Participation of active-site carboxylates of Escherichia coli DNA polymerase I (Klenow fragment) in the formation of a prepolymerase ternary complex.
We have investigated the roles of four active-site carboxylates in the formation of a prepolymerase ternary complex of Escherichia coli DNA polymerase I (Klenow fragment), containing the template-primer and dNTP. The analysis of nine mutant enzymes with conserved and nonconserved substitutions of Asp(705), Glu(710), Asp(882), and Glu(883) clearly shows that both catalytically essential aspartates, Asp(705) and Asp(882), are required for the formation of a stable ternary complex. Of the two glutamates, only Glu(710) is required for ternary complex formation, while Glu(883) does not participate in this process. This investigation also reveals two interesting properties of the Klenow fragment with regard to enzyme-template-primer binary and enzyme-template-primer-dNTP ternary complex formation. These are (a) the significant resistance of enzyme-template-primer-dNTP ternary complexes to the addition of high salt or template-primer challenge and (b) the ability of the Klenow fragment to form ternary complexes in the presence of noncatalytic divalent cations such as Ca(2+), Co(2+), Ni(2+), and Zn(2+).
Gangurde
Rajiv
R
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA.
Modak
Mukund J
MJ
D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002264:Carboxylic Acids / Q000737:chemistry*; D002413:Cations, Divalent / Q000737:chemistry; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic
D002264:Carboxylic Acids; D002413:Cations, Divalent; D017931:DNA Primers; D015226:Dinucleoside Phosphates; D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D001224:Aspartic Acid; D018698:Glutamic Acid; D004256:DNA Polymerase I