title: Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I.
abstract: 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University Medical School, New Haven, CT 06510.
authors.forename: V
authors.initials: V
authors.lastname: Derbyshire
authors.forename: P S
authors.initials: PS
authors.lastname: Freemont
authors.forename: M R
authors.initials: MR
authors.lastname: Sanderson
authors.forename: L
authors.initials: L
authors.lastname: Beese
authors.forename: J M
authors.initials: JM
authors.lastname: Friedman
authors.forename: C M
authors.initials: CM
authors.lastname: Joyce
authors.forename: T A
authors.initials: TA
authors.lastname: Steitz
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003196:Computer Graphics; D003461:Crystallography; D004252:DNA Mutational Analysis; D004256:DNA Polymerase I / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005092:Exonucleases; D008670:Metals; D008958:Models, Molecular; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D008670:Metals; D004256:DNA Polymerase I; D005092:Exonucleases