title: Division of labor among Mycobacterium smegmatis RNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase.
abstract: RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.
authors.affiliation: Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Richa
authors.initials: R
authors.lastname: Gupta
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Debashree
authors.initials: D
authors.lastname: Chatterjee
authors.affiliation: Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA glickmam@mskcc.org.|Division of Infectious Diseases, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Glickman
authors.affiliation: Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism; D012265:Ribonucleotides / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007527:Isoenzymes; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012265:Ribonucleotides; D006861:Hydrogen Peroxide; D016914:Ribonuclease H; C078856:ribonuclease HI