Photoaffinity labeling of the thymidine triphosphate binding domain in Escherichia coli DNA polymerase I: identification of histidine-881 as the site of cross-linking
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), Vol. 26:24
- Country of Publication:
- United States
- Language:
- English
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DNA POLYMERASES
CROSS-LINKING
NUCLEOTIDES
RECEPTORS
AMINO ACID SEQUENCE
PURIFICATION
ESCHERICHIA COLI
HISTIDINE
LIQUID COLUMN CHROMATOGRAPHY
PHOSPHORUS 32
THYMIDINE
AMINO ACIDS
AZINES
AZOLES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ENZYMES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
IMIDAZOLES
ISOTOPES
LIGHT NUCLEI
MEMBRANE PROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEOSIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
POLYMERIZATION
PROTEINS
PYRIMIDINES
RADIOISOTOPES
RIBOSIDES
SEPARATION PROCESSES
TRANSFERASES
550201* - Biochemistry- Tracer Techniques