Kinetic mechanism of DNA polymerase I (Klenow)
The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence, labeled with (/sup 32/P)-nucleotides. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF-DNA/sub n/-dNTP and KF-DNA/sub n+1/-PP/sub i/ complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PP/sub i/ from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.
- Research Organization:
- Pennsylvania State Univ., University Park
- OSTI ID:
- 5446202
- Journal Information:
- Biochemistry; (United States), Vol. 26:25
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DNA POLYMERASES
BIOCHEMICAL REACTION KINETICS
OLIGONUCLEOTIDES
BIOSYNTHESIS
ATP
ESCHERICHIA COLI
LABELLED COMPOUNDS
PHOSPHORUS 32
PHOSPHORYLATION
STOICHIOMETRY
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPES
KINETICS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
REACTION KINETICS
SYNTHESIS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques