Koelmel, W. | Search bioRxiv

bioRxiv2020

How to limit the speed of a motor: The intricate regulation of the XPB ATPase and Translocase in TFIIH

The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. In both processes the ATPase activity of XPB is essential. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on a crystal structure of the full p52/p8 complex and current cryo-EM structures. Importantly, we show that XPBs ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB that acts as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPBs ATPase activity. Our data unravel an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.

biochemistry↗

bioRxiv2019

The TFIIH components p44/p62 act as a damage sensor during nucleotide excision repair

Nucleotide excision repair (NER) protects the genome following exposure to diverse types of DNA damage, including UV light and chemotherapeutics. Mutations in mammalian NER genes lead to diseases such as xeroderma pigmentosum, trichothiodystrophy, and Cockayne syndrome. In eukaryotes, the major transcription factor TFIIH is the central hub of NER. The core components of TFIIH include the helicases XPB, XPD, and five structural subunits. Two of these structural TFIIH proteins, p44 and p62 remain relatively unstudied; p44 is known to regulate the helicase activity of XPD during NER whereas p62s role is thought to be structural. However, a recent cryo-EM structure shows that p44, p62, and XPD make extensive contacts within TFIIH, with part of p62 occupying XPDs DNA binding site. This observation implies a more extensive role in DNA repair beyond the structural integrity of TFIIH. Here, we show that p44 stimulates XPDs ATPase but upon encountering DNA damage, further stimulation is only observed when p62 is part of the ternary complex; suggesting a role for the p44/p62 heterodimer in TFIIHs mechanism of damage detection. Using single molecule imaging, we demonstrate that p44/p62 independently interacts with DNA; it is seen to diffuse, however, in the presence of UV-induced DNA lesions the complex stalls. Combined with the analysis of a recent cryo-EM structure we suggest that p44/p62 acts as a novel DNA-binding entity within TFIIH that is capable of recognizing DNA damage. This revises our understanding of TFIIH and prompts more extensive investigation into the core subunits for an active role during both DNA repair and transcription.

biochemistry↗