Cis-syn Thymidine Dimer Repair by DNA Photolyase in Real Time

Biochemistry. 2003 Jul 22;42(28):8558-68. doi: 10.1021/bi034015w.

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 (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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteria / enzymology*
  • Bacteria / genetics
  • Cell-Free System
  • DNA Repair / physiology*
  • Deoxyribodipyrimidine Photo-Lyase / metabolism*
  • Escherichia coli / enzymology*
  • Kinetics
  • Pyrimidine Dimers / chemistry*
  • Spectrophotometry
  • Thymidine*

Substances

  • Pyrimidine Dimers
  • Deoxyribodipyrimidine Photo-Lyase
  • Thymidine