Received September 12, 2005

Revised Manuscript Received December 14, 2005

Abstract:

The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase (Pol ), and for the homologous domain of DNA polymerase (Pol ). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol ), followed by a -elimination reaction. To better understand the underlying chemistry, we have determined pK_a values for the lysine residues in the Pol lyase domain labeled with [-¹³C]lysine. At neutral pH, the H protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pK_a value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of ~1:1.3, consistent with a dissociation constant of ~1 M. The -proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pK_a values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general increase in the Hill coefficients observed in the complex is consistent with the screening of the interacting lysine residues by the DNA. The pK_a of K312 residue increased to 10.58 in the complex, probably due to salt bridge formation with the 5'-phosphate group of the DNA. The pK_a values obtained for the lyase domain of Pol in the present study are consistent with recent crystallographic studies of Pol complexed with 5-phosphorylated abasic sugar analogues in nicked DNA which reveal an open site with no obvious interactions that would significantly depress the pK value for the active site lysine residue. It is suggested that due to the heterogeneity of the damaged DNA substrates with which Pol as well as other related polymerases may be required to bind, the unexpectedly poor optimization of the lyase catalytic site may reflect a compromise of flexibility with catalytic efficiency.

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