Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase beta by UV cross-linking.
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.
Prasad
R
R
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-0851.
Kumar
A
A
Widen
S G
SG
Casas-Finet
J R
JR
Wilson
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004256:DNA Polymerase I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011071:Poly T / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases; D014357:Trypsin; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D011071:Poly T; D004256:DNA Polymerase I; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin