Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses.
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Torizawa T, Ueda T, Kuramitsu S, Hitomi K, Todo T, Iwai S, Morikawa K, Shimada I
Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses.
J Biol Chem. 2004 Jul 30;279(31):32950-6. Epub 2004 May 28.
- PubMed ID
- 15169780 [ View in PubMed]
- Abstract
The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.