Structure of the Functional Domain of φ29 Replication Organizer

Asensio, Juan Luis; Albert, Armando; Muñoz‐Espín, Daniel; González, Carlos; Hermoso, J.A.; Villar, Laurentino; Jiménez‐Barbero, Jesús; Salas, Margarita; Meijer, Wilfried J. J.

doi:10.1074/jbc.m501687200

Cited by 9 publications

(20 citation statements)

References 35 publications

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“…Extensive evidence exists that p16.7 is responsible for attaching replicating 29 DNA to the membrane of infected cells by binding directly to phage dsDNA (24)(25)(26)(27). The observation that a GFP fusion of the 29 membrane protein p16.7 (p16.7-GFP) displayed a helical localization pattern in non-infected cells suggested that p16.7 might interact with the cytoskeleton.…”

Section: Discussionmentioning

confidence: 99%

“…The right-side early operon contains gene 16.7 that encodes a membrane protein (p16.7) required for optimal in vivo 29 DNA replication (23,24). Additional functional, biochemical and structural studies have provided strong evidence that p16.7 is responsible for attaching 29 DNA to the bacterial membrane (24)(25)(26). Crystallographic resolution of the p16.7 DNA binding domain (p16.7C) in complex with dsDNA revealed that 1 dsDNA binding unit is formed by 3 p16.7C dimers that are arranged in such a way that they form a deep positively charged longitudinal cavity that interacts with the phosphate backbone of dsDNA (27).…”

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The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria

Muñoz‐Espín

Daniel

Kawai

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage 29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the 29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the 29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.Bacillus subtilis ͉ phage 29 G enes of the mreB family encode homologues of eukaryotic actin (1, 2) that form a cytoskeleton in most non-spherical bacteria (3-6). MreB proteins form filamentous structures following a helical path around the inner surface of the cytoplasmic membrane (1). These actin-like filaments are continuously remodelled during cell-cycle progression (7-11). Evidence is accumulating that the bacterial MreB cytoskeleton plays key roles in several important cellular processes such as cell shape determination, chromosome segregation, and cell polarity (1,3,8,(12)(13)(14)(15)(16). Whereas Gram-negative bacteria have a single mreB gene, Gram-positive bacteria often have multiple mreB homologues. Bacillus subtilis encodes 3 MreB isoforms: MreB, Mbl,.For decades, evidence has been provided that replication of phage DNA, like that of other prokaryotic genomes, occurs at the cytoplasmic membrane (for review see 20). However, little is known about the proteins or their organization in membraneassociated replication of viral genomes in bacteria. Phages 29 and SPP1 infect the Gram-positive bacterium B. subtilis, and phage PRD1 infects the Gram-negative bacterium Escherichia coli. Whereas PRD1 and 29 use the protein-primed mechanism of DNA replication, phage SPP1 replicates its DNA initially via the theta mode and later via a rolling circle mode [reviewed in (21)]. Here we show a key role for the MreB cytoskeleton in phages replicating by different modes in the distantly related bacteria E. coli and B. subtilis. Thus, the efficiency of replication of phage PRD1, and that of phages SPP1 and 29, is severely affected in the absence of an intact cytoskeleton.The underlying mechanism by which the cytoskeleton leads to efficient phage DNA replication was analyzed in detail for B. subtilis phage 29, whose DNA replication has been well characterized in vitro. The 29 genome consists of a linear doublestranded DNA (dsDNA) with a terminal protein (TP) covalently linked at each 5Ј end that is the primer for the initiation of phage DNA replication. Hence, initiation of 29 DNA replication occurs via a so-called protein-prim...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria

Muñoz‐Espín

Daniel

Kawai

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Gel retardation and DNase I footprinting assays were performed essentially as described (30,31). A 297-bp DNA fragment corresponding to the ϕ29 right end was amplified by PCR using genomic ϕ29 DNA as template and primers R-25 and R-OUT SUPER.…”

