title: A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.
abstract: The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
authors.forename: P S
authors.initials: PS
authors.lastname: Freemont
authors.forename: D L
authors.initials: DL
authors.lastname: Ollis
authors.forename: T A
authors.initials: TA
authors.lastname: Steitz
authors.forename: C M
authors.initials: CM
authors.lastname: Joyce
mesh_terms: D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D010446:Peptide Fragments; D004247:DNA; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V