Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase beta by UV cross-linking
| Autor: | R, Prasad, A, Kumar, S G, Widen, J R, Casas-Finet, S H, Wilson |
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| Rok vydání: | 1993 |
| Předmět: |
Binding Sites
Poly T Ultraviolet Rays Molecular Sequence Data Osmolar Concentration Serine Endopeptidases DNA Single-Stranded Chromatography Ion Exchange DNA Polymerase I Peptide Fragments Recombinant Proteins Rats Molecular Weight Kinetics Cross-Linking Reagents Escherichia coli Animals Autoradiography Electrophoresis Polyacrylamide Gel Trypsin Amino Acid Sequence Cloning Molecular Phosphorus Radioisotopes |
| Zdroj: | The Journal of biological chemistry. 268(30) |
| ISSN: | 0021-9258 |
| Popis: | Rat DNA polymerase beta (beta-pol) is a 39-kDa monomeric protein, organized in two structurally and functionally distinct domains. The 8-kDa NH2-terminal domain binds single-stranded (ss) DNA, whereas the 31-kDa COOH-terminal domain does not. To facilitate studies on ssDNA binding structure-function relationships of beta-pol, we overexpressed the 8-kDa domain in Escherichia coli, and purified the recombinant protein to homogeneity. Single-stranded nucleic acid binding of the recombinant 8-kDa domain was found to be similar to that previously reported for the 8-kDa fragment prepared by proteolysis of intact beta-pol (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P. Karpel, R. L., and Wilson, S. H. (1990b) J. Biol. Chem. 265, 2124-2131; Casas-Finet, J. R., Kumar, A., Morris, G., Wilson, S. H., and Karpel, R. L. (1991) J. Biol. Chem. 266, 19618-19625). Residues in or near the DNA-binding pocket of the recombinant 8-kDa domain were examined by photochemical cross-linking to [32P] p(dT)16. Cross-linking was localized to a tryptic fragment spanning residues 28 through 35 and a V8 protease fragment spanning residues 27 through 58. Sequence analysis of the various [32P]p(dT)16-labeled proteins indicated that Ser30 and His34 were modified by cross-linking to p(dT)16. Therefore, these residues of the ssDNA-binding domain of beta-pol appear to be in close contact with this nucleic acid probe. |
| Databáze: | OpenAIRE |
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