Dissecting the role of the φ29 terminal protein DNA binding residues in viral DNA replication

Published by Oxford University Press on behalf of Nucleic Acids Research. Phage φ29 DNA replication takes place by a proteinpriming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the φ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the Km for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the φ29 double-stranded DNA binding protein p6 in the reaction, which decreased the Km of the DNA polymerase for dATP about 130-190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification.Spanish Ministry of Economy and Competitiveness [BFU2011–23645] ; Consolider-Ingenio from the Spanish 2800 Nucleic Acids Research, 2015, Vol. 43, No. 5 Ministry of Science and Innovation [CSD2007–00015 to M.S.] ; Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular‘Severo Ochoa’. I.H. was holder of an FPU fellowship from the Spanish Ministry of Education. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2011- 23645]Peer Reviewe

Estudio del dominio de unión a DNA de la proteína terminal del bacteriófago [phi]29 y su papel en la replicación del DNA viral

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-07-2015El mecanismo de replicación iniciada con proteína terminal (TP) del bacteriófago ϕ29 ha sido extensamente estudiado in vitro. Sin embargo, se conoce muy poco sobre la organización espacial y temporal de la replicación de ϕ29 in vivo. En la presente Tesis se ha estudiado la localización subcelular de los principales componentes de la maquinaria replicativa del bacteriófago ϕ29, es decir, la TP y la DNA polimerasa viral. Tanto la TP iniciadora como la TP parental localizan en el nucleoide bacteriano en ausencia de otros componentes virales. Por otra parte, la localización de la DNA polimerasa viral tiene lugar a lo largo de toda la longitud celular cuando es expresada de manera individual, pero localiza en el nucleoide bacteriano cuando es coexpresada junto con la TP. Por tanto, la expresión de la TP determina la localización de la DNA polimerasa viral en el nucleoide bacteriano. Durante el proceso infectivo, ambas proteínas colocalizan con el nucleoide bacteriano, siguiendo su dinámica de segregación. En etapas intermedias del ciclo celular tanto la TP como la DNA polimerasa viral exhiben un patrón de localización tipo helicoidal en células infectadas, patrón que depende de la proteína del citoesqueleto tipo actina de Bacillus subtilis MreB. Además, se ha determinado que el dominio N-terminal de la TP es el responsable de su localización en el nucleoide bacteriano, mostrando que la presencia de este dominio es esencial para que se dé una replicación eficiente del DNA viral durante la infección. Trabajos anteriores determinaron que el dominio N-terminal de la TP de ϕ29 tiene capacidad de unión a DNA in vitro. En la presente Tesis se han estudiado los residuos aminoacídicos del dominio N-terminal de la TP implicados en su unión a DNA. Asimismo, se ha determinado que la TP de ϕ29 se une al DNA genómico de B. subtilis in vivo, encontrándose una correlación entre la capacidad de unión a DNA y la localización en el nucleoide bacteriano. La resolución de la estructura cristalográfica del heterodímero formado por la DNA polimerasa y la TP no permitió determinar la estructura del dominio N-terminal de la TP, al encontrarse desordenado en el cristal. En la presente Tesis se ha analizado la estructura secundaria del dominio N-terminal mediante dicroísmo circular, mostrando que tiene un alto contenido en α-hélice. Por otra parte, se ha estudiado el papel que la unión a DNA de la TP puede tener en la replicación del DNA viral in vitro mediante la caracterización bioquímica de TPs mutantes en residuos básicos del dominio N-terminal, encontrándose que los mutantes defectivos en unión a DNA están afectados en diferentes etapas de este proceso. La proteína viral de unión a DNA de doble banda p6 compensa los defectos de iniciación y transición de estas TPs mutantes. Además, se ha determinado que el dominio N-terminal de la TP es necesario para una unión eficiente a la DNA polimerasa, así como para la amplificación del TP-DNA in vitro. En conjunto, estos resultados nos permiten proponer un papel del dominio N-terminal de la TP en el reconocimiento y apertura del origen de replicación.The protein-priming mechanism of bacteriophage ϕ29 DNA replication has been extensively studied in vitro. However, little is known about the spatial and temporal organization of the ϕ29 replication process in vivo. In this Thesis the subcellular localization of the main components of the bacteriophage ϕ29 replicative machinery has been studied, i.e., the terminal protein (TP) and the viral DNA polymerase. Both the primer and parental TP localize at the bacterial nucleoid in the absence of other viral components. On the other hand, the DNA polymerase localizes along the cellular length when it is expressed individually, but it localizes at the bacterial nucleoid when it is co-expressed together with TP. Therefore, TP expression determines DNA polymerase localization at the bacterial nucleoid. During infection, both proteins colocalize at the bacterial nucleoid following its segregation dynamics. At middle stages of the cell cycle both the TP and the DNA polymerase display a helix-like localization pattern in infected cells, this pattern depending on the Bacillus subtilis actin-like cytoskeleton protein MreB. Furthermore, it has been determined that the TP N-terminal domain is responsible for its nucleoid localization, being the presence of this domain essential for an efficient viral DNA replication during the infective process. Previous works have shown that the TP N-terminal domain possesses DNA binding capacity in vitro. In this Thesis the TP N-terminal domain amino acidic residues involved in DNA binding have been studied. Additionally, it has been determined that ϕ29 TP binds B. subtilis genomic DNA in vivo, finding a correlation between DNA binding capacity and nucleoid localization. The resolution of the crystallographic structure of the heterodimer formed by the DNA polymerase and the TP could not solve the structure of the TP N-terminal domain, as it was disordered in the crystal lattice. In the present work the secondary structure of the TP N-terminal domain has been analysed by circular dichroism, showing that it has a high proportion of α-helix. Additionally, by means of the biochemical characterisation of mutant TPs in basic residues of the N-terminal domain, the possible role of TP DNA binding capacity in viral DNA replication in vitro has been studied. TP mutants defective in DNA binding are affected in performing different stages of viral DNA replication. The viral double-stranded DNA binding protein p6 overcomes the initiation and transition defects of these mutant TPs. In addition, it has been determined that the TP N-terminal domain is necessary for an efficient binding to the DNA polymerase as well as for TP-DNA amplification in vitro. On the whole, these results allow us to propose a role of the TP N-terminal domain in the recognition and unwinding of the replication origi

DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5′ ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3′–5′ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the TP at the bacterial nucleoid, where viral DNA replication takes place. The biochemical properties of the Φ29 DBP and SSB and their function in the initiation and elongation of Φ29 DNA replication, respectively, will be described.This work has been supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2014-52656-P to MS) and (BFU2014-53791-P to MV), ComFuturo Grant from Fundación General CSIC (to MR) and by an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa.”Peer reviewedPeer Reviewe

Nuclear localization signals in phage terminal proteins provide a novel gene delivery tool in mammalian cells

Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. Unexpectedly, we have found functional eukaryotic nuclear localization signals (NLSs) within the TP sequences of bacteriophages from diverse families and hosts. Given the role of bacteriophages as vehicles for horizontal gene transfer (HGT), we postulated that viral genomes that have covalently linked NLS-containing terminal proteins might behave as vectors for HGT between bacteria and the eukaryotic nucleus. To validate this hypothesis, we profited from the in vitro Φ29 amplification system that allows the amplification of heterologous DNAs producing linear molecules of DNA with TP covalently attached to both 5' ends. Interestingly, these in vitrogenerated TP-DNA molecules showed enhanced gene delivery in mammalian cells, supporting a possible role in HGT by transferring genes between prokaryotes and eukaryotes. Moreover, these TP-DNA molecules are a useful tool to amplify and subsequently deliver genes efficiently into the eukaryotic nucleus. Here, we suggest various possible applications and further developments of the technique with biotechnological and therapeutic purposesThis work was supported by Grant BFU 2011-23645 and Consolider-Ingenio Grant 2010 24717 from the Spanish Ministry of Economy and Competitiveness (MINECO) and by an institutional grant from Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa.

