Common TFIIH recruitment mechanism in global genome and transcription-coupled repair subpathways

Abstract

Nucleotide excision repair is initiated by two different damage recognition subpathways, global genome repair (GGR) and transcription-coupled repair (TCR). In GGR, XPC detects DNA lesions and recruits TFIIH via interaction with the pleckstrin homology (PH) domain of TFIIH subunit p62. In TCR, an elongating form of RNA Polymerase II detects a lesion on the transcribed strand and recruits TFIIH by an unknown mechanism. Here, we found that the TCR initiation factor UVSSA forms a stable complex with the PH domain of p62 via a short acidic string in the central region of UVSSA, and determined the complex structure by NMR. The acidic string of UVSSA binds strongly to the basic groove of the PH domain by inserting Phe408 and Val411 into two pockets, highly resembling the interaction mechanism of XPC with p62. Mutational binding analysis validated the structure and identified residues crucial for binding. TCR activity was markedly diminished in UVSSA-deficient cells expressing UVSSA mutated at Phe408 or Val411. Thus, a common TFIIH recruitment mechanism is shared by UVSSA in TCR and XPC in GGR.

The sequential and cooperative action of CSB, CSA and UVSSA targets the TFIIH complex to DNA damage-stalled RNA polymerase II

“…Importantly, we found that UVSSA contains a TFIIH-interacting region (TIR; amino acids 400-500), which is crucial for the association of TFIIH with stalled RNAPIIo. Consistently, it has been shown that the PH domain of p62 (1-108) associates with a small fragment in UVSSA (400-419) in vitro and that mutations within this region causes a defect in recovery of RNA synthesis in vivo 40 . Moreover, we found that the UVSSA ∆CIR mutant was not only unable to associate with CSA, but also with the TFIIH complex.…”

“…Biochemical in vitro experiments have shown that the association of CSB with RNAPII is sufficient to recruit TFIIH 23 . In addition, CSA was shown to associate with the p44 subunit of TFIIH 26 , while UVSSA can interact with the p62 subunit of TFIIH 40 . In agreement, we found that GFP-UVSSA associates with several subunits of the TFIIH complex in a UV-specific manner in vivo.…”

“…CSB and CSA are required for the recruitment of the TFIIH complex It has been shown that CSB, CSA, and UVSSA can each associate with TFIIH 23,26,40 , but which of these proteins is responsible for the recruitment of TFIIH to DNA damagestalled RNAPIIo to initiate repair is currently unknown. To directly asses if CSB and CSA are required for the recruitment of TFIIH, we monitored TFIIH (p62 and p89) recruitment in UVSSA-KO complemented with GFP-UVSSA (WT) in which we additionally knocked out either CSB or CSA.…”

“…It has been reported that UVSSA can interact with TFIIH 16,31,40 , but whether this reflects a constitutive interaction or a UV-induced association is unclear. To gain more insight into the nature of this interaction, we immunoprecipitated GFP-UVSSA from the solubilized chromatin fraction of mock-treated and UV-irradiated cells followed by mass spectrometry (MS).…”

The essential and multifunctional TFIIH complex

“…Potentially, this could preclude XPB action during pol II transcription initiation at some genes. How does TFIIH function during DNA repair? The PH‐like domain of p62 has been shown to interact with several DNA repair factors in a process that is regulated in part by the chromatin remodeler CHD1 . How are these interactions controlled?…”