DNA damage binding protein component DDB1 participates in nucleotide excision repair through DDB2 DNA-binding and cullin 4A ubiquitin ligase activity.
Functional defect in DNA damage binding (DDB) activity has a direct relationship to decreased nucleotide excision repair (NER) and increased susceptibility to cancer. DDB forms a complex with cullin 4A (Cul4A), which is now known to ubiquitylate DDB2, XPC, and histone H2A. However, the exact role of DDB1 in NER is unclear. In this study, we show that DDB1 knockdown in human cells impaired their ability to efficiently repair UV-induced cyclobutane pyrimidine dimers (CPD) but not 6-4 photoproducts (6-4PP). Extensive nuclear protein fractionation and chromatin association analysis revealed that upon irradiation, DDB1 protein is translocated from a loosely bound to a tightly bound in vivo chromatin fraction and the DDB1 translocation required the participation of functional DDB2 protein. DDB1 knockdown also affected the translocation of Cul4A component to the tightly bound form in UV-damaged chromatin in vivo as well as its recruitment to the locally damaged nuclear foci in situ. However, DDB1 knockdown had no effect on DNA damage binding capacity of DDB2. The data indicated that DDB2 can bind to damaged DNA in vivo as a monomer, whereas Cul4A recruitment to damage sites depends on the fully assembled complex. Our data also showed that DDB1 is required for the UV-induced DDB2 ubiquitylation and degradation. In summary, the results suggest that (a) DDB1 is critical for efficient NER of CPD; (b) DDB1 acts in bridging DDB2 and ubiquitin ligase Cul4A; and (c) DDB1 aids in recruiting the ubiquitin ligase activity to the damaged sites for successful commencement of lesion processing by NER.
Li
Jinyou
J
Department of Radiology, The Ohio State University, Columbus, Ohio 43240, USA.
Wang
Qi-En
QE
Zhu
Qianzheng
Q
El-Mahdy
Mohamed A
MA
Wani
Gulzar
G
Praetorius-Ibba
Mette
M
Wani
Altaf A
AA
D044842:Cullin Proteins / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D005839:Gentamicins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014466:Ultraviolet Rays
C114234:CUL4A protein, human; D044842:Cullin Proteins; C091435:DDB1 protein, human; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D005839:Gentamicins; D011994:Recombinant Proteins; C010680:antibiotic G 418; D044767:Ubiquitin-Protein Ligases