SYNTHESIS, CRYSTAL-STRUCTURE, AND ENZYMATIC EVALUATION OF A DNA-PHOTOLESION ISOSTERE

Nucleotide analogues are useful tools for the investigation of interac tions between DNA-binding proteins and DNA at a molecular level. Herei n we describe the synthesis of the DNA-lesion analogue 2, which is req uired to determine the extent to which specific phosphodiesters in the DNA backbone contribute to the recognition of cyclobutane pyrimidine dimer DNA lesion by the dimer-specific repair enzymes DNA photolyases or T4-endonuclease V. The analogue 2 is a close structural mimic of cy clobutane pyrimidine dimers like 1. which are the major lesions induce d upon irradiation of cells with UV light. Instead of the negatively c harged phosphate link in 1, analogue 2 contains an uncharged but isost eric formacetal moiety. The analysis of this and other phosphodiester contacts is hoped to provide insight into the lesion recognition proce ss, which is currently believed to require the nipping of the lesioned base out of the DNA double helix. The lesion analogue 2 is synthetica lly available in large quantities, which allowed us to establish a new , fast and sensitive DNA photolyase assay. A precise X-ray crystal str ucture analysis of the DNA-lesion analogue 2 is also presented. The st ructure underlines the isosteric character of 2 and reveals, in combin ation with the only other available X-ray crystal structure determined from a thymine-dimer triester analogue, interesting structural featur es of cyclobutane pyrimidine dimer lesions. We describe the incorporat ion of the lesion analogue 2 into oligonucleotides by using standard p hosphoramidite chemistry. Initial enzymatic repair studies are reporte d with three different types of DNA photolyases. These studies show th at the lesion analogue 2 is rapidly repaired by photolyases from Anacy stis nidulans, Neurospora crassa and from the marsupial Potorous trida ctylis. The enzymatic investigations indicate that all photolyases, in cluding enzymes from higher organisms (Tridactylis) accept the formace tal dimer as a lesion substrate and therefore could possess a similar DNA-lesion recognition process, in which the interaction with the cent ral phosphate unit is only of limited importance.