Template-primer binding site of E. coli DNA Pol I: identification of ARG-841 as an essential residue

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5904775

Mohan, P M; Basu, A; Abraham, K I; Modak, M J

To identify the template-primer (TP) binding site(s) in E. coli DNA Pol-I, they have used an active site directed reagent, phenylglyoxal (PG), which specifically reacts with arginine residues. Preincubation of DNA Pol-I with PG resulted in the loss of polymerase, 3' - 5' exonuclease and TP-binding functions. Presence of TP but not dNTPs protects the enzyme from PG modification. Labeling studies with (/sup 14/C)-PG indicated that two arginine residues were modified per mol of the enzyme. They have further analyzed the PG modified enzyme by tryptic peptide mapping on reverse phase HPLC in order to identify the site of PG reactivity. Appearance of a new peptide peak in PG modified, but not in DNA protected, enzyme was consistently observed. Furthermore, extent of enzyme modification appeared to be related to the quantitative increase in this peptide. Amino acid composition and sequence analyses of this peptide revealed it to span residues 837-857 in the primary amino acid sequence of Pol-I. Therefore, the region spanning these residues constitutes a part of the DNA binding domain. Arg-841 was further identified as the site of PG-modification, which implicates involvement of this residue in the DNA binding function of E. coli DNA Pol-I.

• Research Organization: New Jersey Medical School, Newark
• OSTI ID: 5904775
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CHROMATOGRAPHY
DNA
DNA POLYMERASES
ENZYMES
ESCHERICHIA COLI
GENETIC MAPPING
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MAPPING
MEMBRANE PROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROTEINS
REACTION KINETICS
REAGENTS
RECEPTORS
RESIDUES
SEPARATION PROCESSES
TRACER TECHNIQUES
TRANSFERASES