Physical and functional interaction between DDB and XPA in nucleotide excision repair.
Damaged DNA-binding protein (DDB), consisting of DDB1 and DDB2 subunits recognizes a wide spectrum of DNA lesions. DDB is dispensable for in vitro nucleotide excision repair (NER) reaction, but stimulates this reaction especially for cyclobutane pyrimidine dimer (CPD). Here we show that DDB directly interacts with XPA, one of core NER factors, mainly through DDB2 subunit and the amino-acid residues between 185 and 226 in XPA are important for the interaction. Interestingly, the point mutation causing the substitution from Arg-207 to Gly, which was previously identified in a XP-A revertant cell-line XP129, diminished the interaction with DDB in vitro and in vivo. In a defined system containing R207G mutant XPA and other core NER factors, DDB failed to stimulate the excision of CPD, although the mutant XPA was competent for the basal NER reaction. Moreover, in vivo experiments revealed that the mutant XPA is recruited to damaged DNA sites with much less efficiency compared with wild-type XPA and fails to support the enhancement of CPD repair by ectopic expression of DDB2 in SV40-transformed human cells. These results suggest that the physical interaction between DDB and XPA plays an important role in the DDB-mediated NER reaction.
Wakasugi
Mitsuo
M
Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-1192, Japan.
Kasashima
Hiromi
H
Fukase
Yuko
Y
Imura
Mayumi
M
Imai
Rika
R
Yamada
Saki
S
Cleaver
James E
JE
Matsunaga
Tsukasa
T
D002460:Cell Line; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D020125:Mutation, Missense; D054730:Protein Interaction Domains and Motifs; D011740:Pyrimidine Dimers / Q000378:metabolism; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; D051760:Xeroderma Pigmentosum Group A Protein