Specificity of damage recognition and catalysis of DNA repair.
A common feature of DNA repair enzymes is their ability to recognize the damage independently of 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 changes the dynamics of DNA bending. The reduced bending force constant can be used by the enzyme to induce DNA bending and facilitate base flipping. We show that a thymine dimer (TD) containing DNA requires less energy to bend, lowering the barrier for base flipping. On the other hand, bending in DNA with U-G mismatch is affected only by a small amount and flipping is not enhanced significantly. T4 endonuclease V (endoV), which recognizes TD, utilizes the reduced barrier for flipping as a specific recognition element. In uracil DNA glycosylase (UDG), which recognizes U-G mismatches, base flipping is not enhanced and recognition is encoded in a highly specific binding pocket for the flipped base. Simulations of UDG and endoV in complex with damaged DNA provide insight into the essential elements of the catalytic mechanism. Calculations of pKas of active site residues in endoV and endoV-DNA complex show that the pKa, of the N-terminus is reduced from 8.01 to 6.52 while that of Glu-23 increases from 1.52 to 7.82. Thus, the key catalytic residues are in their neutral form. The simulations also show that Glu-23 is also H-bonded to O4' of the 5'-TD enhancing the nucleophilic attack on Cl and that 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 pKa increases to 7.1 from 4.9 in the free enzyme. The pKa of Asp-145, the other important catalytic residue, remains around 4.2 in the free enzyme and in the complex. Thus, it can not act as a proton acceptor. In the complex the 3'-phosphate of uracil is stabilized next to Asp-145 by two bridging water molecules. Such a configuration activates one water molecule to act as a proton acceptor to produce a stabilizing hydronium ion and the other as a proton donor to produce the nucleophilic hydroxide. It appears that DNA glycosylases share commonalties in recognition of damage but differ in their catalytic mechanisms.
Osman
R
R
Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029, USA. osman@inka.mssm.edu
Fuxreiter
M
M
Luo
N
N
D020137:Base Pair Mismatch; D002384:Catalysis; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D051981:Uracil-DNA Glycosidase
D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase