Crystallographic Study of Molecular Mechanism of DNA Repair by Photolyase

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Photolyases are 50 to 70 kDa single chain proteins containing two different chromophoric cofactors in equimolar amounts. Photoreactivation comprises several steps : damage recognition and binding of photolyase to DNA,photon absorption, interchromophoric energy transfer and electron transfer from chrompophore to DNA,resulting in the reversal of UV-induced pyrimidine dimers into monomers. The catalytic cofactor FAD is essential for the light-dependent repair process. In addition, a second cofactor is present which acts as a light-harvesting chromophore. Two structures are known for the second cofactor, either 8-hydroxy-5-deazaflavin (8-HDF) present in photolyase from e.g.the cyanobacterium, Anacystis nidulans or 5,10-methenyltetrahydro-folic acid (MTHF) found in E.coli photolyase. The crystal structure of A.nidulans photolyase (53,000 Da) was determined at 1.8 resolution from X-ray diffraction data obtained with synchrotron radiation. The refined model, comprising the residues 1 to 475, the two cofactors and 192 water molecules, has an R-factor of 0.197. The structure is composed of an alpha/beta and a helical domain, which provides binding sites for the 8-HDF and FAD chromophores, respectively. The present crystal structure of 8-HDF type photolyase from Anacystis nidulans showed the similarity of the backbone structure with MTHF type E.coli photolyase but completely different binding site of the light-harvesting cofactor. This is a first example that homologous primary and tertiary structures in closely related proteins recognize two different rtpes of cofactors at different binding-sites.