Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair.
Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH-CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER.
Department of Radiology, The Ohio State University, Columbus, OH 43210, USA. zhu.49@osu.edu
Qianzheng
Q
Zhu
Gulzar
G
Wani
Nidhi
N
Sharma
Altaf
A
Wani
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000473:pathology*; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D059748:Proteolysis; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D051758:Transcription Factor TFIIH / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays / Q000009:adverse effects; D014983:Xeroderma Pigmentosum / Q000235:genetics; D051759:Xeroderma Pigmentosum Group D Protein / Q000235:genetics / Q000378:metabolism*; D000099250:DNA Excision Repair Protein ERCC-5; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D004720:Endonucleases; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D012319:RNA Polymerase II; D051758:Transcription Factor TFIIH; D014157:Transcription Factors; D051759:Xeroderma Pigmentosum Group D Protein; D000099250:DNA Excision Repair Protein ERCC-5; C514215:MNAT1 protein, human; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C072022:XPBC-ERCC-3 protein; C086984:XPC protein, human; C497292:ERCC2 protein, human