Photoaffinity labeling of DNA template-primer binding site in Escherichia coli DNA polymerase I. Identification of involved amino acids.
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.
Pandey
V N
VN
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry, New Jersey Medical School, Newark 07103.
Kaushik
N
N
Modak
M J
MJ
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004256:DNA Polymerase I / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
D000345:Affinity Labels; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D014357:Trypsin