Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase β by UV cross-linking

Rajendra Prasad, Amalendra Kumar, Steven Widen, Jose R. Casas-Finet, Samuel H. Wilson

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Abstract

Rat DNA polymerase β (β-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 β-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 β-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 β-pol appear to be in close contact with this nucleic acid probe.

Original languageEnglish (US)
Pages (from-to)22746-22755
Number of pages10
JournalJournal of Biological Chemistry
Volume268
Issue number30
StatePublished - Oct 25 1993

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Single-Stranded DNA
DNA-Directed DNA Polymerase
Rats
Binding Sites
Nucleic Acid Probes
Proteolysis
Recombinant Proteins
Escherichia coli
Nucleic Acids
Sequence Analysis
Proteins
DNA
glutamyl endopeptidase

ASJC Scopus subject areas

  • Biochemistry

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Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase β by UV cross-linking. / Prasad, Rajendra; Kumar, Amalendra; Widen, Steven; Casas-Finet, Jose R.; Wilson, Samuel H.

In: Journal of Biological Chemistry, Vol. 268, No. 30, 25.10.1993, p. 22746-22755.

Research output: Contribution to journalArticle

Prasad, Rajendra ; Kumar, Amalendra ; Widen, Steven ; Casas-Finet, Jose R. ; Wilson, Samuel H. / Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase β by UV cross-linking. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 30. pp. 22746-22755.
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N2 - Rat DNA polymerase β (β-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 β-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 β-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 β-pol appear to be in close contact with this nucleic acid probe.

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