Heat-activated growth of metastable and length-defined DNA fibers expands traditional polymer assembly.

Biopolymers such as nucleic acids and proteins exhibit dynamic backbone folding, wherein site-specific intramolecular interactions determine overall structure. Proteins then hierarchically assemble into supramolecular polymers such as microtubules, that are robust yet dynamic, constantly growing or shortening to adjust to cellular needs. The combination of dynamic, energy-driven folding and growth with structural stiffness and length control is difficult to achieve in synthetic polymer self-assembly. Here we show that highly charged, monodisperse DNA-oligomers assemble via seeded growth into length-controlled supramolecular fibers during heating; when the temperature is lowered, these metastable fibers slowly disassemble. Furthermore, the specific molecular structures of oligomers that promote fiber formation contradict the typical theory of block copolymer self-assembly. Efficient curling and packing of the oligomers - or 'curlamers' - determine morphology, rather than hydrophobic to hydrophilic ratio. Addition of a small molecule stabilises the DNA fibers, enabling temporal control of polymer lifetime and underscoring their potential use in nucleic-acid delivery, stimuli-responsive biomaterials, and soft robotics.

Structural investigation of Trypanosoma cruzi Akt-like kinase as drug target against Chagas disease.

According to the World Health Organization, Chagas disease (CD) is the most prevalent poverty-promoting neglected tropical disease. Alarmingly, climate change is accelerating the geographical spreading of CD causative parasite, Trypanosoma cruzi, which additionally increases infection rates. Still, CD treatment remains challenging due to a lack of safe and efficient drugs. In this work, we analyze the viability of T. cruzi Akt-like kinase (TcAkt) as drug target against CD including primary structural and functional information about a parasitic Akt protein. Nuclear Magnetic Resonance derived information in combination with Molecular Dynamics simulations offer detailed insights into structural properties of the pleckstrin homology (PH) domain of TcAkt and its binding to phosphatidylinositol phosphate ligands (PIP). Experimental data combined with Alpha Fold proposes a model for the mechanism of action of TcAkt involving a PIP-induced disruption of the intramolecular interface between the kinase and the PH domain resulting in an open conformation enabling TcAkt kinase activity. Further docking experiments reveal that TcAkt is recognized by human inhibitors PIT-1 and capivasertib, and TcAkt inhibition by UBMC-4 and UBMC-6 is achieved via binding to TcAkt kinase domain. Our in-depth structural analysis of TcAkt reveals potential sites for drug development against CD, located at activity essential regions.

Fluorescent molecular rotors detect O6-methylguanine dynamics and repair in duplex DNA.

Alkylation at the O6 position of guanine is a common and highly mutagenic form of DNA damage. Direct repair of O6-alkylguanines by the "suicide" enzyme O6-methylguanine DNA methyltransferase (MGMT, AGT, AGAT) maintains genome stability and inhibits carcinogenesis. In this study, a fluorescent analogue of thymidine containing trans-stilbene (tsT) is quenched by O6-methylguanine residues in the opposite strand of DNA by molecular dynamics that propagate through the duplex with as much as ∼9 Šof separation. Increased fluorescence of tsT or the cytosine analogue tsC resulting from MGMT-mediated DNA repair were distinguishable from non-covalent DNA-protein binding following protease digest. To our knowledge, this is the first study utilizing molecular rotor base analogues to detect DNA damage and repair activities in duplex DNA.

Meeting report: transposable elements at the crossroads of evolution, health and disease 2023.

The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.

Minimalist Design of Wireframe DNA Nanotubes: Tunable Geometry, Size, Chirality, and Dynamics.

