Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Wakamatsu, Taisuke; Kim, Kwang Jin; Uemura, Yuri; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

doi:10.1074/jbc.m110.161596

Cited by 35 publications

(50 citation statements)

References 56 publications

(75 reference statements)

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“…Moreover, when using a 59 33 P-labeled 24-mer RNA substrate, degradation intermediates were clearly visible, confirming a 39-59 exonuclease activity. However, it has been published recently that Mpn140 has an in vitro 59-39 exonuclease activity on 6-mers and 11-mers oligos (Wakamatsu et al 2011). The corresponding specific activities are in the same range as those reported here for the 39-59 degradation of Cy5-labeled nanoRNAs, raising the possibility that Mpn140 may change its polarity with the nature of the substrate.…”

Section: Discussionsupporting

confidence: 52%

Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

Postic¹,

Danchin²,

Mechold³

2011

RNA

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Processive RNases are unable to degrade efficiently very short oligonucleotides, and they are complemented by specific enzymes, nanoRNases, that assist in this process. We previously identified NrnA (YtqI) from Bacillus subtilis as a bifunctional protein with the ability to degrade nanoRNA (RNA oligos #5 nucleotides) and to dephosphorylate 39-phosphoadenosine 59-phosphate (pAp) to AMP. While the former activity is analogous to that of oligoribonuclease (Orn) from Escherichia coli, the latter corresponds to CysQ. NrnA homologs are widely present in bacterial and archaeal genomes. They are found preferably in genomes that lack Orn or CysQ homologs. Here, we characterize NrnA homologs from important human pathogens, Mpn140 from Mycoplasma pneumoniae, and Rv2837c from Mycobacterium tuberculosis. Like NrnA, these enzymes degrade nanoRNA and dephosphorylate pAp in vitro. However, they show dissimilar preferences for specific nanoRNA substrate lengths. Whereas NrnA prefers RNA 3-mers with a 10-fold higher specific activity compared to 5-mers, Rv2837c shows a preference for nanoRNA of a different length, namely, 2-mers. Mpn140 degrades Cy5-labeled nanoRNA substrates in vitro with activities varying within one order of magnitude as follows: 5-mer>4-mer>3-mer>2-mer. In agreement with these in vitro activities, both Rv2837c and Mpn140 can complement the lack of their functional counterparts in E. coli: CysQ and Orn. The NrnA homolog from Streptococcus mutans, SMU.1297, was previously shown to hydrolyze pAp and to complement an E. coli cysQ mutant. Here, we show that SMU.1297 can complement an E. coli orn À mutant, suggesting that having both pAp-phosphatase and nanoRNase activity is a common feature of NrnA homologs.

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Section: Discussionsupporting

confidence: 52%

Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

Postic¹,

Danchin²,

Mechold³

2011

RNA

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“…3). However, the 5′‐phosphorylation does not affect the exonuclease activity of ttNrn against 11‐mer ssDNA [13]. This observation suggests a difference in the detailed mechanism of substrate recognition between mononucleotide and oligoribonucleotide.…”

Section: Resultsmentioning

confidence: 86%

Crystal structure of the ligand‐binding form of nanoRNase from Bacteroides fragilis, a member of the DHH/DHHA1 phosphoesterase family of proteins

et al. 2013

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NanoRNase (Nrn) specifically degrades nucleoside 3′,5′-bisphosphate and the very short RNA, nanoRNA, during the final step of mRNA degradation. The crystal structure of Nrn in complex with a reaction product GMP was determined. The overall structure consists of two domains that are interconnected by a flexible loop and form a cleft. Two Mn2+ ions are coordinated by conserved residues in the DHH motif of the N-terminal domain. GMP binds near the DHHA1 motif region in the C-terminal domain. Our structure enables us to predict the substrate-bound form of Nrn as well as other DHH/DHHA1 phosphoesterase family proteins.

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“…If both matched and mismatched RNA primers exist in a ssDNA template, the replicative DNA polymerases can use the matched primers to assemble the replisome. Moreover, considering that PfRecJ exhibits higher specific activity on ssRNA than RNA/DNA hybrids in vitro , this enzyme might be involved in the degradation of diverse ssRNAs (such as mRNA), as observed with other members of the DHH phosphoesterase superfamily (24). …”

Section: Discussionmentioning

confidence: 87%

“…Hence, the OB-fold domain mainly functions in improving the ssDNA-binding capability of bacterial RecJ (Supplementary Figure S6A). Several members of the DHH phosphoesterase superfamily efficiently digest ssDNA and ssRNA shorter than 5 nt in the 5′–3′ direction (24). This activity of DHH phosphoesterase is proposed to play a role in RNA and DNA recycling.…”

Section: Discussionmentioning

confidence: 99%

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

Liu

Han

et al. 2013

Nucleic Acids Research

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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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Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Cited by 35 publications

References 56 publications

Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

Crystal structure of the ligand‐binding form of nanoRNase from Bacteroides fragilis, a member of the DHH/DHHA1 phosphoesterase family of proteins

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

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