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.