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

Citations Over Time

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.

Related Papers

• → Construction and characterization of a bacteriophage T4 DNA polymerase deficient in 3'-->5' exonuclease activity.(1993)108 cited
• → Contribution of Polar Residues of the J-Helix in the 3‘−5‘ Exonuclease Activity of Escherichia coli DNA Polymerase I (Klenow Fragment): Q677 Regulates the Removal of Terminal Mismatch(2005)13 cited
• → Two DNA Polymerases of Escherichia coli Display Distinct Misinsertion Specificities for 2-Hydroxy-dATP during DNA Synthesis(2000)29 cited
• [Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs].(1990)
• [Acyclic analogs of 2',3'-dideoxy-2',3'-didehydronucleoside-5'-triphosphates--terminators of DNA synthesis, catalyzed by a broad set of DNA polymerases].(1993)