DNA nanotubes (NTs) have attracted extensive interest as artificial cytoskeletons for biomedical, synthetic biology, and materials applications. Here, we report the modular design and assembly of a minimalist yet robust DNA wireframe nanotube with tunable cross-sectional geometry, cavity size, chirality, and length, while using only four DNA strands. We introduce an h-motif structure incorporating double-crossover (DX) tile-like DNA edges to achieve structural rigidity and provide efficient self-assembly of h-motif-based DNA nanotube (H-NT) units, thus producing programmable, micrometer-long nanotubes. We demonstrate control of the H-NT nanotube length via short DNA modulators. Finally, we use an enzyme, RNase H, to take these structures out of equilibrium and trigger nanotube assembly at a physiologically relevant temperature, underlining future cellular applications. The minimalist H-NTs can assemble at near-physiological salt conditions and will serve as an easily synthesized, DNA-economical modular template for biosensors, plasmonics, or other functional materials and as cost-efficient drug-delivery vehicles for biomedical applications.

FIRRM cooperates with FIGNL1 to promote RAD51 disassembly during DNA repair.

Interstrand DNA cross-links (ICLs) represent complex lesions that compromise genomic stability. Several pathways have been involved in ICL repair, but the extent of factors involved in the resolution of ICL-induced DNA double-strand breaks (DSBs) remains poorly defined. Using CRISPR-based genomics, we identified FIGNL1 interacting regulator of recombination and mitosis (FIRRM) as a sensitizer of the ICL-inducing agent mafosfamide. Mechanistically, we showed that FIRRM, like its interactor Fidgetin like 1 (FIGNL1), contributes to the resolution of RAD51 foci at ICL-induced DSBs. While the stability of FIGNL1 and FIRRM is interdependent, expression of a mutant of FIRRM (∆WCF), which stabilizes the protein in the absence of FIGNL1, allows the resolution of RAD51 foci and cell survival, suggesting that FIRRM has FIGNL1-independent function during DNA repair. In line with this model, FIRRM binds preferentially single-stranded DNA in vitro, raising the possibility that it directly contributes to RAD51 disassembly by interacting with DNA. Together, our findings establish FIRRM as a promoting factor of ICL repair.

Structural basis for DNA targeting by the Tn7 transposon.

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.

A dissipative pathway for the structural evolution of DNA fibres.

Biochemical networks interconnect, grow and evolve to express new properties as different chemical pathways are selected during a continuous cycle of energy consumption and transformation. In contrast, synthetic systems that push away from equilibrium usually return to the same self-assembled state, often generating waste that limits system recyclability and prevents the formation of adaptable networks. Here we show that annealing by slow proton dissipation selects for otherwise inaccessible morphologies of fibres built from DNA and cyanuric acid. Using single-molecule fluorescence microscopy, we observe that proton dissipation influences the growth mechanism of supramolecular polymerization, healing gaps within fibres and converting highly branched, interwoven networks into nanocable superstructures. Just as the growth kinetics of natural fibres determine their structural attributes to modulate function, our system of photoacid-enabled depolymerization and repolymerization selects for healed materials to yield organized, robust fibres. Our method provides a chemical route for error-checking, distinct from thermal annealing, that improves the morphologies and properties of supramolecular materials using out-of-equilibrium systems.

Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation.

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes, such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and polo kinase Cdc5. Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, noncatalytic role for Exo1, through noncanonical interaction with polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.

Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division.

Polo-like kinases (Plks) are key cell cycle regulators. They contain a kinase domain followed by a polo-box domain that recognizes phosphorylated substrates and enhances their phosphorylation. The regulatory subunit of the Dbf4-dependent kinase complex interacts with the polo-box domain of Cdc5 (the sole Plk in Saccharomyces cerevisiae) in a phosphorylation-independent manner. We have solved the crystal structures of the polo-box domain of Cdc5 on its own and in the presence of peptides derived from Dbf4 and a canonical phosphorylated substrate. The structure bound to the Dbf4-peptide reveals an additional density on the surface opposite to the phospho-peptide binding site that allowed us to propose a model for the interaction. We found that the two peptides can bind simultaneously and non-competitively to the polo-box domain in solution. Furthermore, point mutations on the surface opposite to the phosphopeptide binding site of the polo-box domain disrupt the interaction with the Dbf4 peptide in solution and cause an early anaphase arrest phenotype distinct from the mitotic exit defect typically observed in cdc5 mutants. Collectively, our data illustrates the importance of non-canonical interactions mediated by the polo-box domain and provide key mechanistic insights into the combinatorial recognition of substrates by Polo-like kinases.

