City College of CUNY
Department of Chemistry
Biochemistry Seminar
Wednesday, April 5, 2000
Room J1027 at 11:15 AM
Roman Osman
Professor of Physiology and Biophysics
Mount Sinai School of Medicine
Specificity of Damage Recognition and Catalysis in DNA Repair

A common feature of DNA repair enzymes is their ability to recognize the damage independently of the sequence in which they are found. The presence of a flipped-out base inserted into the protein in several DNA-enzyme complexes suggests a contribution to enzyme specificity. Molecular simulations of damaged DNA indicate that the damage produces changes in DNA structure and in the dynamics of DNA bending and opening. A Potential of Mean Force analysis shows that a thymine dimer (TD) containing DNA is bent compared to normal DNA and its bending flexibility is increased. The coupling between bending and opening lowers the barrier for base flipping by 3.4 Kcal/mole. On the other hand, bending in DNA with a U-G mismatch is affected only by a small amount compared to a C-G, but the wobble base pair generates a significant opening and increases DNA flexibility. This lowers the barrier by 11.3 Kcal/mole and enhances the flipping of U compared to C. Both T4 endonuclease V (endoV), which recognizes TD, and uracil DNA glycosylase (UDG), which recognizes U-G mismatches, utilize the reduced barrier for flipping as a specific recognition element in damaged DNA.
Simulations of UDG and endoV in complex with damaged DNA provide insight into the essential elements of the catalytic mechanism. Calculations of pK_as of active site residues in endoV and endoV-DNA complex show that the pK_a of the N-terminus is reduced from 8.0 to 6.5 while that of Glu-23 increases from 1.5 to 7.8. Thus, the key catalytic residues are in their neutral form. The simulations also show that Glu-23 is H-bonded to O4 92 of the 5 92-TD enhancing the nucleophilic attack on C1 92. Arg-26 enhances the hydrolysis by electrostatic stabilization but does not participate in proton transfer. In the enzyme-substrate complex of UDG, the role of electrostatic stabilization is played by His-268, whose pK_a increases to 7.1 from 4.9 in the free enzyme. However, after the enzymatic reaction is completed the pK_a of His-268 is reduced to allow proton transfer to the negatively charged base. The pK_a of Asp-145, the other important catalytic residue, remains around 4.2 in the free enzyme and in the complex. Thus, Asp-145 can not act as a general base to activate a water molecule for nucleophilic attack. In the complex, the 3 92-phosphate of uracil is stabilized next to Asp-145 by two bridging water molecules. Such a configuration can activate one water molecule to act as a proton acceptor and the other as a proton donor to produce the nucleophilic hydroxide.