title: Distinct roles of the active-site Mg2+ ligands, Asp882 and Asp705, of DNA polymerase I (Klenow fragment) during the prechemistry conformational transitions.
abstract: DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg(2+) ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp(705) and Asp(882), of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp(882); this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp(882) position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp(705) carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg(2+) into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg(2+) concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Oya
authors.initials: O
authors.lastname: Bermek
authors.forename: Nigel D F
authors.initials: ND
authors.lastname: Grindley
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Joyce
mesh_terms: D001224:Aspartic Acid / Q000502:physiology*; D002384:Catalysis; D020134:Catalytic Domain; D004256:DNA Polymerase I / Q000737:chemistry*; D008024:Ligands; D008274:Magnesium / Q000378:metabolism*; D044367:Phase Transition; D011487:Protein Conformation
chemical_list: D008024:Ligands; D001224:Aspartic Acid; D004256:DNA Polymerase I; D008274:Magnesium