DNA Damage Binding Protein Component DDB1 Participates in Nucleotide Excision Repair through DDB2 DNA-binding and Cullin 4A Ubiquitin Ligase Activity

Abstract

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. (Cancer Res 2006; 66(17): 8590-7)

Accelerated protein evolution analysis reveals genes and pathways associated with the evolution of mammalian longevity

“…DDB1 in particular is involved in many distinct DNA response and DNA repair pathways. For example, DDB1 binds to SKP2 and plays a role in the ubiquitination of CDKN1B, a cyclin-dependent kinase inhibitor (Nishitani et al 2006) and may recruit nucleotide excision repair proteins in order to repair DNA damage (Li et al 2006). Deficiency in DDB1 is associated with xeroderma pigmentosum (Kapetanaki et al 2006).…”

Modulation of Nucleotide Excision Repair by Mammalian SWI/SNF Chromatin-remodeling Complex

“…HeLa cells were transfected with N-terminal GFP-HA-His-tagged XPC (kindly provided by Dr. Jan H. Hoeijamakers, Erasmus University, The Netherlands) and a stably transfected cell line (HeLa-XPC) was established in our laboratory. Li-Fraumeni syndrome fibroblast 041 cell line as described previously (18) was provided by Dr. Michael Tainsky (M.D. Anderson Cancer Center, Austin, TX).…”

The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation

“…In Vivo Ubiquitylation and Phosphorylation Detection-DDB2 ubiquitylation and phosphorylation were detected as described before (43). Briefly, HeLa-DDB2 cells were treated with MG132 (10 M) 30 min before UV irradiation at 40 J/m 2 and kept in the same medium for indicated time periods.…”