Molecular and functional characterization of RecD, a novel member of the SF1 family of helicases, from Mycobacterium tuberculosis [Microbiology]

March 23rd, 2015 by Dewhare, S. S., Umesh, T. G., Muniyappa, K.

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA (ssDNA) and linear duplexes containing 5' overhangs relative to the 3' overhangs, but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having ≥18-nucleotide overhangs, but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared to linear duplex having 3' overhangs. The intrinsic ATPase activity of MtRecD was stimulated by ssDNA. Site-specific mutagenesis of K179 in motif-I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5' overhangs; it could also catalyze significant unwinding of substrates containing 3' overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5′→3′ and weak 3′→5′ unwinding activities. The extent of unwinding of Y-shaped DNA structures was ~3-fold lower compared to duplexes with 5' overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and presents important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.
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