RESUMEN
Fundamental aspects of DNA replication, such as the anatomy of replication stall sites, how replisomes are influenced by gene transcription, and whether the progression of sister replisomes is coordinated, are poorly understood. Available techniques do not allow the precise mapping of the positions of individual replisomes on chromatin. We have developed a method called Replicon-seq that entails the excision of full-length replicons by controlled nuclease cleavage at replication forks. Replicons are sequenced using Nanopore, which provides a single-molecule readout of long DNA. Using Replicon-seq, we found that sister replisomes function autonomously and yet progress through chromatin with remarkable consistency. Replication forks that encounter obstacles pause for a short duration but rapidly resume synthesis. The helicase Rrm3 plays a critical role both in mitigating the effect of protein barriers and with facilitating efficient termination. Replicon-seq provides a high-resolution means of defining how individual replisomes move across the genome.
Asunto(s)
ADN Helicasas , Replicación del ADN , Cromatina/genética , Cromosomas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismoRESUMEN
When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Proteínas de Xenopus/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , ADN/química , ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitinación , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
The convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the convergence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indicating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efficient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These findings identify a eukaryotic pathway for DNA replication termination that is distinct from previously characterized prokaryotic mechanisms.
Asunto(s)
ADN Helicasas/genética , Replicación del ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , ADN-Topoisomerasas/genética , Escherichia coli/genética , Eucariontes/genética , Inestabilidad Genómica , Plásmidos/genéticaRESUMEN
The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin-RING ubiquitin ligase CUL-2LRR-1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC-48 "unfoldase". Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome-associated TIMELESS-TIPIN complex is required for CUL-2LRR-1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS-TIPIN, CUL-2LRR-1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM-7 subunit of CMG. Subsequently, the UBXN-3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4. We show that UBXN-3 is important in vivo for replisome disassembly in the absence of TIMELESS-TIPIN. Correspondingly, co-depletion of UBXN-3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN-3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Complejos Multienzimáticos/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Mutaciones Letales SintéticasRESUMEN
Anomalies in dismantling the machinery of DNA replication can compromise genome integrity and contribute to tumorigenesis and aging. In this issue of Genes & Development, Dewar and colleagues (pp. 275-290) identified an E3 ubiquitin ligase, CUL2LRR2, that modifies a subunit of the replicative CMG (Cdc45, minichromosome maintenance [MCM] subunits 2-7, and the GINS complex) helicase and triggers disassembly of the replication machinery. Their study offers critical insight into the mechanism of DNA replication termination while at the same time raising important questions for future research.
Asunto(s)
ADN Helicasas/genética , Replicación del ADN , Proteínas de Ciclo Celular/genética , Humanos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its Mcm7 subunit and the action of the p97 ATPase. Using a proteomic screen in Xenopus egg extracts, we identified factors that are enriched on chromatin when CMG unloading is blocked. This approach identified the E3 ubiquitin ligase CRL2Lrr1, a specific p97 complex, other potential regulators of termination, and many replisome components. We show that Mcm7 ubiquitylation and CRL2Lrr1 binding to chromatin are temporally linked and occur only during replication termination. In the absence of CRL2Lrr1, Mcm7 is not ubiquitylated, CMG unloading is inhibited, and a large subcomplex of the vertebrate replisome that includes DNA Pol ε is retained on DNA. Our data identify CRL2Lrr1 as a master regulator of replisome disassembly during vertebrate DNA replication termination.
