DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair.
Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.
Wakasugi
Mitsuo
M
Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi, Kanazawa 920-0934, Japan.
Kawashima
Aki
A
Morioka
Hiroshi
H
Linn
Stuart
S
Sancar
Aziz
A
Mori
Toshio
T
Nikaido
Osamu
O
Matsunaga
Tsukasa
T
D002461:Cell Line, Transformed; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011740:Pyrimidine Dimers / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein
C091435:DDB1 protein, human; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C086984:XPC protein, human