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Nucleotide excision repair (NER) is vital for genome integrity. Yet, our understanding of the complex NER protein machinery remains incomplete. Combining cryo-EM and XL-MS data with AlphaFold2 predictions, we build an integrative model of the NER pre-incision complex(PInC). Here TFIIH serves as a molecular ruler, defining the DNA bubble size and precisely positioning the XPG and XPF nucleases for incision. Using simulations and graph theoretical analyses, we unveil PInCs assembly, global motions, and partitioning into dynamic communities. Remarkably, XPG caps XPDs DNA-binding groove and bridges both junctions of the DNA bubble, suggesting a novel coordination mechanism of PInCs dual incision. XPA rigging interlaces XPF/ERCC1 with RPA, XPD, XPB, and 5 ssDNA, exposing XPAs crucial role in licensing the XPF/ERCC1 incision. Mapping disease mutations onto our models reveals clustering into distinct mechanistic classes, elucidating xeroderma pigmentosum and Cockayne syndrome disease etiology.

Transcription factor IIH (TFIIH) is a protein assembly essential for transcription initiation and nucleotide excision repair (NER). Yet, understanding of the conformational switching underpinning these diverse TFIIH functions remains fragmentary. TFIIH mechanisms critically depend on two translocase subunits, XPB and XPD. To unravel their functions and regulation, we build cryo-EM based TFIIH models in transcription- and NER-competent states. Using simulations and graph-theoretical analysis methods, we reveal TFIIH's global motions, define TFIIH partitioning into dynamic communities and show how TFIIH reshapes itself and self-regulates depending on functional context. Our study uncovers an internal regulatory mechanism that switches XPB and XPD activities making them mutually exclusive between NER and transcription initiation. By sequentially coordinating the XPB and XPD DNA-unwinding activities, the switch ensures precise DNA incision in NER. Mapping TFIIH disease mutations onto network models reveals clustering into distinct mechanistic classes, affecting translocase functions, protein interactions and interface dynamics.

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

Many acute and chronic diseases affect the distal lung alveoli. Alveolar epithelial cell (AEC) lines are needed to better model these diseases. We used de-identified human remnant transplant lungs to develop a method to establish AEC lines. The lines grow well in 2-dimensional (2D) culture as epithelial monolayers expressing lung progenitor markers. In 3-dimensional (3D) culture with fibroblasts, Matrigel, and specific media conditions, the cells form alveolar-like organoids expressing mature AEC markers including aquaporin 5 (AQP5), G-protein-coupled receptor class C group 5 member A (GPRC5A), and surface marker HTII280. Single-cell RNA sequencing of an AEC line in 2D versus 3D culture revealed increased cellular heterogeneity and induction of cytokine and lipoprotein signaling in 3D organoids. Our approach yields lung progenitor lines that retain the ability to differentiate along the alveolar cell lineage despite long-term expansion and provides a valuable system to model and study the distal lung in vitro.

Transcription-coupled repair is essential for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA polymerase II. Mutations impairing CSB function cause severe genetic disease. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase to bypass certain lesions while triggering excision of others is incompletely understood. Here we build structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables simulations and graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We identify an allosteric pathway coupling motions of the Rad26 ATPase modules to changes in RNA polymerase and DNA to unveil a structural mechanism for CSB-assisted progression past less bulky lesions. Our models allow functional interpretation of the effects of Cockayne syndrome disease mutations.

Critical for transcription initiation and bulky lesion DNA repair, TFIIH provides an exemplary system to connect molecular mechanisms to biological outcomes due to its strong genetic links to different specific human diseases. Recent advances in structural and computational biology provide a unique opportunity to re-examine biologically relevant molecular structures and develop possible mechanistic insights for the large dynamic TFIIH complex. TFIIH presents many puzzles involving how its two SF2 helicase family enzymes, XPB and XPD, function in transcription initiation and repair: how do they initiate transcription, detect and verify DNA damage, select the damaged strand for incision, coordinate repair with transcription and cell cycle through Cdk-activating-kinase (CAK) signaling, and result in very different specific human diseases associated with cancer, aging, and development from single missense mutations? By joining analyses of breakthrough cryo-electron microscopy (cryo-EM) structures and advanced computation with data from biochemistry and human genetics, we develop unified concepts and molecular level understanding for TFIIH functions with a focus on structural mechanisms. We provocatively consider that TFIIH may have first evolved from evolutionary pressure for TCR to resolve arrested transcription blocks to DNA replication and later added its key roles in transcription initiation and global DNA repair. We anticipate that this level of mechanistic information will have significant impact on thinking about TFIIH, laying a robust foundation suitable to develop new paradigms for DNA transcription initiation and repair along with insights into disease prevention, susceptibility, diagnosis and interventions.

Transcription preinitiation complexes (PICs) are vital assemblies whose function underlies the expression of protein-encoding genes. Cryo-EM advances have begun to uncover their structural organization. Nevertheless, 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 that link core-PIC to TFIIH. p62 rigging interlaces p34, p44 and XPD while capping the DNA-binding and ATP-binding sites of XPD. PIC kinks and locks substrate DNA, creating negative supercoiling within the Pol II cleft to facilitate promoter opening. Mapping disease mutations associated with xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome onto defined communities reveals clustering into three mechanistic classes that affect TFIIH helicase functions, protein interactions and interface dynamics.

AimTo identify functional lung adenocarcinoma (LUAD) risk SNPs.Materials & methodsEighteen validated LUAD risk SNPs (p ≤ 5 × 10-8) and 930 SNPs in high linkage disequilibrium (r2 > 0.5) were integrated with epigenomic information from primary human alveolar epithelial cells. Enhancer-associated SNPs likely affecting transcription factor-binding sites were predicted. Three SNPs were functionally investigated using luciferase assays, expression quantitative trait loci and cancer-specific expression.ResultsForty-seven SNPs mapped to putative enhancers; 11 located to open chromatin. Of these, seven altered predicted transcription factor-binding motifs. Rs6942067 showed allele-specific luciferase expression and expression quantitative trait loci analysis indicates that it influences expression of DCBLD1, a gene that encodes an unknown membrane protein and is overexpressed in LUAD.ConclusionIntegration of candidate LUAD risk SNPS with epigenomic marks from normal alveolar epithelium identified numerous candidate functional LUAD risk SNPs including rs6942067, which appears to affect DCBLD1 expression. Data deposition: Data are provided in GEO record GSE84273.

Human flap endonuclease 1 (FEN1) and related structure-specific 5'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually 'locks' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA-RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB.