DNA photolyase base flipping of thymidine dimers investigated using modified fluorescent nucleobases.

摘要

UV light causes many different DNA photoproducts. The cis-syn cyclobutylpyrimidine dimer (CPD) is the predominant DNA photoproduct upon UV irradiation. The CPD lesions can be repaired in several ways, including direct repair by DNA photolyase and nucleotide excision repair. DNA photolyase is a monomeric protein with two noncovalently bound cofactors, flavin adenine dinucleotide (FAD) and a photoantenna molecule. DNA photolyase repairs the CPD by a light driven electron transfer. We showed previously that the CPD has to be flipped out of the DNA double helix when it binds to DNA photolyase using a fluorescent adenine analogue, 2-aminopurine (2Ap).;We showed that base flipping of thymidine dimers by DNA photolyase causes a large local distortion at both the 5'- and 3'-side of the CPD lesion. However, the distortion at 5'-side of lesion is much greater than that at 3'-side of CPD lesion. This differential distortion is consistent with the latest crystal structure results of DNA photolyase:substrate complex. We also studied the thermal stability of duplexes with both modified nucleobases and/or CPD. The introduction of CPD lowers the melting points of duplexes much more than the natural bases replaced by these analogues.;Another adenine analogue, 4-amino-6-methyl-8-(2-deoxy-beta-d-ribofuranosyl)-7(8 H)-pteridone (6MAP), has also been used to study base flipping by DNA photolyase. Thermal stability studies show that the introduction of 6MAP lowers the melting temperature of 6MAP containing duplexes. 6MAP lowers the thermal stability of 6MAP containing duplexes more than that of the introduction of 2Ap in the corresponding 2Ap containing duplexes. Forster resonance energy transfer (FRET) calculations have been done using the latest crystal structure as a model. FRET studies of 2Ap or 6MAP and oxidized photolyase have been compared.;Finally, a fluorescent cytosine analogue, pyrrolo-dC was used to study how the CPD base flipping by DNA photolyase affects the flanking bases on the non-lesion strand. The results show that CPD base flipping by DNA photolyase in solution has almost no effect on the base stacking of the flanking bases, which is consistent with the results of the crystal structure of DNA photolyase:substrate complex.