Purification of a novel UV-damaged-DNA binding protein highly specific for (6-4) photoproduct.
UV damage-specific binding proteins are considered to play important roles in early responses of cells irradiated with UV, including damage recognition in the DNA repair process. We have surveyed nuclear and cytoplasmic proteins which bind selectively to UV-irradiated DNA using an electrophoretic mobility shift assay. We detected four distinct binding activities with different mobilities in fractions separated from HeLa cells by heparin chromatography. Three of them were found in nuclear extracts and one in cytoplasmic extracts. We purified one of the binding factors from nuclear extracts to homogeneity, which was designated NF-10 (the 10th fraction of nuclear extract on heparin chromatography). It migrated as a 40 kDa polypeptide in SDS-PAGE, and bound to UV-irradiated double- stranded DNA but not to unirradiated DNA. The binding pattern of the NF-10 protein to DNA irradiated with UV corresponded to the induction kinetics of (6-4) photoproduct. Removal of (6-4) photoproducts from UV- irradiated DNA by (6-4) photoproduct-specific photolyase diminished the binding of NF-10 protein. These results suggest that the NF-10 protein binds to UV-damaged DNA through (6-4) photoproduct. Immunoblot analysis using a monoclonal antibody revealed that the NF-10 protein was expressed in cell lines from all complementation groups of xeroderma pigmentosum, indicating that the NF-10 protein is a novel UV-damaged-DNA binding protein.
Wakasugi
M
M
Division of Radiation Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
Abe
Y
Y
Yoshida
Y
Y
Matsunaga
T
T
Nikaido
O
O
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes / Q000235:genetics; D010777:Photochemistry; D011485:Protein Binding; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D004247:DNA