Louise Prakash, and
Satya Prakash*
Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, Texas 77555-1061
Received 12 May 2006/ Returned for modification 14 June 2006/ Accepted 20 June 2006
Human DNA polymerase
(Pol
) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol
with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol
active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N6 of an A or the O6 of a G, here we provide evidence that whereas hydrogen bonding at N6 is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O6 is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O6 and N7 hydrogen bonding to DNA synthesis by Pol
, we have examined its proficiency for replicating through the 6O-methyl guanine and 8-oxoguanine lesions, which affect the O6 and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O6 is an imperative. The dispensability of N6 hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol
with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N6 positions of templating A.
Present address: Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
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