Characterization of the helicase activity and substrate specificity of Mycobacterium tuberculosis UvrD.
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.
Curti
Elena
E
Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Smerdon
Stephen J
SJ
Davis
Elaine O
EO
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases