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Science 8 April 1988:
Vol. 240. no. 4849, pp. 199 - 201
DOI: 10.1126/science.2832946
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Articles

Science, Vol 240, Issue 4849, 199-201
Copyright © 1988 by American Association for the Advancement of Science

articles

Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I

V Derbyshire, PS Freemont, MR Sanderson, L Beese, JM Friedman, CM Joyce, and TA Steitz

Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, CT 06510.

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.


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