Genetic and Crystallographic Studies of the 3′,5′-Exonucleolytic Site of DNA Polymerase I
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- Victoria Derbyshire
- Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, CT 06510.
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- Paul S. Freemont
- Department of Molecular Biophysics and Biodchemistry and Howard Hughes Medical Institute at Yale University, New Haven, CT 06511.
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- Mark R. Sanderson
- Department of Molecular Biophysics and Biodchemistry and Howard Hughes Medical Institute at Yale University, New Haven, CT 06511.
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- Lorena Beese
- Department of Molecular Biophysics and Biodchemistry and Howard Hughes Medical Institute at Yale University, New Haven, CT 06511.
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- Jonathan M. Friedman
- Department of Molecular Biophysics and Biodchemistry and Howard Hughes Medical Institute at Yale University, New Haven, CT 06511.
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- Catherine M. Joyce
- Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, CT 06510.
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- Thomas A. Steitz
- Department of Molecular Biophysics and Biodchemistry and Howard Hughes Medical Institute at Yale University, New Haven, CT 06511.
書誌事項
- 公開日
- 1988-04-08
- DOI
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- 10.1126/science.2832946
- 公開者
- American Association for the Advancement of Science (AAAS)
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説明
<jats:p>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.</jats:p>
収録刊行物
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- Science
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Science 240 (4849), 199-201, 1988-04-08
American Association for the Advancement of Science (AAAS)