Introduction of a tryptophan reporter group into loop 1 of the recA protein. Examination of the conformational states of the recA-ssDNA complex by fluorescence spectroscopy.
Site-directed mutagenesis was used to replace His-163 in the Loop 1 region of the recA protein with a tryptophan residue. The [H163W]recA protein binds single-stranded DNA (ssDNA), catalyzes ssDNA-dependent ATP hydrolysis, and is fully active in the three-strand exchange reaction. In addition, the fluorescence properties of the Trp-163 reporter group are very sensitive to the binding of nucleotide cofactors to the H163W]recA-ssDNA complex. The fluorescence of Trp-163 is modestly quenched by the binding of ADP (21%) and strongly quenched by the nonhydrolyzable ATP analog, ATP gamma S (70%); since ADP and ATP gamma S stabilize the closed and open conformations of the recA-ssDNA complex, respectively, the quenched states observed with these nucleotides likely reflect differences in the fluorescence properties of tryptophan 163 in these two states. ATP has a more complex time-dependent effect on Trp-163 fluorescence. When ATP is added to [H163W]recA-ssDNA complexes, there is an immediate quenching of Trp-163 fluorescence (44%) which is intermediate in intensity between that observed with ADP and ATP gamma S. The ATP-induced quenching gradually decreases with time as the pool of ATP is converted to ADP by the ATP hydrolysis activity of the [H163W]recA protein. These results are discussed with regard to the nucleotide cofactor-dependent conformational transitions of the recA-ssDNA complex.
Stole
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Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205.
Bryant
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FR
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation / Q000187:drug effects; D011938:Rec A Recombinases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D017931:DNA Primers; D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D006639:Histidine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases