Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

• Published in: The Journal of biological chemistry Vol. 290; no. 19; pp. 11948 - 11968
• Main Authors: Dewhare, Shivendra Singh, Umesh, T.G., Muniyappa, K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08-05-2015; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Motifs; Amino Acid Sequence; Atomic Force Microscopy (AFM); ATPase; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bioinformatics; Catalysis; Cloning; Cloning, Molecular; DNA Helicase; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Recombination; DNA, Single-Stranded - metabolism; Enzyme Kinetics; Enzymology; Escherichia coli - enzymology; Escherichia coli Proteins - metabolism; Exodeoxyribonuclease V - metabolism; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Hydrolysis; Infectious Disease; Microscopy, Atomic Force; Molecular Sequence Data; Mutagenesis, Site-Directed; Mycobacteria; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Protein Binding; Protein Multimerization; Recombination, Genetic; Sequence Homology, Amino Acid; Species Specificity; Atomic Force Microscopy (AFM); Mycobacterium tuberculosis; ATPase; Cloning; Mycobacteria; Infectious Disease; Enzyme Kinetics; Bioinformatics; DNA Recombination; DNA Helicase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.619395

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.
Background: The heterotrimeric M. tuberculosis RecBCD complex, or each of its individual subunits, remains uncharacterized.
Results: MtRecD exists as a homodimer in solution, catalyzes ssDNA-dependent ATP hydrolysis, unwinding of DNA replication/recombination intermediates, and interacts with RecA.
Conclusion: MtRecD possesses strong 5′ → 3′- and weak 3′ → 5′-helicase activities.
Significance: These findings provide insights into the mechanism underlying DSB repair and homologous recombination in mycobacteria.