Nucleotide excison repair (NER) is a highly versatile template-based DNA repair mechanism that can address lesions ranging from cyclo butane pyrimdine dimers, to cisplatinum crosslinks, and bulky adducts like acetyl amino flourenes. Eukaryotic NER employs more than thirty different proteins that coordinate and facilitate the search, verification, incision, and DNA re-synthesis of the various lesions with extremely high precision. High resolution structures of key complexes combined with biochemistry and computational biology have greatly enhanced our understanding of the NER process. In this review we will highlight recent discoveries in NER research concerning lesion search and handover of complex assemblies, lesion verification, and potential mechanisms providing the signal for incision with a focus on structural biology.

Nitroalkenes are endogenous products generated by the metabolism of unsaturated fatty acids. They are generated under oxidative stress conditions, mediating important anti-inflammatory signaling activities through covalent modification of protein cysteine thiols. Despite being cytoprotective in benign tissue, nitroalkenes display single-agent anti-proliferative activity in breast cancer cells and sensitize them to multiple DNA-damaging agents. Initial mechanistic evidence suggested that nitroalkene anti-cancer activities are partially mediated by inhibition of homologous recombination (HR) through the recombinase RAD51 at Cys319. However, nitroalkenes are multi-target agents, and thus, it is likely that other important DNA repair targets beyond RAD51 are modified by nitroalkenes, contributing to their anti-cancer effects. We, therefore, conducted a global proteomics analysis to address this question. This analysis led to the identification of the recQ helicase RECQL4 with a nitro-alkylation at Cys1052. This modification was further confirmed by click chemistry-based chemoproteomics and determined to be DNA damage-dependent. Functional analyses demonstrated that nitroalkene modification inhibits RECQL4 ATP-dependent helicase activity and disrupts DSB end resection and downstream homology-dependent repair. Furthermore, experiments with C1052S mutant RECQL4 revealed that RECQL4 is a major mediator of nitroalkene effects on end resection, DSB formation, and repair. The evidence presented here denotes RECQL4 as an important nitroalkene target conferring DSB repair inhibition and supports further evaluation of nitroalkenes as therapeutic agents in RECQL4-amplified cancers.

Germline mutations in the DNA repair helicase XPD can cause the diseases xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). XP patients bear an increased risk of skin cancer including melanoma. This is not observed for TTD patients despite DNA repair defects. To examine whether TTD cells harbor features counteracting tumorigenesis, we developed a TTD melanoma cell model containing the XPD variant R722W. Intriguingly, TTD melanoma cells exhibited reduced proliferation and an increased signature of the melanocyte lineage factor MITF, along with a strong basal upregulation of REDD2, an inhibitor of the mTOR/S6K/4EBP1-dependent messenger RNA (mRNA) translation machinery. REDD2 levels were partially driven by MITF and contributed to reduced melanoma proliferation. In a TTD model for melanocytes-the progenitor cells of melanoma-the MITF gene signature was also increased, but here without affecting REDD2 expression. However, ribosomal protein synthesis was reduced particularly in R722W melanocytes after UV stress, indicating a compromised mRNA translation machinery. Impaired translation was also demonstrated for the TTD XPD variant A725P, but not for an XP variant. Concludingly, the impaired translation and reduced fitness observed in TTD melanocytes and melanoma cells, particularly after UV stress, offer a possible explanation why TTD patients do not develop melanomas.

The super family 2 (SF2) helicase XPD is a central component of the general transcription factor II H (TFIIH) which is essential for transcription and nucleotide excision DNA repair (NER)[1][1]. Within these two processes XPDs helicase function is vital for NER but not for transcription initiation, where XPD only acts as a scaffold for other factors [2][2]. We deciphered one of the most enigmatic steps in XPD helicase action: the active separation of dsDNA and its stalling upon approaching an interstrand crosslink, one of the most severe DNA damages in the cell, using cryo EM. Furthermore, the structure clearly shows how dsDNA is separated and reveals a highly unusual involvement of the Arch domain in active dsDNA separation. Combined with mutagenesis and biochemical analyses, we identify distinct functional residues important for helicase activity. Surprisingly, those areas also affect core TFIIH translocase activity, revealing a yet unencountered function of XPD within the TFIIH scaffold. Importantly, our structure provides a basis for XPD damage recognition and further suggests how the NER bubble could be formed, leading to a model for the location of the XPG nuclease relative to the excised damage.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2