Evaluation of pre- and post-fusion Human metapneumovirus F proteins as subunit vaccine candidates in mice.

Human metapneumovirus (hMPV) is an important respiratory pathogen especially in young children and elderly subjects. Our objective was to assess the immunogenicity and protection conferred by predominant pre- and post-fusion (F) hMPV-F constructs in Balb/C mice. Immunizations without adjuvant were not immunogenic whereas alum-adjuvanted hMPV-F proteins, regardless of their conformations, generated comparable neutralizing antibody titers with undetectable pulmonary viral titers following viral challenge. In conclusion, we found no apparent advantage for mixtures of predominant pre-fusion F proteins over post-fusion conformations for hMPV vaccination in opposite to recent data obtained with the human respiratory syncytial virus.

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility.

Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.

The More the Merrier: When Four Interfaces Are Better Than One.

In this issue of Structure, Zhou et al. (2019) determine the crystal and solution structures of iASPP bound to PP1. The proteins interact through a discontinuous interface defined by four contact points. Association/dissociation of individual contacts enables the transition between multiple conformations and provides mechanistic insight into how ASPPs regulate phosphorylation of p53.

Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate.

Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.

Streptomyces IHF uses multiple interfaces to bind DNA.

BACKGROUND: Nucleoid associated proteins (NAPs) are essential for chromosome condensation in bacterial cells. Despite being a diverse group, NAPs share two common traits: they are small, oligomeric proteins and their oligomeric state is critical for DNA condensation. Streptomyces coelicolor IHF (sIHF) is an actinobacterial-specific nucleoid-associated protein that despite its name, shares neither sequence nor structural homology with the well-characterized Escherichia coli IHF. Like E. coli IHF, sIHF is needed for efficient nucleoid condensation, morphological development and antibiotic production in S. coelicolor. METHODS: Using a combination of crystallography, small-angle X-ray scattering, electron microscopy and structure-guided functional assays, we characterized how sIHF binds and remodels DNA. RESULTS: The structure of sIHF bound to DNA revealed two DNA-binding elements on opposite surfaces of the helix bundle. Using structure-guided functional assays, we identified an additional surface that drives DNA binding in solution. Binding by each element is necessary for both normal development and antibiotic production in vivo, while in vitro, they act collectively to restrain negative supercoils. CONCLUSIONS: The cleft defined by the N-terminal and the helix bundle of sIHF drives DNA binding, but the two additional surfaces identified on the crystal structure are necessary to stabilize binding, remodel DNA and maintain wild-type levels of antibiotic production. We propose a model describing how the multiple DNA-binding elements enable oligomerization-independent nucleoid condensation. GENERAL SIGNIFICANCE: This work provides a new dimension to the mechanistic repertoire ascribed to bacterial NAPs and highlights the power of combining structural biology techniques to study sequence unspecific protein-DNA interactions.

Role of Era in assembly and homeostasis of the ribosomal small subunit.

Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.

Supramolecular arrangement of the full-length Zika virus NS5.

Zika virus (ZIKV), a member of the Flaviviridae family, has emerged as a major public health threat, since ZIKV infection has been connected to microcephaly and other neurological disorders. Flavivirus genome replication is driven by NS5, an RNA-dependent RNA polymerase (RdRP) that also contains a N-terminal methyltransferase domain essential for viral mRNA capping. Given its crucial roles, ZIKV NS5 has become an attractive antiviral target. Here, we have used integrated structural biology approaches to characterize the supramolecular arrangement of the full-length ZIKV NS5, highlighting the assembly and interfaces between NS5 monomers within a dimeric structure, as well as the dimer-dimer interactions to form higher order fibril-like structures. The relative orientation of each monomer within the dimer provides a model to explain the coordination between MTase and RdRP domains across neighboring NS5 molecules and mutational studies underscore the crucial role of the MTase residues Y25, K28 and K29 in NS5 dimerization. The basic residue K28 also participates in GTP binding and competition experiments indicate that NS5 dimerization is disrupted at high GTP concentrations. This competition represents a first glimpse at a molecular level explaining how dimerization might regulate the capping process.

