Abstract:The XPD family of helicases, that includes human disease-related FANCJ, DDX11 and RTEL1, are Superfamily two helicases that contain an iron-sulphur cluster domain, translocate on ssDNA in a 5’−3’ direction and play important roles in genome stability. Consequently, mutations in several of these family members in eukaryotes cause human diseases. Family members in bacteria, such as the DinG helicase from Escherichia coli, are also involved in DNA repair. Here we present crystal structures of complexes of DinG bo… Show more
“…The linker deletion in human PIC has more subtle effects on the lower half of the TFIIH–core-PIC interface as compared to yeast PIC (Figure S4). Furthermore, our model supports p62 regulatory as well as structural roles: one p62 region (residues 274–293) tracks along the DNA path on XPD, based on a recent XPD ortholog structure 45 and another (residues 333–342) caps XPD and could influence DNA binding and ATPase function, respectively (Figure 1i and Figure 1o). Notably, our results highlight a key role of TFIIE (TFIIEα and TFIIEβ) for PIC structural integrity.…”
Transcription pre-initiation complexes (PIC) are vital assemblies whose function underlies protein gene expression. Cryo-EM advances have begun to uncover their structural organization. Yet, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically complete human PIC structural model. This enables simulations that reveal the assembly’s global motions, define PIC partitioning into dynamic communities and delineate how structural modules function together to remodel DNA. We identify key TFIIE–p62 interactions linking core-PIC to TFIIH. P62 rigging interlaces p34, p44 and XPD while capping XPD DNA-binding and ATP-binding sites. PIC kinks and locks substrate DNA, creating negative supercoiling within the Pol II cleft to facilitate promoter opening. Mapping Xeroderma Pigmentosum, Trichothiodystrophy, and Cockayne syndrome disease mutations onto defined communities reveals clustering into three mechanistic classes, affecting TFIIH helicase functions, protein interactions and interface dynamics.
“…The linker deletion in human PIC has more subtle effects on the lower half of the TFIIH–core-PIC interface as compared to yeast PIC (Figure S4). Furthermore, our model supports p62 regulatory as well as structural roles: one p62 region (residues 274–293) tracks along the DNA path on XPD, based on a recent XPD ortholog structure 45 and another (residues 333–342) caps XPD and could influence DNA binding and ATPase function, respectively (Figure 1i and Figure 1o). Notably, our results highlight a key role of TFIIE (TFIIEα and TFIIEβ) for PIC structural integrity.…”
Transcription pre-initiation complexes (PIC) are vital assemblies whose function underlies protein gene expression. Cryo-EM advances have begun to uncover their structural organization. Yet, functional analyses are hindered by incompletely modeled regions. Here we integrate all available cryo-EM data to build a practically complete human PIC structural model. This enables simulations that reveal the assembly’s global motions, define PIC partitioning into dynamic communities and delineate how structural modules function together to remodel DNA. We identify key TFIIE–p62 interactions linking core-PIC to TFIIH. P62 rigging interlaces p34, p44 and XPD while capping XPD DNA-binding and ATP-binding sites. PIC kinks and locks substrate DNA, creating negative supercoiling within the Pol II cleft to facilitate promoter opening. Mapping Xeroderma Pigmentosum, Trichothiodystrophy, and Cockayne syndrome disease mutations onto defined communities reveals clustering into three mechanistic classes, affecting TFIIH helicase functions, protein interactions and interface dynamics.
“…Our results thus suggest that an initial DNA interaction in the presence of MAT1 is possible but does not lead to helicase activity due to Arch domain blocking. This is further supported by a recent study describing the DNA translocation mechanism of DinG, the E. coli homolog of XPD 27 . In this study the ssDNA fragment extends toward the Arch domain of DinG spanning both HD1 and HD2 which is also thought to be a prerogative for triggering the ATPase activity.…”
Section: Discussionsupporting
confidence: 72%
“…7b, c) as has been shown earlier for DinG and archaeal XPD. Combined with the observation that the DinG Arch domain rotates and opens during the ATP driven translocation cycle 27 it is very likely that human XPD utilizes a similar mechanism. In the TFIIH cryo EM structure of Kokic et al a Y-fork DNA substrate was used.…”
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 highresolution 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.
“…10.7554/eLife.44771.024Figure 6—figure supplement 1.Mapping of XPD disease mutations on the TFIIH structure.( A ) Several residues affected by XP mutations localize near the DNA binding surfaces of XPD. This figure uses the same color scheme as Figure 6 (XP mutations purple; TTD yellow; CS-XP orange; helicase elements for DNA binding in blue, nucleotide binding and hydrolysis in green, coupling of nucleotide hydrolysis to DNA translocation in brown (Fairman-Williams et al, 2010); DNA superposed from (Cheng and Wigley, 2018)). ( B ) Mapping of mutations near the catalytic site of XPD.…”
Transcription factor IIH (TFIIH) is a heterodecameric protein complex critical for transcription initiation by RNA polymerase II and nucleotide excision DNA repair. The TFIIH core complex is sufficient for its repair functions and harbors the XPB and XPD DNA-dependent ATPase/helicase subunits, which are affected by human disease mutations. Transcription initiation additionally requires the CdK activating kinase subcomplex. Previous structural work has provided only partial insight into the architecture of TFIIH and its interactions within transcription pre-initiation complexes. Here, we present the complete structure of the human TFIIH core complex, determined by phase-plate cryo-electron microscopy at 3.7 Å resolution. The structure uncovers the molecular basis of TFIIH assembly, revealing how the recruitment of XPB by p52 depends on a pseudo-symmetric dimer of homologous domains in these two proteins. The structure also suggests a function for p62 in the regulation of XPD, and allows the mapping of previously unresolved human disease mutations.
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