Neuronal specification in space and time

International audienceTo understand how neurons assemble to form functional circuits, it is necessary to obtain a detailed knowledge of their diversity and to define the developmental specification programs that give rise to this diversity. Invertebrates and vertebrates appear to share common developmental principles of neuronal specification in which cascades of transcription factors temporally pattern progenitors, while spatial cues modify the outcomes of this temporal patterning. Here, we highlight these conserved mechanisms and describe how they are used in distinct neural structures. We present the questions that remain for a better understanding of neuronal specification. Single-cell RNA profiling approaches will potentially shed light on these questions, allowing not only the characterization of neuronal diversity in adult brains, but also the investigation of the developmental trajectories leading to the generation and maintenance of this diversity

Disclosing the in vivo organization of a viral histone-like protein in Bacillus subtilis mediated by its capacity to recognize the viral genome

Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage φ29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the φ29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA,which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria.Spanish Ministry of Science and Innovation; Spanish Ministries of Education; Fundación Ramón ArecesPeer Reviewe

Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages

A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of ο29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of ο29 TP led us to identify a bona fide NLS within residues 1-37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of ο29 TP attached to the 5′ DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes.Spanish Ministry of Economy and Competitiveness; Fundación Ramón Areces; Spanish Ministry of EducationPeer Reviewe

Uso de señales de localización nuclear de proteínas de bacteriófagos como vehículo para transferencia de genes

[EN] The present invention relates to the use, in medicine, of a terminal protein, preferably ϕ29, Nf, Cp-1, Bam35 or PRD1, of a bacteriophage virus of the family Podoviridae or Tectiviridae with protein-primed DNA replication, characterized in that said protein comprises, first, at least one nuclear localization amino acid sequence, i.e. an amino acid sequence with NLS function, when said protein is expressed in mammalian cells, and, second, at least one amino acid sequence for covalent bonding to the ends of the DNA. The present invention is used for cases in which an exogenous DNA is introduced into the nucleus of cells of a mammal, so that said DNA may have a therapeutic effect. The present invention may also be used for the introduction of DNA fragments into human cells for the expression of heterologous genes for use in gene therapy[ES] La presente invención hace referencia al uso en medicina de una proteína terminal de un virus bacteriófago con replicación de ADN cebada por proteína, preferentemente ϕ29, Nf, Cp-1, Bam35 o PRD1, pertenecientes a la familia Podoviridae o Tectiviridae, caracterizada por que dicha proteína comprende en primer lugar, al menos una secuencia aminoacídica de localización al núcleo celular, es decir con función NLS, cuando dicha proteína se expresa en células de mamífero, y en segundo lugar, al menos una secuencia aminoacídica de unión covalente a los extremos de ADN. La presente invención se aplica a aquellos casos en los que un ADN exógeno se introduce en el núcleo de células de un mamífero, con objeto de que dicho ADN lleve a cabo un efecto terapéutico. Otra aplicación de la presente invención es para la introducción de fragmentos de ADN en células humanas para llevar a cabo la expresión de genes heterólogos para su uso en terapias génicasPeer reviewedConsejo Superior de Investigaciones Científicas, Universidad Autónoma de MadridA2 Solicitud de patente sin informe sobre el estado de la técnic

Phage ϕ29 protein p1 promotes replication by associating with the FtsZ ring of the divisome in Bacillus subtilis

During evolution, viruses have optimized the interaction with host factors to increase the efficiency of fundamental processes such as DNA replication. Bacteriophage ϕ29 protein p1 is a membrane-associated protein that forms large protofilament sheets that resemble eukaryotic tubulin and bacterial filamenting temperature-sensitive mutant Z protein (FtsZ) polymers. In the absence of protein p1, phage ϕ29 DNA replication is impaired. Here we show that a functional fusion of protein p1 to YFP localizes at the medial region of Bacillus subtilis cells independently of other phage-encoded proteins. We also show that ϕ29 protein p1 colocalizes with the B. subtilis cell division protein FtsZ and provide evidence that FtsZ and protein p1 are associated. Importantly, the midcell localization of YFP-p1 was disrupted in a strain that does not express FtsZ, and the fluorescent signal was distributed all over the cell. Depletion of penicillin-binding protein 2B (PBP2B) in B. subtilis cells did not affect the subcellular localization of YFP-p1, indicating that its distribution does not depend on septal wall synthesis. Interestingly, when ϕ29 protein p1 was expressed, B. subtilis cells were about 1.5-fold longer than control cells, and the accumulation of ϕ29 DNA was higher in mutant B. subtilis cells with increased length. We discuss the biological role of p1 and FtsZ in the ϕ29 growth cycle.