Structural and Functional Characterization of an RNase HI Domain from the Bifunctional Protein Rv2228c from
            <i>Mycobacterium tuberculosis</i>

Watkins, Harriet A.; Baker, Edward N.

doi:10.1128/jb.01615-09

Cited by 31 publications

(35 citation statements)

References 50 publications

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“…The properties of M. smegmatis RnhC reported here differ from those ascribed to the M. tuberculosis homolog Rv2228c (14). First, whereas Rv2228c displayed identical activity in degrading RNA:DNA and RNA:RNA duplexes, RnhC is exclusively an RNase H enzyme.…”

Section: Discussioncontrasting

confidence: 40%

“…Watkins and Baker reported that M. tuberculosis Rv2228c cleaved an RNA:RNA duplex in the presence of manganese and that the K m and V max for double-stranded RNase (dsRNase) activity were identical to the K m and V max for RNase H activity (14). In light of their results, we surveyed the substrate specificity of RnhC in the presence of manganese as the metal cofactor.…”

Section: Recombinant Msmeg_4305mentioning

confidence: 99%

“…Two short gaps in the alignment flank the putative linker between an N-terminal RNase H domain and a C-terminal acid phosphatase domain. Four conserved acidic residues, Asp8, Glu49, Asp73, and Asp123, are predicted to coordinate two catalytic metal ions in the RNase H active site (14). We produced full-length wild-type MSMEG_4305 in E. coli as a His 10 -Smt3 fusion and isolated the protein from a soluble extract by nickel-agarose chromatography.…”

Section: Recombinant Msmeg_4305mentioning

confidence: 99%

“…The in vivo impact of ribonucleotide incorporation by mycobacterial polymerases is undoubtedly blunted, if not obscured, by the presence in the M. smegmatis proteome of four different RNase H enzymes: MSMEG_5562/RnhA (13), MSMEG_4305 (14), MSMEG_2442/RnhB, and MSMEG_5849 (15). RNase H enzymes incise the RNA strand of RNA:DNA hybrid duplexes; they are classified as type I (H1) or type II (H2 and H3) (16)(17)(18)(19).…”

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confidence: 99%

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Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

Jacewicz

Shuman

2015

J Bacteriol

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Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3=-OH end and at least one or two ribonucleotides on the 5=-PO 4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. IMPORTANCERNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single-ribonucleotide surveillance. RnhC has a C-terminal acid phosphatase domain that is functionally autonomous of its N-terminal RNase H catalytic domain. RnhC homologs are prevalent in Actinobacteria.T he human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis have a large roster of DNA repair enzymes, including a shared set of eight DNA polymerases (1-5), four of which-LigD-POL, PolD1, PolD2, and DinB2-have the distinctive properties of low fidelity and readily incorporating ribonucleotides in lieu of deoxyribonucleotides during primer extension and gap repair in vitro (4-10). LigD-POL, PolD1, and PolD2 are paralogous members of the AEP (archaeal/eukaryal polymerase/primase) polymerase family (4, 10, 11). They incorporate between one and four sequential ribonucleoside monophosphates (rNMPs) at a DNA primer terminus; after four rNMPs, they cease to elongate (4,8). This effect is attributed to their inability to extend an RNA:DNA hybrid primer terminus with a fully A-form helical conformation (8). DinB2 is a Y family polymerase, and it...

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Section: Discussioncontrasting

confidence: 40%

Section: Recombinant Msmeg_4305mentioning

confidence: 99%

Section: Recombinant Msmeg_4305mentioning

confidence: 99%

mentioning

confidence: 99%

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Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

Jacewicz

Shuman

2015

J Bacteriol

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“…RNase HII activity was monitored in vitro by measuring the increase in fluorescence resulting from the degradation of an artificially synthesized RNA·DNA hybrid as described earlier (57). Concentrations of 100 M (each) custom-synthesized 12-mers, comprising 5=-fluorescein-poly(A) ribooligonucleotide and 3=-dabsyl-poly(dT) deoxyribooligonucleotide (Bioneer, South Korea), were hybridized in annealing buffer (50 mM Tris-Cl, pH 7.5 at RT, 60 mM KCl) by heating the mixture to 95°C for 5 min, followed by slow cooling to room temperature.…”

Section: Methodsmentioning

confidence: 99%

MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

Murdeshwar

Chatterji

2012

J Bacteriol

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Crystal structures of MBP fusion proteins

Waugh

2016

Protein Science

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Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter-domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP-mediated structural distortions are very rare.

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Structural and Functional Characterization of an RNase HI Domain from the Bifunctional Protein Rv2228c from Mycobacterium tuberculosis

Cited by 31 publications

References 50 publications

Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

Crystal structures of MBP fusion proteins

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