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Homogeneous Escherichia coli DNA polymerase I has been found to contain 1.0 ± 0.15 g atom of zinc per mole of enzyme. Removal of zinc from the enzyme resulted in an apoenzyme which contained 0.06 g atom of zinc per mole of enzyme. Polymerase activity of the "zinc-free" apoenzyme was 7% of the polymerase activity of the holoenzyme. Complete restoration of polymerase activity of the apoenzyme was obtained by incubation with zinc ions. Co2+ and Mn2+ restored activity by about 80% and 45%, respectively, while Mg2+, Ni2+, Hg2+, Fe2+, and Cd2+ failed to do so. The Zn2+ on the enzyme was exchanged with radioactive 65Zn. Dialysis against the chelator 1,10-phenanthroline showed that the loss of 65Zn from the enzyme paralleled the loss of activity. These results indicate that the bound Zn2+ is essential for activity. To investigate the function of the enzyme-bound zinc, we studied the interaction of the enzyme with oligonucleotides, nucleoside monophosphates, and triphosphates by nuclear quadrupolar relaxation using 79Br- as a halide probe. The enzyme-bound Zn2+ in DNA polymerase was 80 times as effective as ionic Zn2+ in promoting the transverse nuclear quadrupolar relaxation (1/T2) of 79Br- at two frequencies. Titration of E. coli DNA polymerase I with an oligonucleotide (dT)6–9 by measuring 1/T2 of 79Br- showed that this oligonucleotide bound to the enzyme, forming a 1:1 complex, in which the access of Br- to the enzyme-bound zinc was diminished by a factor of 3. No such reduction in 1/T2 was observed with dTMP or dTTP or in titrating the zinc-free apoenzyme with (dT)6–9. These results are consistent with our previous hypothesis that Zn2+ in DNA polymerase functions by coordinating the 3'-OH terminus or growing point of DNA. |