Asunto(s)
Cromatina/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , ADN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Adenosina Trifosfatasas/metabolismo , Animales , Cromatina/genética , ADN Polimerasa II/metabolismo , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitinación , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMEN
Protein-mediated "chromosome kissing" between two DNA sites in trans (or in cis) is known to facilitate three-dimensional control of gene expression and DNA replication. However, the mechanisms of regulation of the long-range interactions are unknown. Here, we show that the replication terminator protein Fob1 of Saccharomyces cerevisiae promoted chromosome kissing that initiated rDNA recombination and controlled the replicative life span (RLS). Oligomerization of Fob1 caused synaptic (kissing) interactions between pairs of terminator (Ter) sites that initiated recombination in rDNA. Fob1 oligomerization and Ter-Ter kissing were regulated by intramolecular inhibitory interactions between the C-terminal domain (C-Fob1) and the N-terminal domain (N-Fob1). Phosphomimetic substitutions of specific residues of C-Fob1 counteracted the inhibitory interaction. A mutation in either N-Fob1 that blocked Fob1 oligomerization or C-Fob1 that blocked its phosphorylation antagonized chromosome kissing and recombination and enhanced the RLS. The results provide novel insights into a mechanism of regulation of Fob1-mediated chromosome kissing.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , Replicación del ADN/genética , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Mutación , Fosforilación , Estructura Terciaria de Proteína , Recombinación Genética/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the "replication fork trap" region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the nonpermissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15 to 24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.
Asunto(s)
Replicación del ADN , ADN Bacteriano , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli , ADN Bacteriano/biosíntesis , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a "replication fork trap" - found within a chromosomal region where forks are allowed to enter but not leave - is set by the protein-DNA roadblock Tus-Ter. The exact sequence of events by which Tus-Ter blocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein-protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus-Ter interactions at the nonpermissive face - interactions that result in a highly stable "locked" complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus-Ter mechanism - leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA-protein system further.
Asunto(s)
Replicación del ADN , ADN Bacteriano/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Bacterias/química , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/química , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mapas de Interacción de ProteínasRESUMEN
Reb1 ofSchizosaccharomyces pomberepresents a family of multifunctional proteins that bind to specific terminator sites (Ter) and cause polar termination of transcription catalyzed by RNA polymerase I (pol I) and arrest of replication forks approaching the Ter sites from the opposite direction. However, it remains to be investigated whether the same mechanism causes arrest of both DNA transactions. Here, we present the structure of Reb1 as a complex with a Ter site at a resolution of 2.7 Å. Structure-guided molecular genetic analyses revealed that it has distinct and well-defined DNA binding and transcription termination (TTD) domains. The region of the protein involved in replication termination is distinct from the TTD. Mechanistically, the data support the conclusion that transcription termination is not caused by just high affinity Reb1-Ter protein-DNA interactions. Rather, protein-protein interactions between the TTD with the Rpa12 subunit of RNA pol I seem to be an integral part of the mechanism. This conclusion is further supported by the observation that double mutations in TTD that abolished its interaction with Rpa12 also greatly reduced transcription termination thereby revealing a conduit for functional communications between RNA pol I and the terminator protein.
Asunto(s)
ADN de Hongos/química , Proteínas de Unión al ADN/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Regiones Terminadoras Genéticas , Factores de Transcripción/química , Terminación de la Transcripción Genética , Cristalografía por Rayos X , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa I/química , ARN Polimerasa I/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Replication forks in both prokaryotic and eukaryotic systems pause at random sites due to depletion of dNTP pools, DNA damage, tight binding nonhistone proteins or unusual DNA sequences and/or structures, in a mostly non-polar fashion. However, there is also physiologically programmed replication termination at sequence-specific authentic replication termini. Here, the structure and functions of programmed replication termini, their mechanism of action and their diverse physiological functions in prokaryotes and eukaryotes have been reviewed.
Asunto(s)
Replicación del ADN , Sustitución de Aminoácidos , Animales , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , ADN Bacteriano/genética , Silenciador del Gen , Humanos , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched substrates in vitro. Although initially associated with homologous recombination and DNA repair, studies of cells lacking RecG over the past 25 years have led to the suggestion that the protein might be multi-functional and associated with a number of additional cellular processes, including initiation of origin-independent DNA replication, the rescue of stalled or damaged replication forks, replication restart, stationary phase or stress-induced 'adaptive' mutations and most recently, naïve adaptation in CRISPR-Cas immunity. Here we discuss the possibility that many of the phenotypes of recG mutant cells that have led to this conclusion may stem from a single defect, namely the failure to prevent re-replication of the chromosome. We also present data indicating that this failure does indeed contribute substantially to the much-reduced recovery of recombinants in conjugational crosses with strains lacking both RecG and the RuvABC Holliday junction resolvase.
