Enzymologic mechanism of calf thymus DNA polymerase delta.
The catalytic core protomer of calf thymus DNA polymerase delta (pol delta) was purified to apparent homogeneity by a modified procedure, and its enzymologic mechanism was investigated using a combination of steady-state kinetics and semiquantitative sedimentation binding analyses. Like DNA polymerase alpha (pol alpha), in the absence of a primer, pol delta was able to bind single-stranded but not double-stranded DNA. This, in conjunction with the observation of induced substrate (dNTP) inhibition of pol delta in the presence of a correctly base-paired 2',3'-dideoxyribonucleotide-terminated primer, suggests that pol delta follows an ordered sequential ter-reactant mechanism of substrate recognition and binding similar to that elucidated for pol alpha. Pol delta binds template first followed by primer and then template-directed dNTP. With suitable substrates, addition to incubations of proliferating cell nuclear antigen, the pol delta auxiliary factor, leads to a reduction in Km and increase in Vmax. This suggests that proliferating cell nuclear antigen enhances the processivity of pol delta by increasing both the residence time of pol delta on the DNA template-primer and the rate at which individual nucleotides are incorporated.
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Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
Tan
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Downey
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Fisher
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D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002417:Cattle; D002853:Chromatography, Liquid; D004247:DNA / Q000235:genetics; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology*
D004247:DNA; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase