Zinc-free and reduced T4 gene 32 protein binds single-stranded DNA weakly and fails to stimulate UvsX-catalyzed homologous pairing.
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.
Qiu
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Department of Biochemistry and Biophysics, Texas A & M University, College Station 77843-2128.
Kodadek
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Giedroc
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DP
D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000032:analysis; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D008745:Methylation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D001059:Apoproteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D015032:Zinc; D003545:Cysteine; D004229:Dithiothreitol