Structural basis of DNA crossover capture by Escherichia coli DNA gyrase.

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.

What's on the Other Side of the Gate: A Structural Perspective on DNA Gate Opening of Type IA and IIA DNA Topoisomerases.

DNA topoisomerases have an essential role in resolving topological problems that arise due to the double-helical structure of DNA. They can recognise DNA topology and catalyse diverse topological reactions by cutting and re-joining DNA ends. Type IA and IIA topoisomerases, which work by strand passage mechanisms, share catalytic domains for DNA binding and cleavage. Structural information has accumulated over the past decades, shedding light on the mechanisms of DNA cleavage and re-ligation. However, the structural rearrangements required for DNA-gate opening and strand transfer remain elusive, in particular for the type IA topoisomerases. In this review, we compare the structural similarities between the type IIA and type IA topoisomerases. The conformational changes that lead to the opening of the DNA-gate and strand passage, as well as allosteric regulation, are discussed, with a focus on the remaining questions about the mechanism of type IA topoisomerases.

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor-binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain-containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

Structural basis for allosteric regulation of Human Topoisomerase IIα.

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6-7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme's activities and opens perspective for the analysis of post-translational modifications.

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.

The interplay between DNA topoisomerase 2α post-translational modifications and drug resistance.

The type 2 DNA topoisomerases (Top2) are conserved enzymes and biomarkers for cell proliferation. The catalytic activities of the human isoform Top2α are essential for the regulation of DNA topology during DNA replication, transcription, and chromosome segregation. Top2α is a prominent target for anti-cancer drugs and is highly regulated by post-translational modifications (PTM). Despite an increasing number of proteomic studies, the extent of PTM in cancer cells and its importance in drug response remains largely uncharacterized. In this review, we highlight the different modifications affecting the human Top2α in healthy and cancer cells, taking advantage of the structure-function information accumulated in the past decades. We also overview the regulation of Top2α by PTM, the level of PTM in cancer cells, and the resistance to therapeutic compounds targeting the Top2 enzyme. Altogether, this review underlines the importance of future studies addressing more systematically the interplay between PTM and Top2 drug resistance.

Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex.

DNA gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, a detailed architecture of the full-length DNA gyrase from the model organism E. coli is still missing. Herein, we report the complete structure of the E. coli DNA gyrase nucleoprotein complex trapped by the antibiotic gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of two states of the DNA-binding/cleavage domain provides a better understanding of the allosteric movements of the enzyme complex. The local atomic resolution in the DNA-bound area reaching up to 3.0 Å enables the identification of the antibiotic density. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates.

Structural Basis for DNA Gyrase Interaction with Coumermycin A1.

Coumermycin A1 is a natural aminocoumarin that inhibits bacterial DNA gyrase, a member of the GHKL proteins superfamily. We report here the first cocrystal structures of gyrase B bound to coumermycin A1, revealing that one coumermycin A1 molecule traps simultaneously two ATP-binding sites. The inhibited dimers from different species adopt distinct sequence-dependent conformations, alternative to the ATP-bound form. These structures provide a basis for the rational development of coumermycin A1 derivatives for antibiotherapy and biotechnology applications.

Publisher Correction: Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Post-translational modifications in DNA topoisomerase 2α highlight the role of a eukaryote-specific residue in the ATPase domain.

Type 2 DNA topoisomerases (Top2) are critical components of key protein complexes involved in DNA replication, chromosome condensation and segregation, as well as gene transcription. The Top2 were found to be the main targets of anticancer agents, leading to intensive efforts to understand their functional and physiological role as well as their molecular structure. Post-translational modifications have been reported to influence Top2 enzyme activities in particular those of the mammalian Top2α isoform. In this study, we identified phosphorylation, and for the first time, acetylation sites in the human Top2α isoform produced in eukaryotic expression systems. Structural analysis revealed that acetylation sites are clustered on the catalytic domains of the homodimer while phosphorylation sites are located in the C-terminal domain responsible for nuclear localization. Biochemical analysis of the eukaryotic-specific K168 residue in the ATPase domain shows that acetylation affects a key position regulating ATP hydrolysis through the modulation of dimerization. Our findings suggest that acetylation of specific sites involved in the allosteric regulation of human Top2 may provide a mechanism for modulation of its catalytic activity.

A User-Friendly DNA Modeling Software for the Interpretation of Cryo-Electron Microscopy Data.

