Characterization of DNA recognition by the human UV-damaged DNA-binding protein.
The UV-damaged DNA-binding (UV-DDB) protein is the major factor that binds DNA containing damage caused by UV radiation in mammalian cells. We have investigated the DNA recognition by this protein in vitro, using synthetic oligonucleotide duplexes and the protein purified from a HeLa cell extract. When a 32P-labeled 30-mer duplex containing the (6-4) photoproduct at a single site was used as a probe, only a single complex was detected in an electrophoretic mobility shift assay. It was demonstrated by Western blotting that both of the subunits (p48 and p127) were present in this complex. Electrophoretic mobility shift assays using various duplexes showed that the UV-DDB protein formed a specific, high affinity complex with the duplex containing an abasic site analog, in addition to the (6-4) photoproduct. By circular permutation analyses, these DNA duplexes were found to be bent at angles of 54 degrees and 57 degrees in the complexes with this protein. From the previously reported NMR studies and the fluorescence resonance energy transfer experiments in the present study, it can be concluded that the UV-DDB protein binds DNA that can be bent easily at the above angle.
Department of Bioorganic Chemistry, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Y
Y
Fujiwara
C
C
Masutani
T
T
Mizukoshi
J
J
Kondo
F
F
Hanaoka
S
S
Iwai
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D004249:DNA Damage; D015342:DNA Probes / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013379:Substrate Specificity
C091435:DDB1 protein, human; D015342:DNA Probes; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D004255:Deoxyribodipyrimidine Photo-Lyase