title: Effects of conserved residues and naturally occurring mutations on Mycobacterium tuberculosis RecG helicase activity.
abstract: RecG is a helicase that is conserved in nearly all bacterial species. The prototypical Escherichia coli RecG promotes regression of stalled replication forks, participates in DNA recombination and DNA repair, and prevents aberrant replication. Mycobacterium tuberculosis RecG (RecGMtb) is a DNA-dependent ATPase that unwinds a variety of DNA substrates, although its preferred substrate is a Holliday junction. Here, we performed site-directed mutagenesis of selected residues in the wedge domain and motifs Q, I, Ib and VI of RecGMtb. Three of the 10 substitution mutations engineered were detected previously as naturally occurring SNPs in the gene encoding RecGMtb. Alanine substitution mutations at residues Q292, F286, K321 and R627 abolished the RecGMtb unwinding activity, whilst RecGMtb F99A, P285S and T408A mutants exhibited ~25-50 % lower unwinding activity than WT. We also found that RecGMtb bound ATP in the absence of a DNA cofactor.
authors.affiliation: Centre for Molecular Biology and Neuroscience and Department of Microbiology, Oslo University Hospital (Rikshospitalet), Oslo, Norway.|Centre for Molecular Biology and Neuroscience and Department of Microbiology, University of Oslo, Oslo, Norway.
authors.forename: Ephrem Debebe
authors.initials: ED
authors.lastname: Zegeye
authors.affiliation: Centre for Molecular Biology and Neuroscience and Department of Microbiology, Oslo University Hospital (Rikshospitalet), Oslo, Norway.|Centre for Molecular Biology and Neuroscience and Department of Microbiology, University of Oslo, Oslo, Norway.
authors.forename: Seetha V
authors.initials: SV
authors.lastname: Balasingham
authors.affiliation: Bioinformatics Core Facility, Department of Informatics, University of Oslo, Oslo, Norway.|Centre for Molecular Biology and Neuroscience and Department of Microbiology, Oslo University Hospital (Rikshospitalet), Oslo, Norway.|Centre for Molecular Biology and Neuroscience and Department of Microbiology, University of Oslo, Oslo, Norway.
authors.forename: Jon K
authors.initials: JK
authors.lastname: Laerdahl
authors.affiliation: Centre for Molecular Biology and Neuroscience and Department of Microbiology, University of Oslo, Oslo, Norway.
authors.forename: Håvard
authors.initials: H
authors.lastname: Homberset
authors.affiliation: Department of Molecular Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Per E
authors.initials: PE
authors.lastname: Kristiansen
authors.affiliation: Centre for Molecular Biology and Neuroscience and Department of Microbiology, Oslo University Hospital (Rikshospitalet), Oslo, Norway.|Centre for Molecular Biology and Neuroscience and Department of Microbiology, University of Oslo, Oslo, Norway.
authors.forename: Tone
authors.initials: T
authors.lastname: Tønjum
mesh_terms: D000595:Amino Acid Sequence; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D050505:Mutant Proteins; D004265:DNA Helicases