MECHANISM OF PYRIMIDINE DIMER EXCISION REPAIR: STRUCTURAL BASIS OF DAMAGED DNA RECOGNITION. D.G. Vassylyev^*, K. Morikawa^*, T. Kashiwagi^*, Y. Mikami^*, M. Ariyoshi^*, S. Iwai^*, E. Ohtsuka⁺, ^*Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka, 565, Japan; ⁺Faculty of Pharmaceutical Sciences, Hokkaido Univ., Sapporo, Hokkaido, 060, Japan

The crystal structure of T4 endonuclease V (inactive mutant) in complex with DNA substrate (12 bp) has been solved at 2.75 Å resolution. The atomic model of the complex reveals the unique conformation of the B-DNA duplex with a sharp (60 degrees) kink at the central pyrimidine dimer (PD). The adenine base complementary to 5' side of the PD moiety is completely flipped out of the DNA duplex and accomodated into a cavity on the enzyme surface. The kink induces the deformation of the phosphate backbone in the vicinity of PD. This deformation is recognized by the protein, instead of unique architecture of PD base moiety. Such indirect readout of DNA damage may be general for the DNA repair mechanisms with broad specificity. In addition to the contribution to the protein/DNA recognition the flipped out base also generates an empty space inside DNA which allows the access of the catalytically active residues to the target bond of PD. Thus flipped out base is likely to be a common feature of other DNA glycosylases/DNA complexes.

Crystals of the complex belong to the space group P65(a=b=118.8Å, c=36.3Å, Z=6). The structure was solved by MR method combined with solvent flattening and refined at 2.75Å resolution to a final R-factor of 0.152 (R-free = 0.248).