Tripartite DNA Lesion Recognition and Verification by XPC, TFIIH, and XPA in Nucleotide Excision Repair

Abstract

SUMMARY Transcription factor IIH (TFIIH) is essential for both transcription and nucleotide excision repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten-subunit TFIIH and its seven-subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non-genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity in the presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC-dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.

Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition

“…These findings are consistent with the model that we previously suggested based on our data on archaeal XPD, in which XPD employs (simultaneously) a combination of different lesion-sensing approaches (concomitant with different strand preferences) to enhance its target spectrum (12). In support of our model, recent studies on the entire reconstituted human TFIIH complex also suggested recognition of strongly helix-distorting cisplatin as well as Cy3 adducts on the translocated strand (28). Based on crystallographic and biochemical data in combination with mutational analyses, a structural model has been suggested of how XPD interacts with DNA to recognize lesions (10,11,42).…”

“…In addition, bulky lesions (such as fluorescein, Cy3, or cisplatin adducts) may serve as mechanical roadblocks. Such lesions would hence directly hinder DNA translocation of the helicase leading to its stalling at the lesion site on the translocated DNA strand, as suggested previously for archaeal and human XPD (10,12,28). It is worth noting, however, that the diameter of the pore is too large to be blocked by typical bulky DNA lesions so that further interactions between the lesion and protein residues in the proximity of the pore are likely involved in specific lesion interactions.…”

“…In addition, at least in the eukaryotic system, lesion recognition is possibly aided by further protein interactions, e.g. by XPA (28). To disentangle the complex multiprotein, multistep mechanism of NER lesion identification, we investigated the isolated lesion recognition approaches employed by UvrB and XPD.…”

XPA: A key scaffold for human nucleotide excision repair

“…In both pathways, XPA is recruited to the damage site by the transcription factor II H (TFIIH) complex that is responsible for unwinding double-stranded DNA around the damaged nucleotide creating the NER bubble. XPA is generally understood to function in damage-verification and assembly of NER incision complexes 1,25-27 . XPA is recruited at the same time, and functions in coordination with, the eukaryotic ssDNA binding protein replication protein A (RPA).…”

Novel Candidate Biomarkers of Chemoradiosensitivity in Esophageal Squamous Cell Carcinoma: A Systematic Review

“…XPA and XPC are important molecules involved in mediating nucleotide excision for DNA repair [22]. Yang et al [23] used polymerase chain reaction (PCR) to analyze the genomic DNA isolated from peripheral leukocytes collected from 501 ESCC patients and demonstrated that patients with the XPA 5′UTR A/G and XPC K939Q C/C genotypes are at higher risk of mortality after treatment than patients with wild-type homozygous genotypes, and this effect was more pronounced in patients treated with neoadjuvant CRT followed by esophagectomy.…”