Binding of the regulatory domain of MutL to the sliding ß-clamp is species specific.

The ß-clamp is a protein hub central to DNA replication and fork management. Proteins interacting with the ß-clamp harbor a conserved clamp-binding motif that is often found in extended regions. Therefore, clamp interactions have -almost exclusively- been studied using short peptides recapitulating the binding motif. This approach has revealed the molecular determinants that mediate the binding but cannot describe how proteins with clamp-binding motifs embedded in structured domains are recognized. The mismatch repair protein MutL has an internal clamp-binding motif, but its interaction with the ß-clamp has different roles depending on the organism. In Bacillus subtilis, the interaction stimulates the endonuclease activity of MutL and it is critical for DNA mismatch repair. Conversely, disrupting the interaction between Escherichia coli MutL and the ß-clamp only causes a mild mutator phenotype. Here, we determined the structures of the regulatory domains of E. coli and B. subtilis MutL bound to their respective ß-clamps. The structures reveal different binding modes consistent with the binding to the ß-clamp being a two-step process. Functional characterization indicates that, within the regulatory domain, only the clamp binding motif is required for the interaction between the two proteins. However, additional motifs beyond the regulatory domain may stabilize the interaction. We propose a model for the activation of the endonuclease activity of MutL in organisms lacking methyl-directed mismatch repair.

Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage.

Repair of two major forms of DNA damage, single strand breaks and base modifications, are dependent on XRCC1. XRCC1 orchestrates these repair processes by temporally and spatially coordinating interactions between several other repair proteins. Here we show that XRCC1 contains a central DNA binding domain (CDB, residues 219-415) encompassing its first BRCT domain. In contrast to the N-terminal domain of XRCC1, which has been reported to mediate damage sensing in vitro, we demonstrate that the DNA binding module identified here lacks binding specificity towards DNA containing nicks or gaps. Alanine substitution of residues within the CDB of XRCC1 disrupt DNA binding in vitro and lead to a significant reduction in XRCC1 retention at DNA damage sites without affecting initial recruitment. Interestingly, reduced retention at sites of DNA damage is associated with an increased rate of repair. These findings suggest that DNA binding activity of XRCC1 plays a significant role in retention at sites of damage and the rate at which damage is repaired.

The endonuclease domain of Bacillus subtilis MutL is functionally asymmetric.

DNA mismatch repair is an evolutionarily conserved repair pathway that corrects replication errors. In most prokaryotes and all eukaryotes, the mismatch repair protein MutL is a sequence-unspecific endonuclease that nicks the newly synthesized strand and marks it for repair. Although the sequence of the endonuclease domain of MutL is not conserved, eukaryotic MutLα and prokaryotic MutL share four conserved motifs that define the endonuclease site of the protein. Their endonuclease activity is stimulated by the processivity sliding ß-clamp, or its eukaryotic counterpart PCNA, highlighting the functional conservation. Bacterial MutL homologs form homodimers and, therefore, they have two endonuclease sites. However, eukaryotic MutL homologs associate to form heterodimers, where only one of the protomers of the dimer has endonuclease activity. To probe whether bacterial MutL needs its two endonuclease sites, we engineered variants of B. subtilis MutL harboring a single nuclease site and showed that these variants are functional nucleases. We also find that the protomer harboring the nuclease site must be able to bind to the ß-clamp to recapitulate the nicking activity of wild-type MutL. These results demonstrate the functional asymmetry of bacterial MutL and strengthen the similarities with the endonuclease activity of eukaryotic MutL homologs.