Asunto(s)
ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cromosomas Bacterianos , Daño del ADN , Replicación del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genotipo , Recombinación Homóloga , MutaciónRESUMEN
The eukaryotic replicative helicase (CMG complex) is assembled during DNA replication initiation in a highly regulated manner, which is described in depth by other manuscripts in this Issue. During DNA replication, the replicative helicase moves through the chromatin, unwinding DNA and facilitating nascent DNA synthesis by polymerases. Once the duplication of a replicon is complete, the CMG helicase and the remaining components of the replisome need to be removed from the chromatin. Research carried out over the last ten years has produced a breakthrough in our understanding, revealing that replication termination, and more specifically replisome disassembly, is indeed a highly regulated process. This review brings together our current understanding of these processes and highlights elements of the mechanism that are conserved or have undergone divergence throughout evolution. Finally, we discuss events beyond the classic termination of DNA replication in S-phase and go over the known mechanisms of replicative helicase removal from chromatin in these particular situations.
RESUMEN
Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetics, genomics, and transmission electron microscopy, we find that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs; Sen1 specifically acts at telomeres. rrm3 and sen1 genetically interact and fail to terminate replication, exhibiting fragility at termination zones (TERs) and telomeres. sen1rrm3 accumulates RNA-DNA hybrids and X-shaped gapped or reversed converging forks at TERs; sen1, but not rrm3, builds up RNA polymerase II (RNPII) at TERs and telomeres. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of Top1 and Top2 when forks encounter transcription head on or codirectionally, respectively, thus preventing the slowing down of DNA and RNA polymerases. Hence Rrm3 and Sen1 are indispensable to generate permissive topological conditions for replication termination.
Asunto(s)
ADN Helicasas , ARN Helicasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Topological stress can cause converging replication forks to stall during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome fork stalling, suggesting that alternative mechanisms of termination exist. Using proteomics in Xenopus egg extracts, we show that the helicase RTEL1 and the replisome protein MCM10 are highly enriched on chromatin during fork convergence and are crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 cooperate to promote fork convergence and do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 play a general role in promoting progression of stalled forks, including when forks stall during termination. Our data reveal an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.
Asunto(s)
Cromatina , Replicación del ADN , Animales , ADN/metabolismo , Xenopus laevisRESUMEN
A variety of replication fork traps have recently been characterised in Enterobacterales, unveiling two different types of architecture. Of these, the degenerate type II fork traps are commonly found in Enterobacteriaceae such as Escherichia coli. The newly characterised type I fork traps are found almost exclusively outside Enterobacteriaceae within Enterobacterales and include several archetypes of possible ancestral architectures. Dickeya paradisiaca harbours a somewhat degenerate type I fork trap with a unique Ter1 adjacent to tus gene on one side of the circular chromosome and three putative Ter2-4 sites on the other side of the fork trap. The two innermost Ter1 and Ter2 sites are only separated by 18 kb, which is the shortest distance between two innermost Ter sites of any chromosomal fork trap identified so far. Of note, the dif site is located between these two sites, coinciding with a sharp GC-skew flip. Here we examined and compared the binding modalities of E. coli and D. paradisiaca Tus proteins for these Ter sites. Surprisingly, while Ter1-3 were functional, no significant Tus binding was observed for Ter4 even in low salt conditions, which is in stark contrast with the significant non-specific protein-DNA interactions that occur with E. coli Tus. Even more surprising was the finding that D. paradisiaca Tus has a relatively moderate binding affinity to double-stranded Ter while retaining an extremely high affinity to Ter-lock sequences. Our data revealed major differences in the salt resistance and stability between the D. paradisiaca and E. coli Tus protein complexes, suggesting that while Tus protein evolution can be quite flexible regarding the initial Ter binding step, it requires a highly stringent purifying selection for its final locked complex formation.
Asunto(s)
Replicación del ADN , Dickeya/metabolismo , Proteínas de Escherichia coli , Escherichia coli , Cromosomas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.
