R150A mutant of F TraI relaxase domain: reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide.

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  • Author(s): Harley MJ;Harley MJ; Toptygin D; Troxler T; Schildbach JF
  • Source:
    Biochemistry [Biochemistry] 2002 May 21; Vol. 41 (20), pp. 6460-8.
  • Publication Type:
    Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370623 Publication Model: Print Cited Medium: Print ISSN: 0006-2960 (Print) Linking ISSN: 00062960 NLM ISO Abbreviation: Biochemistry Subsets: MEDLINE
    • Publication Information:
      Original Publication: Washington, American Chemical Society.
    • Subject Terms:
      Mutagenesis, Site-Directed*; DNA Helicases/*genetics ; DNA, Single-Stranded/*metabolism ; DNA-Binding Proteins/*metabolism ; Deoxyribonucleases/*genetics ; F Factor/*genetics ; Oligonucleotides/*metabolism; Alanine/genetics ; Arginine/genetics ; DNA Helicases/metabolism ; Deoxyribonucleases/metabolism ; Escherichia coli Proteins ; F Factor/metabolism ; Fluorescence Polarization ; Hydrolysis ; Protein Binding/genetics ; Protein Structure, Tertiary/genetics
    • Abstract:
      F factor TraI is a helicase and a single-stranded DNA nuclease ("relaxase") essential for conjugative DNA transfer. A TraI domain containing relaxase activity, TraI36, was generated previously. Substituting Ala for Arg150 (R150A) of TraI36 reduces in vitro relaxase activity. The mutant has reduced affinity, relative to wild type, for a 3'-TAMRA-labeled 22-base single-stranded oligonucleotide. While both R150A and wild-type TraI36 bind oligonucleotide, only wild type increases steady-state fluorescence anisotropy of the labeled 22-base oligonucleotide upon binding. In contrast, binding by either protein increases steady-state anisotropy of a 3'-TAMRA-labeled 17-base oligonucleotide. Time-resolved intensity data for both oligonucleotides, bound and unbound, require three lifetimes for adequate fits, at least one more than the fluorophore alone. The preexponential amplitude for the longest lifetime increases upon binding. Time-resolved anisotropy data for both oligonucleotides, bound and unbound, require two rotational correlation times for adequate fits. The longer correlation time increases upon protein binding. Correlation times for the protein-bound 17-base oligonucleotide are similar for both proteins, with the longer correlation time in the range of molecular tumbling of the protein-DNA complex. In contrast, protein binding causes less dramatic increases in correlation times for the 22-base oligonucleotide relative to the 17-base oligonucleotide. Binding studies indicate that R150 contributes to recognition of bases immediately 3' to the DNA cleavage site, consistent with the apparent proximity of R150 and the 3' oligonucleotide end. Models in which the R150A substitution alters single-stranded DNA flexibility at the oligonucleotide 3' end or affects fluorophore-DNA or fluorophore-protein interactions are discussed.
    • Grant Information:
      RR01348 United States RR NCRR NIH HHS
    • Accession Number:
      0 (DNA, Single-Stranded)
      0 (DNA-Binding Proteins)
      0 (Escherichia coli Proteins)
      0 (Oligonucleotides)
      94ZLA3W45F (Arginine)
      EC 3.1.- (Deoxyribonucleases)
      EC 3.6.1.- (TraI protein, E coli)
      EC 3.6.4.- (DNA Helicases)
      OF5P57N2ZX (Alanine)
    • Publication Date:
      Date Created: 20020516 Date Completed: 20020628 Latest Revision: 20190613
    • Publication Date:
      20240829
    • Accession Number:
      10.1021/bi011969i
    • Accession Number:
      12009909