THE ROLE OF BASE FLIPPING IN DAMAGE RECOGNITION AND CATALYSIS BY T4 ENDONUCLEASE-V

The process of moving a DNA base extrahelical (base flipping) has been shown in the co-crystal structure of a W-induced pyrimidine dimer-spe cific glycosylase, T4 endonuclease V, with its substrate DNA. Compared with other enzymes known to use base flipping, endonuclease V is uniq ue in that it moves the base opposite the target site extrahelical, ra ther than moving the target base itself. Utilizing substrate analogs a nd catalytically inactive mutants of T4 endonuclease V, this study inv estigates the discrete steps involved in damage recognition by this DN A repair enzyme. Specifically, fluorescence spectroscopy analysis show s that fluorescence changes attributable to base flipping are specific for only the base directly opposite either abasic site analogs or the 5'-thymine of a pyrimidine dimer, and no changes are detected if the S-aminopurine is moved opposite the 3'-thymine of the pyrimidine dimer . Interestingly, base flipping is not detectable with every specific b inding event suggesting that damage recognition can be achieved withou t base flipping. Thus, base flipping does not add to the stability of the specific enzyme-DNA complex but rather induces a conformational ch ange to facilitate catalysis at the appropriate target site, When used in conjunction with structural information, these types of analyses c an yield detailed mechanistic models and critical amino acid residues for extrahelical base movement as a mode of damage recognition.