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RecJ-like protein from Pyrococcus furiosus has 3'-5' exonuclease activity on RNA: implications for proofreading of 3'-mismatched RNA primers in DNA replication.

Yuan H, Liu XP, Han Z, Allers T, Hou JL, Liu JH - Nucleic Acids Res. (2013)

Bottom Line: Pfu DNA polymerase (PolB) cannot elongate the resulting 3'-mismatched RNA primer because it cannot remove the 3'-mismatched ribonucleotide.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China.

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.

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Hydrolysis of the RNA strand of RNA/DNA hybrids by PfRecJ with preference for 3′-mismatches. The 3′–5′ exonuclease of PfRecJ on RNA/DNA hybrids (A, C–E) and ssRNA (B) was determined in a buffer consisting of 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 10 mM KCl, 5 mM DTT, 0.25 mM MnCl2, 100 ng/μl BSA and 4 U Rnsin. (A) Hydrolysis of the RNA/DNA hybrid. RNA/DNA hybrids (50 nM) with 3′-mismatched and 3′-matched ribonucleotides were incubated with 50 nM PfRecJ at 50°C for 30 min. The 3′–5′ exonuclease activity on ssRNA (B) and 3′-mismatched (g/G) RNA/DNA hybrid (C) was assayed in the presence of different RPA concentrations (0, 0.1, 0.5, 1, 2 and 5 μM). The substrates (50 nM) were incubated with 10 nM PfRecJ at 50°C for 30 min. Panel D is the quantitation of panels B and C. (E) Hydrolysis of the RNA/DNA hybrid with all possible base matches/mismatches. Sixteen RNA/DNA hybrids (50 nM) with all possible base matches/mismatches were incubated with 50 nM PfRecJ at 50°C for 10 min in the presence of 1 μM RPA. The cleavage percentages are listed at the bottom of panel E. Lowercase and uppercase denote RNA and DNA, respectively.
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gkt275-F4: Hydrolysis of the RNA strand of RNA/DNA hybrids by PfRecJ with preference for 3′-mismatches. The 3′–5′ exonuclease of PfRecJ on RNA/DNA hybrids (A, C–E) and ssRNA (B) was determined in a buffer consisting of 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 10 mM KCl, 5 mM DTT, 0.25 mM MnCl2, 100 ng/μl BSA and 4 U Rnsin. (A) Hydrolysis of the RNA/DNA hybrid. RNA/DNA hybrids (50 nM) with 3′-mismatched and 3′-matched ribonucleotides were incubated with 50 nM PfRecJ at 50°C for 30 min. The 3′–5′ exonuclease activity on ssRNA (B) and 3′-mismatched (g/G) RNA/DNA hybrid (C) was assayed in the presence of different RPA concentrations (0, 0.1, 0.5, 1, 2 and 5 μM). The substrates (50 nM) were incubated with 10 nM PfRecJ at 50°C for 30 min. Panel D is the quantitation of panels B and C. (E) Hydrolysis of the RNA/DNA hybrid with all possible base matches/mismatches. Sixteen RNA/DNA hybrids (50 nM) with all possible base matches/mismatches were incubated with 50 nM PfRecJ at 50°C for 10 min in the presence of 1 μM RPA. The cleavage percentages are listed at the bottom of panel E. Lowercase and uppercase denote RNA and DNA, respectively.

Mentions: During DNA replication, an RNA primer forms a ds RNA/DNA hybrid with the DNA template (9–11). Therefore, we characterized the 3′ exonuclease activity of PfRecJ on a ds RNA/DNA hybrid with 3′-end recessed RNA. Results show that PfRecJ preferentially acts on the 3′-mismatched ribonucleotide in the ds RNA/DNA hybrid (Figure 4A); the removal of a g/G mismatch was approximately four times more efficient than that of a g/C match. This preference for the 3′ mismatch suggests that PfRecJ has a potential proofreading activity for mismatched RNA primers. Previous reports have shown that GINS, a core subcomplex in archaeal replisome, stimulates 5′ exonuclease activity and physically interacts with archaeal RecJ-like protein (23,31). However, GINS of P. furiosus did not stimulate the 3′ exonuclease activity of PfRecJ on ssRNAs of various lengths (Supplementary Figure S4A), and on the RNA/DNA hybrid (Supplementary Figure S4B); however, we did confirm the stimulation by GINS of 5′ exonuclease activity on ssDNA (Supplementary Figure S4C). Given that the ssDNA template is bound by RPA during DNA replication, the effects of RPA on the activity of PfRecJ were characterized. RPA exhibited differential effects on the 3′ exonuclease of RecJ on the ssRNA and the RNA/DNA hybrid. The activity on the ssRNA was not affected by RPA (Figure 4B), whereas the activity on the RNA/DNA hybrid was markedly stimulated by RPA (Figure 4C), which elevated cleavage of the RNA strand of the RNA/DNA hybrid by ∼3.5 times (Figure 4D). We then characterized 16 RNA/DNA hybrids with all possible base matches/mismatches in the presence of 1 μM RPA. All 3′-mismatched RNA/DNA hybrids were hydrolyzed with a higher efficiency than the 3′-matched hybrids (Figure 4E). These results indicate that PfRecJ can remove mismatched ribonucleotides incorporated by primase (Figure 1), and that RPA stimulates the ability of RecJ to proofread 3′-mismatched ribonucleotides by binding to the ssDNA template.Figure 4.


