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Journal of Bacteriology, March 2007, p. 1542-1555, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01421-06

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

Elena Curti,^1, Stephen J. Smerdon,² and Elaine O. Davis^1*

Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom,¹ Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom²

Received 7 September 2006/ Accepted 29 November 2006

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.

Published ahead of print on 8 December 2006.

Present address: Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD.