RESUMEN
Accurate DNA replication and transcription elongation are crucial for preventing the accumulation of unreplicated DNA and genomic instability. Cells have evolved multiple mechanisms to deal with impaired replication fork progression, challenged by both intrinsic and extrinsic impediments. The bacterium Bacillus subtilis, which adopts multiple forms of differentiation and development, serves as an excellent model system for studying the pathways required to cope with replication stress to preserve genomic stability. This review focuses on the genetics, single molecule choreography, and biochemical properties of the proteins that act to circumvent the replicative arrest allowing the resumption of DNA synthesis. The RecA recombinase, its mediators (RecO, RecR, and RadA/Sms) and modulators (RecF, RecX, RarA, RecU, RecD2, and PcrA), repair licensing (DisA), fork remodelers (RuvAB, RecG, RecD2, RadA/Sms, and PriA), Holliday junction resolvase (RecU), nucleases (RnhC and DinG), and translesion synthesis DNA polymerases (PolY1 and PolY2) are key functions required to overcome a replication stress, provided that the fork does not collapse.
Asunto(s)
Bacillus subtilis , Proteínas de Escherichia coli , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Replicación del ADN/genética , ADN/metabolismo , Proteínas de Escherichia coli/genéticaRESUMEN
DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA-ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth.
Asunto(s)
Proteínas Bacterianas , Rec A Recombinasas , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Rec A Recombinasas/metabolismoRESUMEN
DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN/fisiología , Resolvasas de Unión Holliday/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Rotura Cromosómica , ADN Bacteriano/metabolismo , ADN Cruciforme/metabolismo , Proteínas de Unión al ADN/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Escherichia coli/genética , Magnesio/metabolismoRESUMEN
Virus infection causes major rearrangements in the subcellular architecture of eukaryotes, but its impact in prokaryotic cells was much less characterized. Here, we show that infection of the bacterium Bacillus subtilis by bacteriophage SPP1 leads to a hijacking of host replication proteins to assemble hybrid viral-bacterial replisomes for SPP1 genome replication. Their biosynthetic activity doubles the cell total DNA content within 15 min. Replisomes operate at several independent locations within a single viral DNA focus positioned asymmetrically in the cell. This large nucleoprotein complex is a self-contained compartment whose boundaries are delimited neither by a membrane nor by a protein cage. Later during infection, SPP1 procapsids localize at the periphery of the viral DNA compartment for genome packaging. The resulting DNA-filled capsids do not remain associated to the DNA transactions compartment. They bind to phage tails to build infectious particles that are stored in warehouse compartments spatially independent from the viral DNA. Free SPP1 structural proteins are recruited to the dynamic phage-induced compartments following an order that recapitulates the viral particle assembly pathway. These findings show that bacteriophages restructure the crowded host cytoplasm to confine at different cellular locations the sequential processes that are essential for their multiplication.
Asunto(s)
Bacillus subtilis/virología , Compartimento Celular , Virosis/patología , Bacillus subtilis/ultraestructura , Bacteriófagos/fisiología , Bacteriófagos/ultraestructura , Cápside/metabolismo , Replicación del ADN , ADN Viral/biosíntesis , ADN Polimerasa Dirigida por ADN , Interacciones Huésped-Patógeno , Complejos Multienzimáticos , Factores de Tiempo , Virión/metabolismoRESUMEN
Changes in the location of γ-tubulin ensure cell survival and preserve genome integrity. We investigated whether the nuclear accumulation of γ-tubulin facilitates the transport of proliferating cell nuclear antigen (PCNA) between the cytosolic and the nuclear compartment in mammalian cells. We found that the γ-tubulin meshwork assists in the recruitment of PCNA to chromatin. Also, decreased levels of γ-tubulin reduce the nuclear pool of PCNA. In addition, the γ-tubulin C terminus encodes a PCNA-interacting peptide (PIP) motif, and a γ-tubulin-PIP-mutant affects the nuclear accumulation of PCNA. In a cell-free system, PCNA and γ-tubulin formed a complex. In tumors, there is a significant positive correlation between TUBG1 and PCNA expression. Thus, we report a novel mechanism that constitutes the basis for tumor growth by which the γ-tubulin meshwork maintains indefinite proliferation by acting as an opportune scaffold for the transport of PCNA from the cytosol to the chromatin.
Asunto(s)
Cromatina/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Tubulina (Proteína)/fisiología , Ciclo Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Humanos , Transporte de Proteínas , Origen de RéplicaRESUMEN
The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3', 5'-diadenosine monophosphate (cdi-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Replicación del ADN/fisiología , ADN/metabolismo , Bacillus subtilis/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genéticaRESUMEN
Natural chromosomal transformation (CT) plays a major role in prokaryote evolution, yet factors that govern the integration of DNA from related species remain poorly understood. We show that in naturally competent Bacillus subtilis cells the acquisition of homeologous sequences is governed by sequence divergence (SD). Integration initiates in a minimal efficient processing segment via homology-directed CT, and its frequency decreases log-linearly with increased SD up to 15%. Beyond this and up to 23% SD the interspecies boundaries prevail, the CT frequency marginally decreases, and short (<10-nucleotides) segments are integrated via homology-facilitated micro-homologous integration. Both mechanisms are RecA dependent. We identify the other recombination proteins required for the acquisition of homeologous DNA. The absence of AddAB, RecF, RecO, RuvAB or RecU, crucial for repair-by-recombination, did not affect CT. However, dprA, radA, recJ, recX or recD2 inactivation strongly decreased intraspecies and interspecies CT. Interspecies CT was not detected beyond ~8% SD in ΔdprA, ~10% in ΔrecJ, ΔradA, ΔrecX and ~14% in ΔrecD2 cells. We propose that DprA, RecX, RadA/Sms, RecJ and RecD2 accessory proteins are important for the generation of genetic diversity. Together with RecA, they facilitate gene acquisition from bacteria of related species.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/genética , ADN Bacteriano/genética , Recombinación Genética , Bacillus subtilis/enzimología , Proteínas Bacterianas/genética , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismoRESUMEN
Ubiquitous RarA AAA+ ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of ΔrecA, ΔrecO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H2O2- or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (ΔaddAB, ΔrecJ, ΔrecQ, ΔrecS) or branch migration (ΔruvAB, ΔrecG, ΔradA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When ΔrarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in ΔrarA ΔrecU and ΔrarA ΔrecX double mutant cells, and was blocked in ΔrarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly.
RESUMEN
A proteolyzed bacteriophage (phage) might release its DNA into the environment. Here, we define the recombination functions required to resurrect an infective lytic phage from inactive environmental viral DNA in naturally competent Bacillus subtilis cells. Using phage SPP1 DNA, a model that accounts for the obtained data is proposed (i) the DNA uptake apparatus takes up environmental SPP1 DNA, fragments it, and incorporates into the cytosol different linear single-stranded (ss) DNA molecules shorter than genome-length; (ii) the SsbA-DprA mediator loads RecA onto any fragmented linear SPP1 ssDNA, but negative modulators (RecX and RecU) promote a net RecA disassembly from these ssDNAs not homologous to the host genome; (iii) single strand annealing (SSA) proteins, DprA and RecO, anneal the SsbA- or SsbB-coated complementary strands, yielding tailed SPP1 duplex intermediates; (iv) RecA polymerized on these tailed intermediates invades a homologous region in another incomplete molecule, and in concert with RecD2 helicase, reconstitutes a complete linear phage genome with redundant regions at the ends of the molecule; and (v) DprA, RecO or viral G35P SSA, may catalyze the annealing of these terminally redundant regions, alone or with the help of an exonuclease, to produce a circular unit-length duplex viral genome ready to initiate replication.
Asunto(s)
Bacillus subtilis/genética , Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/genética , ADN Viral/genética , Recombinación Genética/genética , Bacillus subtilis/virología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Rec A Recombinasas/genéticaRESUMEN
Bacterial RarA is thought to play crucial roles in the cellular response to blocked replication forks. We show that lack of Bacillus subtilis RarA renders cells very sensitive to H2O2, but not to methyl methane sulfonate or 4-nitroquinoline-1-oxide. RarA is epistatic to RecA in response to DNA damage. Inactivation of rarA partially suppressed the DNA repair defect of mutants lacking translesion synthesis polymerases. RarA may contribute to error-prone DNA repair as judged by the reduced frequency of rifampicin-resistant mutants in ΔrarA and in ΔpolY1 ΔrarA cells. The absence of RarA strongly reduced the viability of dnaD23ts and dnaB37ts cells upon partial thermal inactivation, suggesting that ΔrarA cells are deficient in replication fork assembly. A ΔrarA mutation also partially reduced the viability of dnaC30ts and dnaX51ts cells and slightly improved the viability of dnaG40ts cells at semi-permissive temperature. These results suggest that RarA links re-initiation of DNA replication with repair-by-recombination by controlling the access of the replication machinery to a collapsed replication fork.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Reparación del ADN/genética , Replicación del ADN/genética , Recombinación Genética , Bacillus subtilis/efectos de los fármacos , Daño del ADN , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Mutagénesis , Rec A Recombinasas/metabolismo , Recombinación Genética/efectos de los fármacosRESUMEN
RarA is a widely conserved protein proposed to be involved in recombination-dependent replication. We present a cell biological approach to identify functional connections between RarA and other proteins using single molecule tracking. We found that 50% of RarA molecules were static, mostly close to replication forks and likely DNA-bound, while the remaining fraction was highly dynamic throughout the cells. RarA alternated between static and dynamic states. Exposure to H2O2 increased the fraction of dynamic molecules, but not treatment with mitomycin C or with methyl methanesulfonate, which was exacerbated by the absence of RecJ, RecD2, RecS and RecU proteins. The ratio between static and dynamic RarA also changed in replication temperature-sensitive mutants, but in opposite manners, dependent upon inhibition of DnaB or of DnaC (pre)primosomal proteins, revealing an intricate function related to DNA replication restart. RarA likely acts in the context of collapsed replication forks, as well as in conjunction with a network of proteins that affect the activity of the RecA recombinase. Our novel approach reveals intricate interactions of RarA, and is widely applicable for in vivo protein studies, to underpin genetic or biochemical connections, and is especially helpful for investigating proteins whose absence does not lead to any detectable phenotype.
