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Photoaffinity labeling of the thymidine triphosphate binding domain in Escherichia coli DNA polymerase I: identification of histidine-881 as the site of cross-linking

Journal Article · · Biochemistry; (United States)
DOI:https://doi.org/10.1021/bi00398a031· OSTI ID:5303697
Using the technique of ultraviolet-mediated cross-linking of substrate deoxynucleoside triphosphates (dNTPs) to their acceptor site, the authors have labeled the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) with (..cap alpha..-/sup 32/P)dTTP. Covalent cross-linking of (..cap alpha..-/sup 32/P)dTTP to the Klenow fragment is shown to be at the substrate binding site by the following criteria; (a) the cross-linking reaction requires dTTP in its metal chelate form; (b) dTTP is readily competed out by other dNTPs as well as by substrate binding site directed reagents; (c) labeling with dTTP occurs at a single site as judged by peptide mapping. Under optimal conditions, a modification of approximately 20% of the enzyme was achieved. Following tryptic digestion of the (..cap alpha..-/sup 32/P)dTTP-labeled Klenow fragment, reverse-phase high-performance liquid chromatography demonstrated that 80% of the radioactivity was contained within a single peptide. The amino acid composition and sequence of this peptide identified it as the peptide spanning amino acid residues 876-890 in the primary sequence of E. coli Pol I. Chymotrypsin and Staphylococcus aureus V8 protease digestion of the labeled tryptic peptide in each case yielded a single smaller fragment that was radioactive. Amino acid analysis and sequencing of these small peptides further narrowed the dTTP cross-linking site to within the region spanning residues 876-883. They concluded that histidine-881 is the primary attachment site for dTTP in E. coli DNA Pol I, since during amino acid sequencing analysis of all three radioactive peptides loss of the histidine residue at the expected cycle is observed.
Research Organization:
Univ. of Medicine and Dentistry of New Jersey, Newark
OSTI ID:
5303697
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:24; ISSN BICHA
Country of Publication:
United States
Language:
English

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