RESUMEN
Prokaryotes have evolved intricate innate immune systems against phage infection1-7. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB8. GajA functions as a DNA endonuclease that is inactive in the presence of ATP9. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg2+. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.
Asunto(s)
Bacillus cereus , Proteínas Bacterianas , Bacteriófagos , Microscopía por Crioelectrón , Inmunidad Innata , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/ultraestructura , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Apoproteínas/química , Apoproteínas/inmunología , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Bacteriófagos/inmunología , ADN/metabolismo , ADN/química , División del ADN , Magnesio/química , Magnesio/metabolismo , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Viabilidad Microbiana , Bacillus cereus/química , Bacillus cereus/inmunología , Bacillus cereus/metabolismo , Bacillus cereus/ultraestructura , Estructura Cuaternaria de Proteína , ADN Primasa/química , ADN Primasa/metabolismo , ADN Primasa/ultraestructura , ADN-Topoisomerasas/química , ADN-Topoisomerasas/metabolismo , ADN-Topoisomerasas/ultraestructuraRESUMEN
Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN21,2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand3 and TPP1 (in complex with TIN24) recruiting telomerase via interaction with telomerase reverse transcriptase5 (TERT). The telomere DNA ends are replicated and maintained by telomerase6, for the G-strand, and subsequently DNA polymerase α-primase7,8 (PolαPrim), for the C-strand9. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN110-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.
Asunto(s)
ADN Primasa , Complejo Shelterina , Telomerasa , Tetrahymena , Microscopía por Crioelectrón , ADN/genética , ADN/metabolismo , ADN Primasa/química , ADN Primasa/metabolismo , ADN Primasa/ultraestructura , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestructura , Unión Proteica , Complejo Shelterina/química , Complejo Shelterina/metabolismo , Complejo Shelterina/ultraestructura , Telomerasa/química , Telomerasa/metabolismo , Telomerasa/ultraestructura , Telómero/genética , Telómero/metabolismo , Tetrahymena/química , Tetrahymena/enzimología , Tetrahymena/metabolismo , Tetrahymena/ultraestructuraRESUMEN
Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.
Asunto(s)
ADN Primasa/metabolismo , ADN Primasa/fisiología , Cartilla de ADN/química , Animales , Sitios de Unión , ADN , ADN Primasa/ultraestructura , Cartilla de ADN/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Nucleótidos , Conformación Proteica , Elementos Estructurales de las Proteínas/fisiologíaRESUMEN
The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.
Asunto(s)
Bacteriófago T7/genética , ADN Primasa/genética , Replicación del ADN , ADN Viral/genética , Bacteriófago T7/metabolismo , Bacteriófago T7/ultraestructura , ADN/biosíntesis , ADN/genética , ADN Primasa/metabolismo , ADN Primasa/ultraestructura , ADN Viral/metabolismo , ADN Viral/ultraestructura , Cinética , Imagen Individual de Molécula/métodos , Imagen de Lapso de Tiempo/métodosRESUMEN
DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.
Asunto(s)
Proteínas Bacterianas/química , ADN Helicasas/química , ADN Primasa/química , Adenosina Difosfato/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Bacillus cereus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , ADN/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , ADN Primasa/genética , ADN Primasa/metabolismo , ADN Primasa/ultraestructura , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleótidos/metabolismo , Modelos Moleculares , Mutación , Estructura Terciaria de ProteínaRESUMEN
The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA-DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA-DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.
Asunto(s)
ADN Polimerasa I/química , ADN Polimerasa I/ultraestructura , ADN Primasa/química , ADN Primasa/ultraestructura , Secuencia de Aminoácidos , ADN Polimerasa I/metabolismo , ADN Primasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Subunidades de Proteína/química , ARN/química , Saccharomyces cerevisiae/enzimologíaRESUMEN
Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence of either dTTP or beta,gamma-methylene dTTP whereas the ratio between hexamers and heptamers is nearly the converse in the presence of dTDP. When formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di- and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio.
Asunto(s)
Bacteriófago T7/enzimología , ADN Primasa/química , ADN Primasa/metabolismo , Arginina/metabolismo , ADN Primasa/ultraestructura , ADN de Cadena Simple/metabolismo , Histidina/metabolismo , Hidrólisis , Modelos Biológicos , Nucleótidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade. Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode. The transient nature of the helicase-primase (DnaB-DnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function. Taking advantage of the stable DnaB-DnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that connects the N- and C-terminal domains of DnaB. Furthermore, atomic force microscopy (AFM) imaging experiments show that binding of the primase to the helicase induces predominantly a 3-fold symmetric morphology to the hexameric ring. Overall, three DnaG molecules appear to interact with the hexameric ring helicase but a small number of complexes with two and even one DnaG molecule bound to DnaB were also detected. The structural/functional significance of these data is discussed and a speculative structural model for this complex is suggested.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Primasa/metabolismo , Geobacillus stearothermophilus/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/ultraestructura , ADN Primasa/ultraestructura , Replicación del ADN , AdnB Helicasas , Geobacillus stearothermophilus/genética , Microscopía de Fuerza Atómica , Modelos Moleculares , Mutación/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Gene 4 of bacteriophage T7 encodes a protein (gp4) that can translocate along single-stranded DNA, couple the unwinding of duplex DNA with the hydrolysis of dTTP, and catalyze the synthesis of short RNA oligoribonucleotides for use as primers by T7 DNA polymerase. Electron microscopic studies have shown that gp4 forms hexameric rings, and X-ray crystal structures of the gp4 helicase domain and of the highly homologous RNA polymerase domain of Escherichia coli DnaG have been determined. Earlier biochemical studies have shown that when single-stranded DNA is bound to the hexameric ring, the primase domain remains accessible to free DNA. Given these results, a model was suggested in which the primase active site in the gp4 hexamer is located on the outside of the hexameric ring. We have used electron microscopy and single-particle image analysis to examine T7 gp4, and have determined that the primase active site is located on the outside of the hexameric ring, and therefore provide direct structural support for this model.
Asunto(s)
Bacteriófago T4/enzimología , ADN Primasa/química , ADN Primasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , ADN Primasa/ultraestructura , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Escherichia coli/enzimología , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.