Abstract
We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).
Highlights
Helicases play important roles in nucleic acid transactions
Lhr is a monomeric nucleic acid-dependent ATPase that uses the energy of nucleotide hydrolysis to drive 3Ј-to-5Ј translocation along single strand DNA and to unwind duplex DNA or an RNA:DNA hybrid en route
The ATPase is more active in the presence of calcium than magnesium, a feature that distinguishes Lhr from previously characterized mycobacterial helicases. (To wit, UvrD1 is inactive when magnesium is replaced by calcium [4]; RqlH is more active with magnesium than calcium [12]; and SftH is active with magnesium and calcium [13].) Lhr has no appreciable ATPase activity in the absence of nucleic acid; its phosphohydrolase function is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for the DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence
Summary
Results: Mycobacterial Lhr uses the energy of ATP hydrolysis to drive 3Ј-to-5Ј translocation along single strand DNA and to unwind duplexes en route. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3-to-5 translocation along single strand DNA and to unwind duplexes en route. Differences in the roster of DNA helicases between taxa offer useful clues to the evolution and diversification of replication/ repair strategies and, where the rosters diverge in animals versus infectious pathogens, they can suggest anti-infective drug targets With this in mind, we are focused on the ensemble of DNA helicases in Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Key points of interest for this study were as follows. (i) Is Lhr a nucleic acid-dependent phosphohydrolase and, if so, what is its substrate and cofactor specificity? (ii) Can Lhr couple NTP hydrolysis to mechanical work, especially duplex unwinding? (iii) Do the biochemical activities of Lhr point to a role in DNA or RNA transactions? (iv) Is the C-terminal domain of Lhr required for its activities?
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