Damaged DNA-binding protein DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and replication protein A.
Human cells contain a protein that binds to UV-irradiated DNA with high affinity. This protein, damaged DNA-binding protein (DDB), is a heterodimer of two polypeptides, p127 and p48. Recent in vivo studies suggested that DDB is involved in global genome repair of cyclobutane pyrimidine dimers (CPDs), but the mechanism remains unclear. Here, we show that in vitro DDB directly stimulates the excision of CPDs but not (6-4)photoproducts. The excision activity of cell-free extracts from Chinese hamster AA8 cell line that lacks DDB activity was increased 3-4-fold by recombinant DDB heterodimer but not p127 subunit alone. Moreover, the addition of XPA or XPA + replication protein A (RPA), which themselves enhanced excision, also enhanced the excision in the presence of DDB. DDB was found to elevate the binding of XPA to damaged DNA and to make a complex with damaged DNA and XPA or XPA + RPA as judged by both electrophoretic mobility shift assays and DNase I protection assays. These results suggest that DDB assists in the recognition of CPDs by core NER factors, possibly through the efficient recruitment of XPA or XPA.RPA, and thus stimulates the excision reaction of CPDs.
Wakasugi
M
M
Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan.
Shimizu
M
M
Morioka
H
H
Linn
S
S
Nikaido
O
O
Matsunaga
T
T
D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000502:physiology*; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D011740:Pyrimidine Dimers; D016601:RNA-Binding Proteins / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D051716:Replication Protein A; D051760:Xeroderma Pigmentosum Group A Protein
C091435:DDB1 protein, human; C117669:DDB2 protein, human; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; D016601:RNA-Binding Proteins; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein