Send to

Choose Destination
J Biol Chem. 2011 Feb 4;286(5):3755-66. doi: 10.1074/jbc.M110.167593. Epub 2010 Nov 16.

Distinct roles of the active-site Mg2+ ligands, Asp882 and Asp705, of DNA polymerase I (Klenow fragment) during the prechemistry conformational transitions.

Author information

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.


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.

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center