the RNA strand in RNA/DNA hybrids, and Pfu DNA polymerase can extend the 3'-mismatched RNA primer after the 3'-mismatched ribonucleotide is removed by PfRecJ. Finally, we reconstituted the primer-proofreading reaction of a 3'-mismatched ribonucleotide RNA/DNA hybrid using PfRecJ, replication protein A, Proliferating cell nuclear antigen (PCNA) and PolB. Given that PfRecJ is associated with the GINS complex, a central nexus in archaeal DNA replication fork, we speculate that PfRecJ proofreads the RNA primer in vivo.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Yuan
authors.forename: Xi-Peng
authors.initials: XP
authors.lastname: Liu
authors.forename: Zhong
authors.initials: Z
authors.lastname: Han
authors.forename: Thorsten
authors.initials: T
authors.lastname: Allers
authors.forename: Jing-Li
authors.initials: JL
authors.lastname: Hou
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Liu
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism*; D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005095:Exoribonucleases / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; C034477:RNA primers; D012313:RNA; D004247:DNA; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D005095:Exoribonucleases