RecJ-like protein from Pyrococcus furiosus has 3'-5' exonuclease activity on RNA: implications for proofreading of 3'-mismatched RNA primers in DNA replication.

Yuan H, Liu XP, Han Z, Allers T, Hou JL, Liu JH - Nucleic Acids Res. (2013)

Hydrolysis of the RNA strand of RNA/DNA hybrids by PfRecJ with preference for 3′-mismatches. The 3′–5′ exonuclease of PfRecJ on RNA/DNA hybrids (A, C–E) and ssRNA (B) was determined in a buffer consisting of 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 10 mM KCl, 5 mM DTT, 0.25 mM MnCl2, 100 ng/μl BSA and 4 U Rnsin. (A) Hydrolysis of the RNA/DNA hybrid. RNA/DNA hybrids (50 nM) with 3′-mismatched and 3′-matched ribonucleotides were incubated with 50 nM PfRecJ at 50°C for 30 min. The 3′–5′ exonuclease activity on ssRNA (B) and 3′-mismatched (g/G) RNA/DNA hybrid (C) was assayed in the presence of different RPA concentrations (0, 0.1, 0.5, 1, 2 and 5 μM). The substrates (50 nM) were incubated with 10 nM PfRecJ at 50°C for 30 min. Panel D is the quantitation of panels B and C. (E) Hydrolysis of the RNA/DNA hybrid with all possible base matches/mismatches. Sixteen RNA/DNA hybrids (50 nM) with all possible base matches/mismatches were incubated with 50 nM PfRecJ at 50°C for 10 min in the presence of 1 μM RPA. The cleavage percentages are listed at the bottom of panel E. Lowercase and uppercase denote RNA and DNA, respectively.
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Related In: Results  -  Collection

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gkt275-F4: Hydrolysis of the RNA strand of RNA/DNA hybrids by PfRecJ with preference for 3′-mismatches. The 3′–5′ exonuclease of PfRecJ on RNA/DNA hybrids (A, C–E) and ssRNA (B) was determined in a buffer consisting of 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 10 mM KCl, 5 mM DTT, 0.25 mM MnCl2, 100 ng/μl BSA and 4 U Rnsin. (A) Hydrolysis of the RNA/DNA hybrid. RNA/DNA hybrids (50 nM) with 3′-mismatched and 3′-matched ribonucleotides were incubated with 50 nM PfRecJ at 50°C for 30 min. The 3′–5′ exonuclease activity on ssRNA (B) and 3′-mismatched (g/G) RNA/DNA hybrid (C) was assayed in the presence of different RPA concentrations (0, 0.1, 0.5, 1, 2 and 5 μM). The substrates (50 nM) were incubated with 10 nM PfRecJ at 50°C for 30 min. Panel D is the quantitation of panels B and C. (E) Hydrolysis of the RNA/DNA hybrid with all possible base matches/mismatches. Sixteen RNA/DNA hybrids (50 nM) with all possible base matches/mismatches were incubated with 50 nM PfRecJ at 50°C for 10 min in the presence of 1 μM RPA. The cleavage percentages are listed at the bottom of panel E. Lowercase and uppercase denote RNA and DNA, respectively.
Mentions: During DNA replication, an RNA primer forms a ds RNA/DNA hybrid with the DNA template (9–11). Therefore, we characterized the 3′ exonuclease activity of PfRecJ on a ds RNA/DNA hybrid with 3′-end recessed RNA. Results show that PfRecJ preferentially acts on the 3′-mismatched ribonucleotide in the ds RNA/DNA hybrid (Figure 4A); the removal of a g/G mismatch was approximately four times more efficient than that of a g/C match. This preference for the 3′ mismatch suggests that PfRecJ has a potential proofreading activity for mismatched RNA primers. Previous reports have shown that GINS, a core subcomplex in archaeal replisome, stimulates 5′ exonuclease activity and physically interacts with archaeal RecJ-like protein (23,31). However, GINS of P. furiosus did not stimulate the 3′ exonuclease activity of PfRecJ on ssRNAs of various lengths (Supplementary Figure S4A), and on the RNA/DNA hybrid (Supplementary Figure S4B); however, we did confirm the stimulation by GINS of 5′ exonuclease activity on ssDNA (Supplementary Figure S4C). Given that the ssDNA template is bound by RPA during DNA replication, the effects of RPA on the activity of PfRecJ were characterized. RPA exhibited differential effects on the 3′ exonuclease of RecJ on the ssRNA and the RNA/DNA hybrid. The activity on the ssRNA was not affected by RPA (Figure 4B), whereas the activity on the RNA/DNA hybrid was markedly stimulated by RPA (Figure 4C), which elevated cleavage of the RNA strand of the RNA/DNA hybrid by ∼3.5 times (Figure 4D). We then characterized 16 RNA/DNA hybrids with all possible base matches/mismatches in the presence of 1 μM RPA. All 3′-mismatched RNA/DNA hybrids were hydrolyzed with a higher efficiency than the 3′-matched hybrids (Figure 4E). These results indicate that PfRecJ can remove mismatched ribonucleotides incorporated by primase (Figure 1), and that RPA stimulates the ability of RecJ to proofread 3′-mismatched ribonucleotides by binding to the ssDNA template.Figure 4.

Bottom Line: Pfu DNA polymerase (PolB) cannot elongate the resulting 3'-mismatched RNA primer because it cannot remove the 3'-mismatched ribonucleotide.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China.

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.

Show MeSH