NMR study of repair mechanism of DNA photolyase by FAD-induced paramagnetic relaxation enhancement.
Cyclobutane pyrimidine dimer (CPD) photolyases, which contain FAD as a cofactor, use light to repair CPDs. We performed structural analyses of the catalytic site of the Thermus thermophilus CPD photolyase-DNA complex, using FAD-induced paramagnetic relaxation enhancement (PRE). The distances between the tryptophan residues and the FAD calculated from the PRE agree well with those observed in the x-ray structure (with an error of <3 A). Subsequently, a single-stranded DNA containing 13C-labeled CPD was prepared, and the FAD-induced PRE of the NMR resonances from the CPD lesion in complex with the CPD photolyase was investigated. The distance between the FAD and the CPD calculated from the PRE is 16 +/- 3 A. The FAD-induced PRE was also observed in the CPD photolyase-double-stranded DNA complex. Based on these results, a model of the CPD photolyase-DNA complex was constructed, and the roles of Arg-201, Lys-240, Trp-247, and Trp-353 in the CPD-repair reaction are discussed.
Graduate School of Pharmaceutical Sciences, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Takumi
T
Ueda
Akira
A
Kato
Yuuta
Y
Ogawa
Takuya
T
Torizawa
Seiki
S
Kuramitsu
Shigenori
S
Iwai
Hiroaki
H
Terasawa
Ichio
I
Shimada
D001483:Base Sequence; D020134:Catalytic Domain; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D014364:Tryptophan; D004255:Deoxyribodipyrimidine Photo-Lyase