TRANSLOCATION OF A UV-DAMAGED DNA-BINDING PROTEIN INTO A TIGHT ASSOCIATION WITH CHROMATIN AFTER TREATMENT OF MAMMALIAN-CELLS WITH UV-LIGHT

A UV-damaged DNA binding protein (UV-DDB) is the major source of UV-da maged DNA binding activity in mammalian cell extracts. This activity i s defective in at least some xeroderma pigmentosum group E (XP-E) pati ents; microinjection of the UV-DDB protein into their fibroblasts corr ects nucleotide excision repair (NER). In an in vitro reconstituted NE R system, small amounts of UV-DDB stimulate repair synthesis a few fol d. After exposure to UV,mammalian cells show an early dose-dependent i nhibition of the extractable UV-DDB activity; this inhibition may refl ect a tight association of the binding protein with UV-damaged genomic DNA. To investigate the dynamics and location of UV-DDB with respect to damaged chromatin in vivo, we utilized nuclear fractionation and sp ecific antibodies and detected translocation of the p127 component of UV-DDB from a loose to a tight association with chromatinized DNA imme diately after UV treatment. A similar redistribution was found for oth er NER proteins, i.e. XPA, RP-A and PCNA, suggesting their tighter ass ociation with genomic DNA after UV. These studies revealed a specific protein-protein interaction between UV-DDB/p127 and RP-A that appears to enhance binding of both proteins to W-damaged DNA in vitro, providi ng evidence for the involvement of UV-DDB in the damage-recognition st ep of NER. Moreover, the kinetics of the reappearance of extractable U V-DDB activity after UV treatment of human cells with differing repair capacities positively correlate with the cell's capacity to repair 6- 4 pyrimidine dimers (6-4 PD) in the whole genome, a result consistent with an in vivo role for UV-DDB in recognizing this type of UV lesion.