DNA binding domain of Escherichia coli DNA polymerase I: identification of arginine-841 as an essential residue
To identify the DNA binding site(s) in Escherichia coli DNA polymerase I (pol I) (Klenow fragment), the authors 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 DNA binding functions. Furthermore, the presence of DNA but not deoxynucleoside triphosphates protected the enzyme from inactivation. Labeling studies with (7-/sup 14/C)PG indicated that two arginine residues were modified per mole of enzyme. In order to locate the site of PG modification, we digested the PG-treated enzyme with trypsin and V-8 protease. The resulting peptides from each digest were then resolved on reverse-phase hydrophobic columns. As appearance of a new peptide peak was observed in both tryptic and V-8 protease digests. Since inclusion of template-primer during PG modification of enzyme blocks the appearance of these peaks, these peptides were concluded to represent the template-primer binding domain of pol I. Indeed, the extent of inactivation of enzyme by PG treatment correlated very well with the quantitative increase in the new tryptic peptide peak. Amino acid composition analysis of both tryptic peptide and V-8 peptide revealed that two peptides were derived from the same general region; tryptic peptide spanned between residues 837 and 857 while V-8 peptide spanned between residues 841 and 870 in the primary sequence of pol I. Sequence analysis of tryptic peptide further identified arginine-841 as the site of PG modifications, which implicates this residue in the DNA binding function of pol I.
- Research Organization:
- UMDNJ-New Jersey Medical School, Newark
- OSTI ID:
- 5446328
- Journal Information:
- Biochemistry; (United States), Vol. 27:1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CARBON 14 COMPOUNDS
CONFIGURATION INTERACTION
DNA
DNA POLYMERASES
AMINO ACID SEQUENCE
ARGININE
ESCHERICHIA COLI
GLYOXAL
ALDEHYDES
AMINO ACIDS
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
LABELLED COMPOUNDS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
TRANSFERASES
550201* - Biochemistry- Tracer Techniques