Cis−Syn Thymidine Dimer Repair by DNA Photolyase in Real Time

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Abstract
DNA photolyase (PL) is a monomeric flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) via photoinduced electron transfer from a reduced flavin adenine dinucleotide cofactor (FADH-) to the bound CPD. We have used subpicosecond UV transient absorption spectroscopy to measure the electron-transfer and repair kinetics of Anacystis nidulans DNA photolyase with dimeric and pentameric oligothymidine substrates. Here we show that the electron-transfer lifetime is 32 ± 20 ps for the pentameric substrate. Repair of the carbon−carbon double bonds (CC) in the CPD is initiated in ∼60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two CC bonds proceeds sequentially and that the first bond scission has a much lower activation barrier than the second. Our experiments also suggest that the semiquinone FADH• cofactor is not reduced to its catalytically active FADH- state by substrate after repair but remains in the semiquinone state. In contrast to the longer substrate, the dinucleotide substrate produced a mixture of kinetics representing bound and unbound substrate.

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