Abstract
The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold.
Highlights
The XPD helicase is a central component of the general transcription factor transcription factor II H (TFIIH) which plays major roles in transcription and nucleotide excision repair (NER)
We further investigated the interaction between C. thermophilum XPD (ctXPD) and ctMAT1 fragments via native polyacrylamide gel electrophoresis (PAGE) analysis and observed here as well that the ctMAT1Archway variant displayed a reduced affinity toward ctXPD compared to the ctMAT1Lock variant as indicated by the reduced complex formation at similar concentrations (84% unbound ctMAT1Archway to 12.8% unbound ctMAT1Lock, Supplementary Fig. 2c)
The K370E (K369 in ctXPD) variant, which displays a severely impaired helicase activity in both, the human and the C. thermophilum system (Figs. 3c and 5c), is able to interact with MAT1; the resulting TFIIH is able to phosphorylate RNA pol II and RNA synthesis can be initiated (Figs. 3g and 5d, e)
Summary
The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). 1234567890():,; The super family 2 (SF2) helicase XPD is a 5′–3′ helicase and a founding member of the group of iron sulfur cluster (FeS) containing helicases[1] These helicases all share a common fold that consists of two RecA like motor domains (HD1 and HD2), the FeS domain and an additional Arch domain. The path of the translocated single stranded DNA (ssDNA) on XPD has been identified by a combination of structural and biochemical methods It extends from a high affinity binding site in HD2 to HD1. XPD anchors and positions the CAK module, thereby only being important as a scaffolding protein, whereas its full enzymatic activity is indispensable for NER20 to accomplish unwinding of the DNA around the lesion and to participate in damage verification[21,22,23]
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