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-damaged DNA binding activity in mammalian cell extracts. This activity is defective in at least some xeroderma pigmentosum group E (XP-E) patients; microinjection of the UV-DDB protein into their fibroblasts corrects nucleotide excision repair (NER). In an in vitro reconstituted NER system, small amounts of UV-DDB stimulate repair synthesis a few fold. After exposure to UV, mammalian cells show an early dose-dependent inhibition of the extractable UV-DDB activity; this inhibition may reflect 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 specific antibodies and detected translocation of the p127 component of UV-DDB from a loose to a tight association with chromatinized DNA immediately after UV treatment. A similar redistribution was found for other NER proteins, i.e. XPA, RP-A and PCNA, suggesting their tighter association 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 UV-damaged DNA in vitro, providing evidence for the involvement of UV-DDB in the damage-recognition step of NER. Moreover, the kinetics of the reappearance of extractable UV-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.
Otrin
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Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
McLenigan
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M
Takao
M
M
Levine
A S
AS
Protić
M
M
D000818:Animals; D001693:Biological Transport, Active; D002460:Cell Line; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000378:metabolism* / Q000528:radiation effects*; D002999:Clone Cells; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000528:radiation effects*; D006801:Humans; D007700:Kinetics; D051716:Replication Protein A; D014466:Ultraviolet Rays / Q000009:adverse effects*; D014983:Xeroderma Pigmentosum / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein
D002843:Chromatin; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein