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 Journal Article
| Authors: | Jacewicz, A.; Shuman, S. |
| Article Title: | 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 |
| Abstract: | 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<inf>4</inf> 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 H<inf>2</inf>, 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. © 2015, American Society for Microbiology. |
| Keywords: | unclassified drug; nonhuman; protein domain; steady state; amino acid substitution; carboxy terminal sequence; enzyme activity; double stranded dna; amino acid sequence; amino terminal sequence; escherichia coli; excision repair; okazaki fragment; enzyme specificity; rna cleavage; dna cleavage; mycobacterium smegmatis; acid phosphatase; ribonuclease h; dna rna hybridization; dna strand; biochemical analysis; primer rna; priority journal; article; ribonuclease h c; turnover number |
| Journal Title: | Journal of Bacteriology |
| Volume: | 197 |
| Issue: | 15 |
| ISSN: | 0021-9193 |
| Publisher: | American Society for Microbiology |
| Date Published: | 2015-08-01 |
| Start Page: | 2489 |
| End Page: | 2498 |
| Language: | English |
| DOI: | 10.1128/jb.00268-15 |
| PROVIDER: | scopus |
| PUBMED: | 25986906 |
| PMCID: | PMC4518832 |
| DOI/URL: | |
| Notes: | Export Date: 3 August 2015 -- Source: Scopus |
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