G-quadruplex-mediated genomic instability drives SNVs in cancer.

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.

Programmable DNA Interstrand Crosslinking by Alkene-Alkyne [2 + 2] Photocycloaddition.

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.

Structural basis for CDK7 activation by MAT1 and Cyclin H.

Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein.

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.

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

Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five 'structural' subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD's DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD's affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62's high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.

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. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB's 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 acting 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 XPB's ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.

The novel KIT exon 11 germline mutation K558N is associated with gastrointestinal stromal tumor, mastocytosis, and seminoma development.

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.

Structural basis of assembly chaperone- mediated snRNP formation.

Small nuclear ribonucleoproteins (snRNPs) represent key constituents of major and minor spliceosomes. snRNPs contain a common core, composed of seven Sm proteins bound to snRNA, which forms in a step-wise and factor-mediated reaction. The assembly chaperone pICln initially mediates the formation of an otherwise unstable pentameric Sm protein unit. This so-called 6S complex docks subsequently onto the SMN complex, which removes pICln and enables the transfer of pre-assembled Sm proteins onto snRNA. X-ray crystallography and electron microscopy was used to investigate the structural basis of snRNP assembly. The 6S complex structure identifies pICln as an Sm protein mimic, which enables the topological organization of the Sm pentamer in a closed ring. A second structure of 6S bound to the SMN complex components SMN and Gemin2 uncovers a plausible mechanism of pICln elimination and Sm protein activation for snRNA binding. Our studies reveal how assembly factors facilitate formation of RNA-protein complexes in vivo.

The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH.

The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.

FANCM interacts with PCNA to promote replication traverse of DNA interstrand crosslinks.

FANCM is a highly conserved DNA remodeling enzyme that promotes the activation of the Fanconi anemia DNA repair pathway and facilitates replication traverse of DNA interstrand crosslinks. However, how FANCM interacts with the replication machinery to promote traverse remains unclear. Here, we show that FANCM and its archaeal homolog Hef from Thermoplasma acidophilum interact with proliferating cell nuclear antigen (PCNA), an essential co-factor for DNA polymerases in both replication and repair. The interaction is mediated through a conserved PIP-box; and in human FANCM, it is strongly stimulated by replication stress. A FANCM variant carrying a mutation in the PIP-box is defective in promoting replication traverse of interstrand crosslinks and is also inefficient in promoting FANCD2 monoubiquitination, a key step of the Fanconi anemia pathway. Our data reveal a conserved interaction mode between FANCM and PCNA during replication stress, and suggest that this interaction is essential for FANCM to aid replication machines to traverse DNA interstrand crosslinks prior to post-replication repair.

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA).

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a ß-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

In TFIIH, XPD helicase is exclusively devoted to DNA repair.

The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.

Selectivity of Pyridone- and Diphenyl Ether-Based Inhibitors for the Yersinia pestis FabV Enoyl-ACP Reductase.

The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold → 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156.

Functional and structural studies of the nucleotide excision repair helicase XPD suggest a polarity for DNA translocation.

The XPD protein is a vital subunit of the general transcription factor TFIIH which is not only involved in transcription but is also an essential component of the eukaryotic nucleotide excision DNA repair (NER) pathway. XPD is a superfamily-2 5'-3' helicase containing an iron-sulphur cluster. Its helicase activity is indispensable for NER and it plays a role in the damage verification process. Here, we report the first structure of XPD from Thermoplasma acidophilum (taXPD) in complex with a short DNA fragment, thus revealing the polarity of the translocated strand and providing insights into how the enzyme achieves its 5'-3' directionality. Accompanied by a detailed mutational and biochemical analysis of taXPD, we define the path of the translocated DNA strand through the protein and identify amino acids that are critical for protein function.

Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome.

SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.

Crystallizing the 6S and 8S spliceosomal assembly intermediates: a complex project.

The small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6 and U5 are major constituents of the pre-mRNA processing spliceosome. They contain a common RNP core that is formed by the ordered binding of Sm proteins onto the single-stranded Sm site of the snRNA. Although spontaneous in vitro, assembly of the Sm core requires assistance from the PRMT5 and SMN complexes in vivo. To gain insight into the key steps of the assembly process, the crystal structures of two assembly intermediates of U snRNPs termed the 6S and 8S complexes have recently been reported. These multimeric protein complexes could only be crystallized after the application of various rescue strategies. The developed strategy leading to the crystallization and solution of the 8S crystal structure was subsequently used to guide a combination of rational crystal-contact optimization with surface-entropy reduction of crystals of the related 6S complex. Conversely, the resulting high-resolution 6S crystal structure was used during the restrained refinement of the 8S crystal structure.

The crystal structure of the protein-disulfide isomerase family member ERp27 provides insights into its substrate binding capabilities.

About one-third of all cellular proteins pass through the secretory pathway and hence undergo oxidative folding in the endoplasmic reticulum (ER). Protein-disulfide isomerase (PDI) and related members of the PDI family assist in the folding of substrates by catalyzing the oxidation of two cysteines and isomerization of disulfide bonds as well as by acting as chaperones. In this study, we present the crystal structure of ERp27, a redox-inactive member of the PDI family. The structure reveals its substrate-binding cleft, which is homologous to PDI, but is able to adapt in size and hydrophobicity. Isothermal titration calorimetry experiments demonstrate that ERp27 is able to distinguish between folded and unfolded substrates, only interacting with the latter. ERp27 is up-regulated during ER stress, thus presumably allowing it to bind accumulating misfolded substrates and present them to ERp57 for catalysis.

Specialization among iron-sulfur cluster helicases to resolve G-quadruplex DNA structures that threaten genomic stability.

G-quadruplex (G4) DNA, an alternate structure formed by Hoogsteen hydrogen bonds between guanines in G-rich sequences, threatens genomic stability by perturbing normal DNA transactions including replication, repair, and transcription. A variety of G4 topologies (intra- and intermolecular) can form in vitro, but the molecular architecture and cellular factors influencing G4 landscape in vivo are not clear. Helicases that unwind structured DNA molecules are emerging as an important class of G4-resolving enzymes. The BRCA1-associated FANCJ helicase is among those helicases able to unwind G4 DNA in vitro, and FANCJ mutations are associated with breast cancer and linked to Fanconi anemia. FANCJ belongs to a conserved iron-sulfur (Fe S) cluster family of helicases important for genomic stability including XPD (nucleotide excision repair), DDX11 (sister chromatid cohesion), and RTEL (telomere metabolism), genetically linked to xeroderma pigmentosum/Cockayne syndrome, Warsaw breakage syndrome, and dyskeratosis congenita, respectively. To elucidate the role of FANCJ in genomic stability, its molecular functions in G4 metabolism were examined. FANCJ efficiently unwound in a kinetic and ATPase-dependent manner entropically favored unimolecular G4 DNA, whereas other Fe-S helicases tested did not. The G4-specific ligands Phen-DC3 or Phen-DC6 inhibited FANCJ helicase on unimolecular G4 ∼1000-fold better than bi- or tetramolecular G4 DNA. The G4 ligand telomestatin induced DNA damage in human cells deficient in FANCJ but not DDX11 or XPD. These findings suggest FANCJ is a specialized Fe-S cluster helicase that preserves chromosomal stability by unwinding unimolecular G4 DNA likely to form in transiently unwound single-stranded genomic regions.

Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis.

In histidine and tryptophan biosynthesis, two related isomerization reactions are generally catalyzed by two specific single-substrate enzymes (HisA and TrpF), sharing a similar (ß/α)(8)-barrel scaffold. However, in some actinobacteria, one of the two encoding genes (trpF) is missing and the two reactions are instead catalyzed by one bisubstrate enzyme (PriA). To unravel the unknown mechanism of bisubstrate specificity, we used the Mycobacterium tuberculosis PriA enzyme as a model. Comparative structural analysis of the active site of the enzyme showed that PriA undergoes a reaction-specific and substrate-induced metamorphosis of the active site architecture, demonstrating its unique ability to essentially form two different substrate-specific actives sites. Furthermore, we found that one of the two catalytic residues in PriA, which are identical in both isomerization reactions, is recruited by a substrate-dependent mechanism into the active site to allow its involvement in catalysis. Comparison of the structural data from PriA with one of the two single-substrate enzymes (TrpF) revealed substantial differences in the active site architecture, suggesting independent evolution. To support these observations, we identified six small molecule compounds that inhibited both PriA-catalyzed isomerization reactions but had no effect on TrpF activity. Our data demonstrate an opportunity for organism-specific inhibition of enzymatic catalysis by taking advantage of the distinct ability for bisubstrate catalysis in the M. tuberculosis enzyme.

XPD stalled on cross-linked DNA provides insight into damage verification.

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.