Asunto(s)
Adenosina Trifosfatasas/genética , Bacillus subtilis/genética , ADN Helicasas/genética , Reparación del ADN/genética , Replicación del ADN/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Metilmetanosulfonato/farmacología , Mitomicina/farmacología , Rec A Recombinasas/genética , Imagen Individual de MoléculaRESUMEN
Bacillus subtilis bacteriophage SPP1 is a lytic siphovirus first described 50 years ago [1]. Its complete DNA sequence was reported in 1997 [2]. Here we present an updated annotation of the 44,016 bp SPP1 genome and its correlation to different steps of the viral multiplication process. Five early polycistronic transcriptional units encode phage DNA replication proteins and lysis functions together with less characterized, mostly non-essential, functions. Late transcription drives synthesis of proteins necessary for SPP1 viral particles assembly and for cell lysis, together with a short set of proteins of unknown function. The extensive genetic, biochemical and structural biology studies on the molecular mechanisms of SPP1 DNA replication and phage particle assembly rendered it a model system for tailed phages research. We propose SPP1 as the reference species for a new SPP1-like viruses genus of the Siphoviridae family.
Asunto(s)
Fagos de Bacillus/genética , Bacillus subtilis/virología , Genoma Viral , Replicación del ADN , ADN Viral/genética , Evolución Molecular , Genes Virales , Transcripción Genética , Ensamble de Virus/genéticaRESUMEN
The ubiquitous RarA/Mgs1/WRNIP protein plays a crucial, but poorly understood role in genome maintenance. We show that Bacillus subtilis RarA, in the apo form, preferentially binds single-stranded (ss) over double-stranded (ds) DNA. SsbA bound to ssDNA loads RarA, and for such recruitment the amphipathic C-terminal domain of SsbA is required. RarA is a DNA-dependent ATPase strongly stimulated by ssDNA-dsDNA junctions and SsbA, or by dsDNA ends. RarA, which may interact with PriA, does not stimulate PriA DNA unwinding. In a reconstituted PriA-dependent DNA replication system, RarA inhibited initiation, but not chain elongation. The RarA effect was not observed in the absence of SsbA, or when the host-encoded preprimosome and the DNA helicase are replaced by proteins from the SPP1 phage with similar function. We propose that RarA assembles at blocked forks to maintain genome integrity. Through its interaction with SsbA and with a preprimosomal component, RarA might impede the assembly of the replicative helicase, to prevent that recombination intermediates contribute to pathological DNA replication restart.
Asunto(s)
Adenosina Trifosfatasas/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Genoma Bacteriano/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , Especificidad por SustratoRESUMEN
The histone-like protein HU is a conserved nucleoid-associated protein that is involved in the maintenance of the bacterial chromosome architecture. It is the only known nucleoid-associated protein in Streptococcus pneumoniae, but it has not been studied. The pneumococcal gene encoding this protein, hlp, is shown herein to be essential for cell viability. Its disruption was only possible either when it was duplicated in the chromosome and its expression induced from the P Zn promoter, or when hlp was cloned into a plasmid under the control of the inducible P mal promoter. In vitro assays indicated that pneumococcal HU shows a preference for binding to supercoiled DNA rather than to linear or nicked DNA. In vivo experiments in which the amount of HU was manipulated showed a relationship between the amount of HU and the level of DNA supercoiling. A twofold reduction in the amount of HU triggered a 21% increase in DNA relaxation in untreated cells. However, in cells treated with novobiocin, a drug that relaxes DNA by inhibiting DNA gyrase, a 35% increase in DNA relaxation was observed, instead of the expected 20% in cells with a constitutive HU amount. Conversely, a fourfold HU increase caused only 14% of DNA relaxation in the presence of novobiocin. Taken together, these results support an essential role for HU in the maintenance of DNA supercoiling in S. pneumoniae.
RESUMEN
Natural transformation and viral-mediated transduction are the main avenues of horizontal gene transfer in Firmicutes. Bacillus subtilis SPP1 is a generalized transducing bacteriophage. Using this lytic phage as a model, we have analyzed how viral replication and recombination systems contribute to the transfer of plasmid-borne antibiotic resistances. Phage SPP1 DNA replication relies on essential phage-encoded replisome organizer (G38P), helicase loader (G39P), hexameric replicative helicase (G40P), recombinase (G35P) and in less extent on the partially dispensable 5'â3' exonuclease (G34.1P), the single-stranded DNA binding protein (G36P) and the Holliday junction resolvase (G44P). Correspondingly, the accumulation of linear concatemeric plasmid DNA, and the formation of transducing particles were blocked in the absence of G35P, G38P, G39P, and G40P, greatly reduced in the G34.1P, G36P mutants, and slightly reduced in G44P mutants. In contrast, establishment of injected linear plasmid DNA in the recipient host was independent of viral-encoded functions. DNA homology between SPP1 and the plasmid, rather than a viral packaging signal, enhanced the accumulation of packagable plasmid DNA. The transfer efficiency was also dependent on plasmid copy number, and rolling-circle plasmids were encapsidated at higher frequencies than theta-type replicating plasmids.
RESUMEN
The mechanisms that allow to circumvent replicative stress, and to resume DNA synthesis are poorly understood in Bacillus subtilis. To study the role of the diadenylate cyclase DisA and branch migration translocase (BMT) RadA/Sms in restarting a stalled replication fork, we nicked and broke the circular chromosome of an inert mature haploid spore, damaged the bases, and measured survival of reviving spores. During undisturbed ripening, nicks and breaks should be repaired by pathways that do not invoke long-range end resection or genetic exchange by homologous recombination, after which DNA replication might be initiated. We found that DNA damage reduced the viability of spores that lacked DisA, BMT (RadA/Sms, RuvAB or RecG), the Holliday junction resolvase RecU, or the translesion synthesis DNA polymerases (PolY1 or PolY2). DisA and RadA/Sms, in concert with RuvAB, RecG, RecU, PolY1 or PolY2, are needed to bypass replication-blocking lesions. DisA, which binds to stalled or reversed forks, did not apparently affect initiation of PriA-dependent DNA replication in vitro. We propose that DisA is necessary to coordinate responses to replicative stress; it could help to circumvent damaged template bases that otherwise impede fork progression.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Esporas Bacterianas/enzimología , Bacillus subtilis/fisiología , Daño del ADN , Replicación del ADN , ADN Bacteriano/metabolismo , Esporas Bacterianas/fisiologíaRESUMEN
Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA 'initiation primer' on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Bacteriófagos/genética , ADN Polimerasa III/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Bacillus subtilis/virología , Bacteriófagos/metabolismo , Secuencia de Bases , ADN Primasa/metabolismo , ADN de Cadena Simple/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Agar , Unión Proteica , Origen de Réplica/genéticaRESUMEN
DNA-single strand annealing proteins (SSAPs) are recombinases frequently encoded in the genome of many bacteriophages. As SSAPs can promote homologous recombination among DNA substrates with an important degree of divergence, these enzymes are involved both in DNA repair and in the generation of phage mosaicisms. Here, analysing Sak and Sak4 as representatives of two different families of SSAPs present in phages infecting the clinically relevant bacterium Staphylococcus aureus, we demonstrate for the first time that these enzymes are absolutely required for phage reproduction. Deletion of the genes encoding these enzymes significantly reduced phage replication and the generation of infectious particles. Complementation studies revealed that these enzymes are required both in the donor (after prophage induction) and in the recipient strain (for infection). Moreover, our results indicated that to perform their function SSAPs require the activity of their cognate single strand binding (Ssb) proteins. Mutational studies demonstrated that the Ssb proteins are also required for phage replication, both in the donor and recipient strain. In summary, our results expand the functions attributed to the Sak and Sak4 proteins, and demonstrate that both SSAPs and Ssb proteins are essential for the life cycle of temperate staphylococcal phages.
Asunto(s)
Bacteriófagos/fisiología , Recombinasas/fisiología , Staphylococcus aureus/virología , Proteínas Virales/fisiología , Replicación Viral , Replicación del ADN , Proteínas de Unión al ADN/fisiología , MutaciónRESUMEN
UNLABELLED: Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. IMPORTANCE: Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.