Characterization of the Helicase Activity and Substrate Specificity of Mycobacterium tuberculosis UvrD

Abstract

UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3-single-stranded tailed duplex substrates over 5-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3-to-5 polarity. A 3 single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.

UvrD2 Is Essential in Mycobacterium tuberculosis, but Its Helicase Activity Is Not Required

“…3). This unwinding was strictly ATP dependent and required magnesium in the buffer, as was seen for UvrD1 (4). DNA unwinding increased with increasing UvrD2 concentrations, and plotting the initial unwinding rate against protein concentration showed a linear increase, again as seen with UvrD1.…”

“…tuberculosis and other mycobacterial species possess two homologues of UvrD, annotated UvrD1 and UvrD2 (3). Previously, we have shown that UvrD1 of M. tuberculosis is a DNA helicase with 3Ј-5Ј polarity and with an unwinding preference for nicked DNA resembling an NER intermediate and for substrates resembling stalled replication forks (4). Furthermore, an M. tuberculosis uvrD1 mutant strain exhibited increased sensitivity to DNA damaging agents commonly processed by NER (J. Houghton, C. Güthlein, B. Springer, E. C. Böttger, and E. O. Davis, unpublished data), similar to a Mycobacterium smegmatis uvrD1 mutant strain (10).…”

Important Role for Mycobacterium tuberculosis UvrD1 in Pathogenesis and Persistence apart from Its Function in Nucleotide Excision Repair

“…UvrD homologues can also act as translocases, and in this way they are able to displace proteins bound to DNA (1). It is possible that translocase activity of M. tuberculosis UvrD1 could contribute to clearing RecA from the DNA at fork structures (9,46). This behavior would be similar to that of the E. coli RecG helicase, which has been shown to be involved in the recovery of stalled replication forks (41).…”

“…However, biochemical studies of M. tuberculosis UvrD1 have shown it to be a helicase with 3=-5= polarity and with an unwinding preference for nicked DNA duplexes that resemble that of NER intermediates in addition to substrates resembling stalled replication forks (9), and a recent study has implicated a role for M. smegmatis UvrD1 in recombination (22).…”

“…UvrD2 also differs from most UvrD proteins in possessing a HRDC domain more commonly associated with RecQ family helicases (47). Furthermore, purified M. tuberculosis UvrD1 protein has been shown to be highly active on a substrate resembling an NER intermediate in vitro (9). Therefore, we hypothesized that UvrD1 is most likely functional in NER.…”