The structural modeling of a macromolecular machine is like a "Lego" approach that is challenged when blocks, like proteins imported from the Protein Data Bank, are to be assembled with an element adopting a serpentine shape, such as DNA templates. DNA must then be built ex nihilo, but modeling approaches are either not user-friendly or very long and fastidious. In this method chapter we show how to use GraphiteLifeExplorer, a software with a simple graphical user interface that enables the sketching of free forms of DNA, of any length, at the atomic scale, as fast as drawing a line on a sheet of paper. We took as an example the nucleoprotein complex of DNA gyrase, a bacterial topoisomerase whose structure has been determined using cryo-electron microscopy (Cryo-EM). Using GraphiteLifeExplorer, we could model in one go a 155 bp long and twisted DNA duplex that wraps around DNA gyrase in the cryo-EM map, improving the quality and interpretation of the final model compared to the initially published data.

A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family.

PURPOSE: The purpose of this study was to identify mutations that cause non-syndromic male infertility using whole exome sequencing of family cases. METHODS: We recruited a consanguineous Turkish family comprising nine siblings with male triplets; two of the triplets were infertile as well as one younger infertile brother. Whole exome sequencing (WES) performed on two azoospermic brothers identified a mutation in the melanoma antigen family B4 (MAGEB4) gene which was confirmed via Sanger sequencing and then screened for on control groups and unrelated infertile subjects. The effect of the mutation on messenger RNA (mRNA) and protein levels was tested after in vitro cell transfection. Structural features of MAGEB4 were predicted throughout the conserved MAGE domain. RESULTS: The novel single-base substitution (c.1041A>T) in the X-linked MAGEB4 gene was identified as a no-stop mutation. The mutation is predicted to add 24 amino acids to the C-terminus of MAGEB4. Our functional studies were unable to detect any effect either on mRNA stability, intracellular localization of the protein, or the ability to homodimerize/heterodimerize with other MAGE proteins. We thus hypothesize that these additional amino acids may affect the proper protein interactions with MAGEB4 partners. CONCLUSION: The whole exome analysis of a consanguineous Turkish family revealed MAGEB4 as a possible new X-linked cause of inherited male infertility. This study provides the first clue to the physiological function of a MAGE protein.

Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex.

Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest. The identified interlinked peptides suggest a strong network of interaction between GCN5, ADA2B and ADA3 subunits; SGF29 is interacting with GCN5 and ADA3 but not with ADA2B. These restraint data were combined to molecular modeling and a low-resolution interacting model for the human SAGA HAT subcomplex could be proposed, illustrating the potential of an integrative strategy using cross-linking and mass spectrometry for addressing the structural architecture of multiprotein complexes.

Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase.

Type 2A DNA topoisomerases (Topo2A) remodel DNA topology during replication, transcription and chromosome segregation. These multisubunit enzymes catalyze the transport of a double-stranded DNA through a transient break formed in another duplex. The bacterial DNA gyrase, a target for broad-spectrum antibiotics, is the sole Topo2A enzyme able to introduce negative supercoils. We reveal here for the first time the architecture of the full-length Thermus thermophilus DNA gyrase alone and in a cleavage complex with a 155 bp DNA duplex in the presence of the antibiotic ciprofloxacin, using cryo-electron microscopy. The structural organization of the subunits of the full-length DNA gyrase points to a central role of the ATPase domain acting like a 'crossover trap' that may help to sequester the DNA positive crossover before strand passage. Our structural data unveil how DNA is asymmetrically wrapped around the gyrase-specific C-terminal ß-pinwheel domains and guided to introduce negative supercoils through cooperativity between the ATPase and ß-pinwheel domains. The overall conformation of the drug-induced DNA binding-cleavage complex also suggests that ciprofloxacin traps a DNA pre-transport conformation.

Computational tools in protein crystallography.

Protein crystallography emerged in the early 1970s and is, to this day, one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. It is also an extremely powerful tool for drug design. This field has evolved together with developments in computer science and molecular biology, allowing faster three-dimensional structure determination of complex biological assemblies. In recent times, structural genomics initiatives have pushed the development of methods to further speed up this process. The algorithms initially defined in the last decade for structure determination are now more and more elaborate, but the computational tools have evolved toward simpler and more user-friendly packages and web interfaces. We present here a modest overview of the popular software packages that have been developed for solving protein structures, and give a few guidelines and examples for structure determination using the two most popular methods, molecular replacement and multiple anomalous dispersion.

Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction.

The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the ß-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP ß-subunit. We determined the 2.9-Å resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-ß1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP.