IDENTIFICATION OF RESIDUES IN THE SINGLE-STRANDED DNA-BINDING SITE OFTHE 8-KDA DOMAIN OF RAT DNA POLYMERASE-BETA BY UV CROSS-LINKING

Rat DNA polymerase beta (beta-pol) is a 39-kDa monomeric protein, orga nized in two structurally and functionally distinct domains. The 8-kDa NH-2-terminal domain binds single-stranded (ss) DNA, whereas the 31-k Da COOH-terminal domain does not. To facilitate studies on ssDNA bindi ng 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 recombina nt 8-kDa domain was found to be similar to that previously reported fo r 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 Wil son, 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-l inking to [P-32] p(dT)16. Cross-linking was localized to a tryptic fra gment spanning residues 28 through 35 and a V8 protease fragment spann ing residues 27 through 58. Sequence analysis of the various [P-32]p(d T)16-labeled proteins indicated that Ser30 and His34 were modified by cross-linking to p(dT)16. Therefore, these residues of the ssDNA-bindi ng domain of beta-pol appear to be in close contact with this nucleic acid probe.