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Papers In Press, published online ahead of print December 26, 2001
J. Biol. Chem, 10.1074/jbc.M107286200
Submitted on July 31, 2001
Revised on December 20, 2001
Accepted on December 25, 2001
Laboratory of Structural Biology, NIEHS, NIH, Research Triangle Park, NC 27709
Corresponding Author: wilson5{at}niehs.nih.gov
Structures of DNA polymerases bound with DNA reveal that the 5’-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, 
-helix N of DNA polymerase 
is observed to form one face of the binding pocket for the new base pair. Asp276 and Lys280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys280, site-directed mutants were constructed at this position, and the proteins were expressed, purified, and their catalytic efficiency and fidelity assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G ) was strongly diminished relative to wild-type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys280 “stabilizes” templating purines through van der Waals interactions.
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