Section: Methodsmentioning

confidence: 99%

Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

Muñoz‐Espín

Holguera

Ballesteros-Plaza

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.Bacillus subtilis | phage ø29 | DNA polymerase | DNA-binding | bacterial cytoskeleton P rotein-primed DNA replication is a mechanism used to initiate DNA synthesis in a variety of prokaryotic and eukaryotic organisms (1). Phages such as ϕ29 (infecting Bacillus subtilis) and PRD1 (infecting Escherichia coli) (1, 2), animal viruses such as adenoviruses (1, 3), bacterial species from the Streptomyces genus (1, 4), and viruses infecting Archaea (5-7) possess replication origins at their linear chromosomes constituted by inverted terminal repetitions with a terminal protein (TP) linked to both 5′ genome ends. TPs also have been reported in linear plasmids isolated from bacteria, yeast, fungi, and higher plants (1) and in animal and plant RNA viruses (1).The development of an in vitro replication system with purified proteins and DNA from the B. subtilis phage ϕ29 laid the foundations for investigating the protein-primed mechanism of DNA replication and makes ϕ29 a paradigm for studying this process (1, 2). A schematic overview of the in vitro ϕ29 DNA replication mechanism is shown in Fig. S1. The ϕ29 genome consists of a 19,285-bp linear dsDNA, with a TP of 31 kDa (the parental TP) covalently linked to each 5′ end. DNA replication starts with the recognition of the TP-containing DNA ends by a heterodimer formed by the ϕ29 DNA polymerase and a free TP molecule (the primer TP) (8). The DNA polymerase then catalyzes the formation of a covalent bond between deoxyAMP and the hydroxyl group Ser 232 of the primer TP. Replication is coupled to strand displacement, and continuous elongation of the DNA polymerase from both DNA ends generates replication intermediates that finally converge in the complete duplication of the parental strands (reviewed in ref.2). Phage ϕ29 DNA transcription is divided into early and late stages (9). Fig. S1 shows a genetic and a transcriptional map. Genes 2 and 3, encoding phage DNA polymerase and TP, respectively, are located i...

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“…In vitro analyses of a soluble p16.7 derivative lacking the membrane anchor revealed that (i) it is a dimer with unspecific DNA binding activity, (ii) it has affinity for the 29 terminal protein, and (iii) it is able to form higher-order multimers upon DNA binding (36,37). The solution and crystal structures of the dimeric functional C-terminal half of p16.7, p16.7C (residues 63 to 130) (32), as well as the crystal structure of p16.7C complexed with double-stranded DNA have been recently solved (1,2). Based on its properties we considered the possibility that p16.7 has a role in the pull step of DNA ejection.…”

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The Phage φ29 Membrane Protein p16.7, Involved in DNA Replication, Is Required for Efficient Ejection of the Viral Genome

et al. 2007

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It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of Bacillus subtilis bacteriophage 29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step ϳ65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection.Despite being a key step in the phage life cycle, the process whereby phages eject their genome into the cell cytoplasm during the early steps of infection has been one of the major unsolved problems. The current understanding of the phage DNA ejection process has recently been reviewed by Molineux (29). The ejection process of phages that infect gram-negative bacteria is now beginning to be understood at a detailed molecular level (24,30,33). This process is far from being a passive mechanism, just driven by the release of the pressure built inside the capsid. The cell cytoplasm has a pressure of several atmospheres with respect to the external medium. Therefore, the pressure-based mechanism would be responsible for ejection of only part of the phage genome; i.e., ejection of phage DNA by pressure will stop when the decreasing packing forces in the mature virion equal those inside the cytoplasm. Thus, internalization of the remaining part of the phage genome requires additional mechanisms, and diverse strategies have been described for different phages. For instance, some Escherichia coli phages eject their genomes into the cell largely through enzyme-catalyzed, energy-requiring processes. Phage T5 DNA ejection takes place in two steps (23). In the first step, 8% of the genome enters the cytoplasm. Then, there is a pause of about 4 min during which two proteins encoded by this DNA fragment, A1 and A2, are synthesized. The transfer of the remaining DNA (92%) takes place in a second step only if these proteins are synthesized. A2 is a DNA binding protein, thought to pull DNA into the cell (39). Phage T7 is the only one for which it has been clearly demonstrated that DNA internalization is coupled to transcription (reviewed in reference 29). About 850 bp of the left end of the T7 genome are ejected into the host (11). A molecular motor formed by viral proteins gp16 and gp15 has been proposed to control the amount of DNA that enters the cell (21). Transcription from promoters located on this short DNA sequence by the host RNA polymerase provides the force to internalize about 20% of the genome into the cell. T7 RNA polymerase is the...

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Structure of the Functional Domain of φ29 Replication Organizer

Cited by 9 publications

References 35 publications

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria

Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

The Phage φ29 Membrane Protein p16.7, Involved in DNA Replication, Is Required for Efficient Ejection of the Viral Genome

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