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NAR Breakthrough Article Published online 29 November 2016 Nucleic Acids Research, 2017, Vol. 45, No. 1 1–14 doi: 10.1093/nar/gkw1046 NAR Breakthrough Article 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 Richa Gupta1, Debashree Chatterjee2, Michael S. Glickman1,3,* and Stewart Shuman2,* 1Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA, 2Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA and 3Division of Infectious Diseases, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA Received September 14, 2016; Revised October 16, 2016; Editorial Decision October 19, 2016; Accepted October 20, 2016 ABSTRACT pathogenic mycobacteria, as a candidate drug dis- covery target for tuberculosis and leprosy. RNase H enzymes sense the presence of ribonu- cleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of INTRODUCTION an RNA:DNA hybrid. Mycobacterium smegmatis en- codes four RNase H enzymes: RnhA, RnhB, RnhC There is rising interest in the biological impact of ribonu- cleotides embedded in bacterial chromosomes during DNA and RnhD. Here, we interrogate the biochemical ac- replication and repair, and in the pathways of ribonu- tivity and nucleic acid substrate specificity of RnhA. cleotide surveillance that deal with such ‘lesions’ (1). Bacte- We report that RnhA (like RnhC characterized pre- rial polymerases display a range of fidelities with respect to viously) is an RNase H1-type magnesium-dependent discrimination of dNTP and rNTP substrates. The roster of endonuclease with stringent specificity for RNA:DNA DNA repair polymerases of the human pathogen Mycobac- hybrid duplexes. Whereas RnhA does not incise terium tuberculosis and its avirulent relative M. smegma- an embedded mono-ribonucleotide, it can efficiently tis includes four enzymes––LigD-POL, PolD1, PolD2 and cleave within tracts of four or more ribonucleotides DinB2––that have the distinctive properties of low fidelity in duplex DNA. We gained genetic insights to the di- and of readily incorporating ribonucleotides in lieu of de- vision of labor among mycobacterial RNases H by oxyribonucleotides during primer extension and gap repair deleting the rnhA, rnhB, rnhC and rnhD genes, in- in vitro (2–9). We are interested in the connections in my- cobacteria between ribonucleotide utilization and replica- dividually and in various combinations. The salient tive quiescence, which is central to the long-term carriage of conclusions are that: (i) RNase H1 activity is essen- M. tuberculosis in a clinically dormant state. Cells that are tial for mycobacterial growth and can be provided not replicating their DNA may have reduced dNTP pools by either RnhC or RnhA; (ii) the RNase H2 enzymes compared to actively dividing cells. We speculate that DNA RnhB and RnhD are dispensable for growth and (iii) repair with a ‘ribo patch’ by polymerase utilization of avail- RnhB and RnhA collaborate to protect M. smegma- able rNTPs provides a strategy for quiescent cells to avoid tis against oxidative damage in stationary phase. otherwise deadly chromosome damage. Our findings highlight RnhC, the sole RNase H1 in The in vivo impact of ribonucleotide incorporation by mycobacterial polymerases may be obscured by the presence in the M. smegmatis proteome of four *To whom correspondence should be addressed. Tel: +1 212 639 7145; Email: [email protected] Correspondence may also be addressed to Michael S. Glickman. Email: [email protected] Present address: Richa Gupta, Department of Natural Sciences, LaGuardia Community College, Long Island City, NY 11101, USA. C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Nucleic Acids Research, 2017, Vol. 45, No. 1 ribonuclease H enzymes: MSMEG 5562/RnhA (10); ciently cleave a 4-nucleotide ribo tract embedded in duplex MSMEG 4305/RnhC (11); MSMEG 2442/RnhB (12) DNA, but not an embedded di-ribonucleotide or mono- and MSMEG 5849 (13). RNase H enzymes incise the ribonucleotide. Thus, RnhA is a type I RNase H. Whereas RNA strand of RNA:DNA hybrid duplexes; they are M. smegmatis has two type I RNase H enzymes in RnhA classified as type I (H1) or type II (H2 and H3)(14–17). and RnhC, the M. tuberculosis proteome is devoid of an RNase H1 requires an oligoribonucleotide tract and is RnhA homolog. unable to incise a single ribonucleotide embedded in duplex We extend our biochemical findings by querying the ge- DNA. RNase H2 is uniquely capable of incising a single netics of RNase H in M. smegmatis, via constructing and embedded rNMP. Of the four M. smegmatis RNase H characterizing deletion mutants of rnhA, rnhB, rnhC and enzymes, only RnhC has been characterized with respect to rnhD, singly and