Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses.
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.
Torizawa
Takuya
T
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Ueda
Takumi
T
Kuramitsu
Seiki
S
Hitomi
Kenichi
K
Todo
Takeshi
T
Iwai
Shigenori
S
Morikawa
Kosuke
K
Shimada
Ichio
I
D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry*; D019281:Dimerization; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000737:chemistry*; D013053:Spectrophotometry; D020349:Surface Plasmon Resonance; D016963:Thermus thermophilus / Q000378:metabolism; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D011743:Pyrimidines; D004247:DNA; D004255:Deoxyribodipyrimidine Photo-Lyase