title: A conformational change in E. coli DNA polymerase I (Klenow fragment) is induced in the presence of a dNTP complementary to the template base in the active site.
abstract: It is well established that the insertion of a nucleotide into a growing DNA chain requires a conformational change in the structure of a DNA polymerase. These enzymes have been shown to bind a primer-template in the open conformation and then upon binding of a complementary dNTP undergo a conformational rearrangement to the closed ternary complex. This movement results in the positioning of the incoming nucleotide in the proper geometry for the nucleophilic attack by the 3'-hydroxyl of the primer. In this work, tryptic digestion experiments were performed to detect this conformational change in the structure of the exonuclease-deficient DNA polymerase I (Klenow fragment). Three distinct digestion patterns were observed: one for the polymerase alone, one for the binary complex with the primer-template, and one for the ternary polymerase-DNA-dNTP complex. The latter conformational change leads to a stable ternary closed complex formation only when the correct nucleotide is present in the reaction mixture. Positioning of nucleotides with incorrect geometry in the protein active site inhibits or eliminates formation of the closed complex. Similarly, this conformational change is inhibited when the primer terminus of the DNA molecule is altered by the presence of the 2'-hydroxyl.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
authors.forename: L
authors.initials: L
authors.lastname: Dzantiev
authors.forename: L J
authors.initials: LJ
authors.lastname: Romano
mesh_terms: D001665:Binding Sites; D004256:DNA Polymerase I / Q000737:chemistry*; D015224:Dideoxynucleosides / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D012265:Ribonucleotides / Q000494:pharmacology; D013698:Templates, Genetic; D014357:Trypsin
chemical_list: D015224:Dideoxynucleosides; D012265:Ribonucleotides; D004256:DNA Polymerase I; D014357:Trypsin