Abstract
Transcription factor II H (TFIIH) is comprised of core TFIIH and Cdk-activating kinase (CAK) complexes. Here, we investigated the molecular and cellular manifestation of the TFIIH compositional changes by XPG truncation mutations. We showed that both core TFIIH and CAK are rapidly recruited to damage sites in repair-proficient cells. Chromatin immunoprecipitation against TFIIH and CAK components revealed a physical engagement of CAK in nucleotide excision repair (NER). While XPD recruitment to DNA damage was normal, CAK was not recruited in severe XP-G and XP-G/CS cells, indicating that the associations of CAK and XPD to core TFIIH are differentially affected. A CAK inhibition approach showed that CAK activity is not required for assembling pre-incision machinery in vivo or for removing genomic photolesions. Instead, CAK is involved in Ser5-phosphorylation and UV-induced degradation of RNA polymerase II. The CAK inhibition impaired transcription from undamaged and UV-damaged reporter, and partially decreased transcription of p53-dependent genes. The overall results demonstrated that a) XP-G/CS mutations affect the disassembly state of TFIIH resulting in the dissociation of CAK, but not XPD from core TFIIH, and b) CAK activity is not essential for global genomic repair but involved in general transcription and damage-induced RNA polymerase II degradation.
Highlights
The genome of eukaryotic cells is vulnerable to many DNAdamaging agents, which cause devastating cellular consequences
We first determined which form of transcription factor II H (TFIIH) is engaged in cellular Nucleotide excision repair (NER) by examining the in vivo recruitment of core TFIIH and Cdkactivating kinase (CAK) to subnuclear spots where DNA damage is locally generated by micropore UV irradiation
A recent study from Tanaka and Egly laboratories has reported that XPG mutations in xeroderma pigmentosum (XP)-G/Cockayne syndrome (CS) cells affect the assembly states of TFIIH, and that the dissociation of CAK and XPD from core TFIIH leads to a disturbed transactivation of nuclear receptors [33]
Summary
The genome of eukaryotic cells is vulnerable to many DNAdamaging agents, which cause devastating cellular consequences. Cells utilize several repair pathways to overcome the deleterious effects of DNA damage and maintain their genome integrity. Nucleotide excision repair (NER) removes a broad variety of double-helix-distorting DNA lesions, including UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) [1]. NER consists of two sub-pathways: global genomic repair (GGR), which removes DNA damage from the entire genome; and transcription-coupled repair (TCR), which eliminates lesions located on actively transcribed genes [2]. A generally accepted NER model includes damage recognition, dual incision, and gap-filling DNA synthesis steps [4,5]. Other NER factors, such as XPA and RPA, are believed to join the TFIIH-containing repair complex to verify the nature of DNA structure alteration [14]. Subsequent gap-filling DNA synthesis is performed by the concerted action of pol d or pol e, and the cofactors PCNA, RF-C and RPA
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