in combination. We find that rnhA, its RNA requirements (11), a relevant issue given that the rnhB, rnhC and rnhD single mutants are viable, as are ribo-utilizing mycobacterial polymerases differ with respect rnhA rnhB, rnhB rnhC and rnhB rnhD double to how many sequential ribonucleotides they can embed. mutants, and a rnhA rnhB rnhD triple mutant. We The 365-aa RnhC polypeptide consists of two autonomous conclude that there is no essential role for M. smegmatis catalytic domains: an N-terminal 140-aa RNase H1 mod- RNase H2-type enzymes under standard laboratory growth ule and a C-terminal 211-aa acid phosphatase module. conditions. However, we find that rnhA rnhB cells in sta- [The homologous bifunctional RnhC in M. tuberculosis tionary phase are highly sensitized to killing by hydrogen is Rv2228c (18).] The M. smegmatis RnhC endonuclease peroxide and display enhanced sensitivity to UV irradia- is stringently specific for RNA:DNA hybrid duplexes, but tion. RnhC does not selectively recognize and cleave at DNA– In agreement with the recent data of Minias et al. (19), RNA or RNA–DNA junctions in duplex nucleic acid. we were unable to simultaneously delete the rnhA and rnhC RnhC can incise tracts of four or more ribonucleotides loci, unless a second copy of rnhC or rnhA was introduced embedded in duplex DNA, leaving two or more residual elsewhere on the M. smegmatis chromosome. We proceeded ribonucleotides at the cleaved 3-OHendandatleast to discriminate whether the synthetic lethality reflected the one or two ribonucleotides on the 5 -PO4 end. However, absence of an RNase H1-type protein, an RNase H1 en- RnhC cannot incise an embedded mono-ribonucleotide or donuclease activity, or simultaneous absence of an RNase di-ribonucleotide in duplex DNA (11). H1 activity and the acid phosphatase activity of RnhC, by These biochemical characteristics of RnhC seem to rule complementation with biochemically confirmed ‘catalytic- it out as an agent of ribonucleotide excision repair (RER) dead’ mutant alleles of rnhA and rnhC. We show that: (i) of single ribonucleotides embedded during DNA replica- RNase H1 activity is essential for viability of M. smegmatis tion or repair. This RER function, to the extent that it and (ii) the acid phosphatase activity of RnhC is not. We is operative in mycobacteria, would likely default to one discuss how these findings fortify the case for RNase H1as or both of the putative type II RNase H enzymes: RnhB a therapeutic target for human mycobacterial diseases. and MSMEG 5849 (referred to henceforth as RnhD). Nei- ther M. smegmatis RnhB (272-aa) nor its M. tuberculosis MATERIALS AND METHODS counterpart Rv2902c (264-aa), both of which are single- Recombinant RnhA proteins domain proteins homologous to Escherichia coli RNase H2, has been characterized biochemically. Deletion of the M. A 480-nt DNA fragment comprising the rnhA open read- smegmatis rnhB gene has no impact on bacterial growth ing frame (ORF) was amplified from M. smegmatis ge- in liquid culture, sensitivity to hydroxyurea or spontaneous nomic DNA by PCR with primers that introduced a NdeI mutation rate (12). Mycobacterium smegmatis RnhD (alias site at the start codon and a BglII site immediately af- RHII-RSD) is a 567-aa bifunctional enzyme composed of ter the stop codon. The PCR product was digested with an N-terminal RNase H2-like domain and a C-terminal NdeI and BglII and inserted between the NdeI and BamHI (p)ppGpp synthetase domain (13). Full-length RnhD, but sites of pET16b-His10 to generate expression plasmid pET- not an isolated N-terminal domain (aa 1–280), displayed His10•RnhA. Missense mutations E50Q and D72N were RNase H activity in vitro (13). The counterpart in M. tu- introduced into the rnhC ORF by PCR with mutagenic berculosis is Rv0776c, a 259-aa protein homologous to the primers. The inserts in each plasmid were sequenced to ver- N-terminal segment of M. smegmatis RnhD, but lacking the ify the fusion junctions and to ensure that no unwanted (p)ppGpp synthetase module. coding changes were introduced during amplification and Mycobacterium smegmatis RnhA (159-aa) was identi- cloning. fied in 1995 by Mizrahi et al., who cloned the rnhA gene, The RnhA expression plasmids were transfected into E. produced the protein in E. coli as a maltose-binding pro- coli BL21 (DE3) cells. Cultures (2-liter) amplified from sin- tein (MBP)–RnhA fusion, and showed that the affinity- gle transformants were grown at 37◦C in Terrific Broth con- purified MBP–RnhA displayed endoribonuclease activity taining 100 ␮g/ml ampicillin until the A600 reached 0.8. The on an RNA:DNA hybrid substrate (10).
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