Abstract:
:Binding of deoxyribonucleoside 5'-triphosphates to DNA polymerase I of Escherichia coli was measured by using a microscale nonequilibrium dialysis method. It allowed rapid and economic measurement of dissociation constants, with negligible interfering side reactions. A stoichiometry of 1 mol of nucleoside 5'-triphosphate/mol of DNA polymerase was measured, and the occurrence of a single binding site was established, for which the nucleotides competed in the binary complex with the polymerase. Binding affinities decreased in the order dGTP greater than or equal to dATP greater than dCTP congruent to dTTP. These results are in agreement with previous findings [Englund, P. T., Huberman, J. A., Jovin, T. M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038-3044] except that, in a few cases, values of dissociation constants were smaller by factors of 2-3. The cations Mg2+ and Mn2+, as well as spermine, slightly enhanced complex stability at low levels and decreased it at high concentrations, while NaCl and Hg2+ had only destabilizing effects. Recognition between nucleoside 5'-triphosphates and nucleotide templates was studied by titration of the polymerase-[3H]dGTP complex with polynucleotide homopolymers. Complementary poly(dC) did not affect binding of dGTP, and non-complementary templates caused rejection of the nucleotide. Rejection of dGTP followed a saturation dependence with an equivalence of 110 +/- 10 monomer units of polynucleotides bound per molecule of DNA polymerase. The results favor a model by which recognition arises chiefly from the stereogeometrical fit of complementary template and nucleoside 5'-triphosphate into a rigid binding site.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Muise O,Holler Edoi
10.1021/bi00335a033subject
Has Abstractpub_date
1985-07-02 00:00:00pages
3618-22issue
14eissn
0006-2960issn
1520-4995journal_volume
24pub_type
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