The superfamily 2 helicase XPD is a central component of the general transcription factor II H (TFIIH), which is essential for transcription and nucleotide excision DNA repair (NER). Within these two processes, the helicase function of XPD is vital for NER but not for transcription initiation, where XPD acts only as a scaffold for other factors. Using cryo-EM, we deciphered one of the most enigmatic steps in XPD helicase action: the active separation of double-stranded DNA (dsDNA) and its stalling upon approaching a DNA interstrand cross-link, a highly toxic form of DNA damage. The structure shows how dsDNA is separated and reveals a highly unusual involvement of the Arch domain in active dsDNA separation. Combined with mutagenesis and biochemical analyses, we identified distinct functional regions important for helicase activity. Surprisingly, those areas also affect core TFIIH translocase activity, revealing a yet unencountered function of XPD within the TFIIH scaffold. In summary, our data provide a universal basis for NER bubble formation, XPD damage verification and XPG incision. Here, using cryo-EM and biochemistry, the authors delineate how the XPD helicase unorthodoxly uses its Arch domain to separate double-stranded DNA upon approaching a DNA lesion, promoting our understanding of NER bubble formation and damage verification.

G-quadruplex (G4s) DNA structures have been implicated in inducing genomic instability and contributing to cancer development. However, the relationship between G4s and cancer-related single nucleotide variants (cSNVs) in clinical settings remains unclear. In this large-scale study, we integrated experimentally validated G4s with genomic cSNVs from 13480 cancer patients to investigate the spatial association of G4s with the cellular cSNV landscape. Our findings demonstrate an increase in local genomic instability with increasing local G4 content in cancer patients, suggesting a potential role for G4s in driving cSNVs. Notably, we observed distinct spatial patterns of cSNVs and common single nucleotide variants (dbSNVs) in relation to G4s, implying different mechanisms for their generation and accumulation. We further demonstrate large, cancer-specific differences in the relationship of G4s and cSNVs, which could have important implications for a new class of G4-stabilizing cancer therapeutics. Moreover, we show that high G4-content can serve as a prognostic marker for local cSNV density and patient survival rates. Our findings underscore the importance of considering G4s in cancer research and highlight the need for further investigation into the underlying molecular mechanisms of G4-mediated genomic instability, especially in the context of cancer.

Trk (NTRK) receptor and NTRK gene fusions are oncogenic drivers of a wide variety of tumors. Although Trk receptors are typically activated at the cell surface, signaling of constitutive active Trk and diverse intracellular NTRK fusion oncogenes is barely investigated. Here, we show that a high intracellular abundance is sufficient for neurotrophin-independent, constitutive activation of TrkB kinase domains. In HEK293 cells, constitutive active TrkB kinase and an intracellular NTRK2-fusion oncogene (SQSTM1-NTRK2) reduced actin filopodia dynamics, phosphorylated FAK, and altered the cell morphology. Atypical cellular responses could be mimicked with the intracellular kinase domain, which did not activate the Trk-associated MAPK/ERK pathway. In glioblastoma-like U87MG cells, expression of TrkB or SQSTM1-NTRK2 reduced cell motility and caused drastic changes in the transcriptome. Clinically approved Trk inhibitors or mutating Y705 in the kinase domain, blocked the cellular effects and transcriptome changes. Atypical signaling was also seen for TrkA and TrkC. Moreover, hallmarks of atypical pTrk kinase were found in biopsies of Nestin-positive glioblastoma. Therefore, we suggest Western blot-like immunoassay screening of NTRK-related (brain) tumor biopsies to identify patients with atypical panTrk or phosphoTrk signals. Such patients could be candidates for treatment with NTRK inhibitors such as Larotrectinhib or Entrectinhib.

A study in Molecular Cell by Ramadhin et al.1 and two studies in Cell by van den Heuvel et al.2 and by Mevissen et al.3 show that STK19 is a key protein in transcription-coupled nucleotide excision repair (TC-NER) in mammalian cells by mediating the initial repair protein complex formation and subsequent TFIIH recruitment, thereby enabling the vital damage verification step.

Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical, and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2 '-deoxyuridine (PhedU). The crosslinking ability of PhedU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2 + 2] cycloaddition within the DNA duplex, resulting in efficient formation of a PhedU dimer after short irradiation times of a few seconds. A PhedU-dimer-containing DNA was shown to efficiently bind a helicase complex, but the covalent crosslink completely prevented DNA unwinding, suggesting possible applications in biochemistry or structural biology.