RESUMEN
In preparation for cell division, the genome must be copied with high fidelity. However, replisomes often encounter obstacles, including bulky DNA lesions caused by reactive metabolites and chemotherapeutics, as well as stable nucleoprotein complexes. Here, we discuss recent advances in our understanding of TRAIP, a replisome-associated E3 ubiquitin ligase that is mutated in microcephalic primordial dwarfism. In interphase, TRAIP helps replisomes overcome DNA interstrand crosslinks and DNA-protein crosslinks, whereas in mitosis it triggers disassembly of all replisomes that remain on chromatin. We describe a model to explain how TRAIP performs these disparate functions and how they help maintain genome integrity.
Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , Mitosis , Ubiquitina-Proteína Ligasas/metabolismo , Animales , ADN Helicasas/química , Humanos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Xenopus laevisRESUMEN
Bacillus thuringiensis serovar israelensis is the most widely used natural biopesticide against mosquito larvae worldwide. Its lineage has been actively studied and a plasmid-free strain, B. thuringiensis serovar israelensis BGSC 4Q7 (4Q7), has been produced. Previous sequencing of the genome of this strain has revealed the persistent presence of a 235 kb extrachromosomal element, pBtic235, which has been shown to be an inducible prophage, although three putative chromosomal prophages have been lost. Moreover, a 492 kb region, potentially including the standard replication terminus, has also been deleted in the 4Q7 strain, indicating an absence of essential genes in this area. We reanalysed the genome coverage distribution of reads for the previously sequenced variant strain, and sequenced two independently maintained samples of the 4Q7 strain. A 553 kb area, close to the 492 kb deletion, was found to be duplicated. This duplication presumably restored the equal sizes of the replichores, and a balanced functioning of replication termination. An analysis of genome assembly graphs revealed a transient association of the host chromosome with the pBtic235 element. This association may play a functional role in the replication of the bacterial chromosome, and the termination of this process in particular. The genome-restructuring events detected may modify the genetic status of cytotoxic or haemolytic toxins, potentially influencing strain virulence. Twelve of the single-nucleotide variants identified in 4Q7 were probably due to the procedure used for strain construction or were present in the precursor of this strain. No sequence variants were found in pBtic235, but the distribution of the corresponding 4Q7 reads indicates a significant difference from counterparts in natural B. thuringiensis serovar israelensis strains, suggesting a duplication or over-replication in 4Q7. Thus, the 4Q7 strain is not a pure plasmid-less offshoot, but a highly genetically modified derivative of its natural ancestor. In addition to potentially influencing virulence, genome-restructuring events can modify the replication termination machinery. These findings have potential implications for the conclusions of virulence studies on 4Q7 as a model, but they also raise interesting fundamental questions about the functioning of the Bacillus genome.
Asunto(s)
Bacillus thuringiensis/genética , Secuencias Invertidas Repetidas , Secuenciación Completa del Genoma/métodos , Bacillus thuringiensis/clasificación , Cromosomas Bacterianos/genética , Replicación del ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Plásmidos/genética , Profagos/genética , Selección Genética , SerogrupoRESUMEN
Protoplasts of Enterococcus faecalis did not divide but enlarged in Difco Marine Broth containing penicillin. Our previous studies have demonstrated that transcription and translation were essential for bacterial cell enlargement. However, it was uncertain whether replication was also essential. In this study, we measured the amount of DNA in E. faecalis cells during the course of enlargement using quantitative polymerase chain reaction. The growth of normally divided cells (native forms) of E. faecalis exhibited a log phase before 6 h of incubation was reached. Although a difference in quantitation cycle (Cq) values between the replication initiation and termination regions was observed in the log phase, it was not present in the stationary growth phase. On the other hand, the amount of DNA in E. faecalis protoplasts increased during the cell enlargement incubation. The difference of Cq values between the protoplasts at 0 and 96 h of incubation was 8-9, indicating that the DNA amount at 96 h was 200-500 times higher than that at 0 h. The Cq values differed between the replication initiation and termination regions, indicating that the replication level was high. When novobiocin, a DNA replication inhibitor, was added to the medium at 24 h of incubation, DNA replication and cell enlargement were almost stopped. Thus, replication plays an important role in the enlargement of E. faecalis protoplasts.