title: Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis.
abstract: 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 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 with linear duplex having 3' overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 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 with 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 present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.
authors: From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
authors: Shivendra Singh
authors: SS
authors: Dewhare
authors: From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
authors: T G
authors: TG
authors: Umesh
authors: From the Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India kmbc@biochem.iisc.ernet.in.
authors: K
authors: K
authors: Muniyappa
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism*; D043211:Exodeoxyribonuclease V / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011485:Protein Binding; D055503:Protein Multimerization; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D004265:DNA Helicases
keywords: ATPase; Atomic Force Microscopy (AFM); Bioinformatics; Cloning; DNA Helicase; DNA Recombination; Enzyme Kinetics; Infectious Disease; Mycobacteria; Mycobacterium tuberculosis