The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To ...elucidate how capsids maintain stability in an extreme environment, we use cryoelectron microscopy to determine the capsid structure of thermostable phage P74-26 to 2.8-Å resolution. We find P74-26 capsids exhibit an overall architecture very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid to expand during genome packaging, yet maintain structural stability. The P74-26 capsid has T = 7 geometry despite being twice as large as mesophilic homologs. Capsid capacity is increased with a larger, flatter major capsid protein. Given these results, we predict decreased icosahedral complexity (i.e. T ≤ 7) leads to a more stable capsid assembly.
How a DNA Polymerase Clamp Loader Opens a Sliding Clamp Kelch, Brian A.; Makino, Debora L.; O'Donnell, Mike ...
Science (American Association for the Advancement of Science),
12/2011, Letnik:
334, Številka:
6063
Journal Article
Recenzirano
Odprti dostop
Processive chromosomal replication relies on sliding DNA clamps, which are loaded onto DNA by pentameric clamp loader complexes belonging to the AAA+ family of adenosine triphosphatases (ATPases). We ...present structures for the ATP-bound state of the clamp loader complex from bacteriophage T4, bound to an open clamp and primer-template DNA. The clamp loader traps a spiral conformation of the open clamp so that both the loader and the clamp match the helical symmetry of DNA. One structure reveals that ATP has been hydrolyzed in one subunit and suggests that clamp closure and ejection of the loader involves disruption of the ATP-dependent match in symmetry. The structures explain how synergy among the loader, the clamp, and DNA can trigger ATP hydrolysis and release of the closed clamp on DNA.
DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to ...be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5′-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a “limited change/induced fit” mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.
Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and ...lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5'-3' directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.
Tail tube assembly is an essential step in the lifecycle of long-tailed bacteriophages. Limited structural and biophysical information has impeded an understanding of assembly and stability of their ...long, flexible tail tubes. The hyperthermophilic phage P74-26 is particularly intriguing as it has the longest tail of any known virus (nearly 1 μm) and is the most thermostable known phage. Here, we use structures of the P74-26 tail tube along with an in vitro system for studying tube assembly kinetics to propose the first molecular model for the tail tube assembly of long-tailed phages. Our high-resolution cryo-EM structure provides insight into how the P74-26 phage assembles through flexible loops that fit into neighboring rings through tight “ball-and-socket”-like interactions. Guided by this structure, and in combination with mutational, light scattering, and molecular dynamics simulations data, we propose a model for the assembly of conserved tube-like structures across phage and other entities possessing tail tube–like proteins. We propose that formation of a full ring promotes the adoption of a tube elongation-competent conformation among the flexible loops and their corresponding sockets, which is further stabilized by an adjacent ring. Tail assembly is controlled by the cooperative interaction of dynamic intraring and interring contacts. Given the structural conservation among tail tube proteins and tail-like structures, our model can explain the mechanism of high-fidelity assembly of long, stable tubes.
Deaminase activity mediated by the human APOBEC3 family of proteins contributes to genomic instability and cancer. APOBEC3A is by far the most active in this family and can cause rapid cell death ...when overexpressed, but in general how the activity of APOBEC3s is regulated on a molecular level is unclear. In this study, the biochemical and structural basis of APOBEC3A substrate binding and specificity is elucidated. We find that specific binding of single-stranded DNA is regulated by the cooperative dimerization of APOBEC3A. The crystal structure elucidates this homodimer as a symmetric domain swap of the N-terminal residues. This dimer interface provides insights into how cooperative protein-protein interactions may affect function in the APOBEC3 enzymes and provides a potential scaffold for strategies aimed at reducing their mutation load.
Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to ...the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are ...common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader replication factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC’s central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC’s external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.
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