PMID:8063826

Citation	Pandey, VN, Kaushik, N and Modak, MJ (1994) Photoaffinity labeling of DNA template-primer binding site in Escherichia coli DNA polymerase I. Identification of involved amino acids. J. Biol. Chem. 269:21828-34
Abstract	We have used two self-annealing template-primers (TPs) to covalently cross-link the Klenow fragment of Escherichia coli DNA polymerase I in its polymerase mode. The specificity of cross-linking is demonstrated by the observation that other template-primers, but not the template or primer alone, readily compete with self-annealing TPs. The enzyme-TP covalent complex is catalytically active and can incorporate one nucleotide on the primer terminus of the immobilized template-primer. Using a peptide mapping approach, we have identified a 17-amino acid tryptic peptide spanning residues 759-775 as a major constituent of the TP binding domain. Amino acid sequence analysis further revealed that Ile-765, Tyr-766 in the O-helix and Ser-769, Phe-771 in the O1-helix of the three-dimensional crystal structure of the Klenow fragment constitute the attachment site for TP.
Links	PubMed
Keywords	Affinity Labels; Amino Acid Sequence; Base Sequence; Binding, Competitive; Cations, Divalent/pharmacology; Cross-Linking Reagents; DNA Polymerase I/metabolism; DNA Primers/metabolism; DNA-Binding Proteins/metabolism; Dose-Response Relationship, Radiation; Escherichia coli/enzymology; Exodeoxyribonuclease V; Exodeoxyribonucleases/metabolism; Molecular Sequence Data; Osmolar Concentration; Peptide Fragments/chemistry; Sequence Analysis; Trypsin/metabolism; Ultraviolet Rays
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