Nucleotide excision repair (NER) is unique in its ability to identify and remove vastly different lesions from DNA. Recent advances in the structural characterization of complexes involved in detection, verification, and excision of damaged DNA have reshaped our understanding of the molecular architecture of this efficient and accurate machinery. Initial damage recognition achieved through transcription coupled repair (TC-NER) or global genome repair (GG-NER) has been addressed by complexes of RNA Pol II with different TC-NER factors and XPC/RAD23B/Centrin-2 with TFIIH, respectively. Moreover, transcription factor IIH (TFIIH), one of the core repair factors and a central NER hub was resolved in different states, providing important insights how this complex facilitates DNA opening and damage verification. Combined, these recent advances led to a highly improved understanding of the molecular landscape of NER core processes, sharpening our view on how NER is successfully achieved.

Familial gastrointestinal stromal tumors (GIST) are dominant genetic disorders that are caused by germline mutations of the type III receptor tyrosine kinase KIT. While sporadic mutations are frequently found in mastocytosis and GISTs, germline mutations of KIT have only been described in 39 families until now. We detected a novel germline mutation of KIT in exon 11 (p.Lys-558-Asn; K558N) in a patient from a kindred with several GISTs harboring different secondary somatic KIT mutations. Structural analysis suggests that the primary germline mutation alone is not sufficient to release the autoinhibitory region of KIT located in the transmembrane domain. Instead, the KIT kinase module becomes constitutively activated when K558N combines with different secondary somatic mutations. The identical germline mutation in combination with an additional somatic KIT mutation was detected in a second patient of the kindred with seminoma while a third patient within the family had a cutaneous mastocytosis. These findings suggest that the K558N mutation interferes with the juxtamembranous part of KIT, since seminoma and mastocystosis are usually not associated with exon 11 mutations.

The general transcription factor II H (TFIIH) plays an essential role in transcription and nucleotide excision DNA repair (NER). TFIIH is a complex 10 subunit containing molecular machine that harbors three enzymatic activities while the remaining subunits assume regulatory and/or structural functions. Intriguingly, the three enzymatic activities of the CDK7 kinase, the XPB translocase, and the XPD helicase exert different impacts on the overall activities of TFIIH. While the enzymatic function of the XPD helicase is exclusively required in NER, the CDK7 kinase is deeply involved in transcription, whereas XPB is essential to both processes. Recent structural and biochemical endeavors enabled unprecedented details towards the molecular basis of these different TFIIH functions and how the enzymatic activities are regulated within the entire complex. Due to its involvement in two fundamental processes, TFIIH has become increasingly important as a target in cancer therapy and two of the three enzymes have already been addressed successfully. Here we explore the possibilities of recent high resolution structures in the context of TFIIH druggability and shed light on the functional consequences of the different approaches towards TFIIH inhibition.

The successful elimination of bulky DNA damages via the nucleotide excision repair (NER) system is largely determined by the damage recognition step. This step consists of primary recognition and verification of the damage. The TFIIH helicase XPD plays a key role in the verification step during NER. To date, the mechanism of damage verification is not sufficiently understood and requires further detailed research. This study is a systematic investigation of the interaction of ctXPD (Chaetomium thermophilum) as well as ctXPD-ctp44 with model DNAs, which contain structurally different bulky lesions with previously estimated NER repair efficiencies. We have used ATPase and DNA binding studies to assess the interaction of ctXPD with damaged DNA. The result of the analysis of ctXPD-ctp44 binding to DNA containing fluorescent and photoactivatable lesions demonstrates the relationship between the affinity of XPD for DNAs containing bulky damages and the ability of the NER system to eliminate the damage. Photo-cross-linking of ctXPD with DNA probes containing repairable and unrepairable photoactivatable damages reveals differences in the DNA interaction efficiency in the presence and absence of ctp44. In general, the results obtained indicate the ability of ctXPD-ctp44 to interact with a damage and suggest a significant role for ctp44 subunit in the verification process.

Protein-protein interactions play critical roles in biology, but despite decades of effort, the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions that have not yet been identified. Here, we take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes, as represented within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of S. cerevisiae proteins and build models for strongly predicted protein assemblies with two to five components. Comparison to existing interaction and structural data suggests that these predictions are likely to be quite accurate. We provide structure models spanning almost all key processes in Eukaryotic cells for 104 protein assemblies which have not been previously identified, and 608 which have not been structurally characterized. One-sentence summary We take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes.

Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a "quick-soak"-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy.

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.

Significance The fundamental processes of cell-cycle regulation and transcription are linked by the heterotrimeric CDK-activating kinase (CAK) complex. We solved the crystal structure of the active CAK complex and provide a molecular rationale for CAK activation, regulation, and substrate recognition. Our data thus highly advance our understanding of this essential factor which is also a proven target for cancer therapy.