Cis-syn thymidine dimer repair by DNA photolyase in real time.
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 (C=C) in the CPD is initiated in approximately 60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two C=C 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.
MacFarlane
Alexander W
AW
201 Beury Hall, Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122, USA.
Stanley
Robert J
RJ
D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D002474:Cell-Free System; D004260:DNA Repair / Q000502:physiology*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D011740:Pyrimidine Dimers / Q000737:chemistry*; D013053:Spectrophotometry; D013936:Thymidine
D011740:Pyrimidine Dimers; D004255:Deoxyribodipyrimidine Photo-Lyase; D013936:Thymidine