Abstract
The functional role of Zn(II) binding by T4 gene 32 protein (gp32), a single-stranded DNA-binding protein, has been investigated by assessing the capacity of a well-characterized metal-free gp32 derivative to function in vitro as an accessory protein of T4 uvsX-catalyzed homologous pairing. Metal-free gp32 was prepared upon reaction of cysteine thiolates with methylmethanethiol-sulfonate to form the mixed disulfide Cys-SSCH3 or S-methylated species. Far and near ultraviolet circular dichroism spectroscopy suggest a moderate but easily detected change in the far UV region, accompanied by only a minor alteration in the near UV region, relative to the Zn(II)-containing protein. Restoration of the wild-type spectral features is accomplished upon the addition of 2 mM dithiothreitol and excess Zn(II) but not dithiothreitol alone. Unlike wild-type gp32, apo S-methylated gp32 shows weak binding to the recombination substrate, single-stranded M13mp19, and fails to stimulate homologous pairing with a linear M13mp19 duplex substrate by uvsX protein. Complete reactivation of the apo S-methylated protein as a recombination-accessory protein is achievable in situ in the presence of reducing agent and sufficient exogenous Zn(II), but not one or the other alone. Analogous results are obtained with S-methylated C166S (Cys166-->Ser) gp32, revealing that only the metal-liganding cysteines participate in the reconstitution. These findings suggest that formation of the Zn(II) chelate is directly linked to single-stranded DNA binding and functional efficacy of gp32 in DNA metabolism.
Highlights
From the Department of Biochemistry and Biophysics, lkxas A & M University, College Station, lkxas 77843-2128and the $Department of Chemistry and Biochemistry, University of l k m s, Austin, lkxas 78712
The functional role of Zn(II) binding by T4 gene 32 filamentbut plays an important role in postsynaptic protein,a single-stranded DNA-binding protein, phases of recombination, where it is poised to capture the duhas been investigated by assessing the capacity of a plex strand which is displaced upon uvsX-mediated pairing well-characterized metal-freegp32 derivative to func- (Hashimoto and Yonesaki, 1991;Kodadek, 1990).In vitro, gp32 tion in vitroasan accessory proteinT4ofuvsX-catalyzed inhibits the ATPase activity of uvsX, by competing with the homologous pairing
These findings suggest that formation of pends on the nucleotide number within the bound oligonucleothe Zn(II) chelate is directly linked to single-stranded tide (Giedrocet al.,1991).The original model for Zn(I1) coordi
Summary
Metal-free S-methylated derivative and the wild-type protein 2.5 p and detectable inhibition at high ( 2 4 p ~c)oncentration appear to have different relative affinities for ssDNA.Identical (data not shown).
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