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.

The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3′-5′ exonuclease that associates with a DNA replication complex

“…In contrast, TaRecJ2 showed quite different properties from TaRecJ1. TaRecJ2 formed a stable complex with GINS, as reported in S. solfataricus, T. kodakarensis, and P. furiosus (9,22,23), and exhibited 3Ј-5Ј exonuclease activity for both DNA and RNA. TaGINS stimulated the exonuclease activity of TaRecJ2 only at acidic pH values ϽpH 7.0.…”

“…Thermococcus kodakarensis RecJ (GAN, GINS-associated nuclease) has the DNA-specific 5Ј-3Ј exonuclease activity, which is stimulated by the interaction with GINS, and GAN may be involved in Okazaki fragment processing (22). Pyrococcus furiosus RecJ (PfRecJ) reportedly cleaved DNA in the 5Ј-3Ј direction and degraded RNA in the 3Ј-5Ј direction (23). PfRecJ was proposed to be involved in the proofreading of mismatched RNA primers in DNA replication.…”

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

“…6) (20). Although bacterial RecJ is a 5′-3′ exonuclease, binding polarities can switch between homologous proteins over the course of evolution; along these lines, in the archaeal organism Pyrococcus furiosus, the RecJ/Cdc45 homolog has been reported to be a 3′-5′ exonuclease (44). Given that the archaeal RecJ homologs of Cdc45 associate with GINS (45,46), it will be informative to understand in more depth how the repair and replication functions of these proteins are coordinated.…”

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

“…The latter RecJ-like protein was reported as the GINS-associated nuclease (GAN), and may be involved in lagging strand processing (52). Furthermore, characterization of the GAN ortholog from P. furiosus revealed the 3Ј-5Ј exoribonuclease activity, which may function in proofreading for primer synthesis (53). The work sharing the two RecJ-like proteins, GAN and HAN, in Archaea is an interesting subject for future analyses.…”