The Putative Coiled Coil Domain of the φ29 Terminal Protein Is a Major Determinant Involved in Recognition of the Origin of Replication

Serna-Rico, Alejandro; Illana, Belén; Salas, Margarita; Meijer, Wilfried J. J.

doi:10.1074/jbc.m007855200

Cited by 24 publications

(25 citation statements)

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“…Once both ϕ29 TP and DNA polymerase (shown in cyan) are synthesized, they form a heterodimer that associates with the bacterial nucleoid through the N-terminal DNA-binding domain of the priming TP (shown in red) (Fig. 5B) and recognizes the replication origins at both DNA ends by means of specific interactions with the parental TP (14,15). It seems likely that the association of the ϕ29 TP-DNA to a specific bacterial compartment also might help stabilize the interaction with the heterodimeric complex.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…5B). Gp3 is known to interact with versatile molecules other than the DNA polymerase or DNA: in addition to gp1, gp16.7 (viral gene 16.7 product), cellular membrane, and gp3 itself were reported to interact with gp3 (Bravo and Salas, 1997;Bravo et al, 2000;Serna-Rico et al, 2000. In the gp3 variants, interactions of gp3 with these components could be affected.…”

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confidence: 99%

“…First, interaction between the protein primer (gp3 associated with the DNA polymerase) and the terminal protein (gp3 covalently bound to the ϕ29 DNA) could be affected. This interaction is considered to be important for the initiation of ϕ29 DNA replication (Serna-Rico et al, 2000). Alternatively, subcellular localization of gp3 variants could be affected.…”

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Functional linkages between replication proteins of genes 1, 3 and 5 of <i>Bacillus subtilis</i> phage <b>φ</b>29

2012

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Gene 1 product (gp1) of Bacillus subtilis phage ϕ29 has been shown to be involved in viral DNA replication in vivo, but the essential role is still unknown. As part of an ongoing effort to understand the role of gp1 in viral DNA replication, we investigated genetic interaction between gene 1 and other viral genes. Because ϕ29 mutants which do not produce functional gp1 show temperaturesensitive growth, we isolated temperature-resistant phages from the ϕ29 gene 1 mutants, and eventually, obtained nine extragenic suppressors. These suppressor mutations were located in two essential genes for ϕ29 DNA replication in vivo: gene 3 encoding terminal/primer protein (gp3) or gene 5 encoding viral singlestranded DNA binding protein (gp5). Most of these mutations resulted in single amino acid substitutions in the products. By trans-complementation assay, we confirmed that the absence of gp1 at non-permissive temperature can be compensated by the suppressors which have the single amino acid substitution in either gp5 or gp3. These results indicate that gp1 has functional relationship to gp5 and gp3. From the positions of amino acid substitutions in gp3, we propose its new regulatory subdomain at which other molecules including gp1 would interact with and regulate functions of gp3.

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“…Glycerol Gradients-Twenty g of protein p16.7A was subjected to a 15-30% linear glycerol gradient containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 7 mM ␤-mercaptoethanol and run as described before (14). After fractionation of the gradient, aliquots of each fraction were analyzed by SDS-PAGE and gel retardation assays.…”

Section: Methodsmentioning

confidence: 99%

“…29 Protein-primed Initiation, TP-DNA Replication, and Amplification Assays-These assays were performed as described before (14). The reaction mixtures of the 29 TP-DNA replication assays contained the indicated amount of protein p16.7A or SSB p5.…”

Section: Methodsmentioning

confidence: 99%

The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

Serna-Rico¹,

Salas

Meijer³

2002

Journal of Biological Chemistry

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The functional role of the 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA Studies on DNA replication and related processes have provided detailed insights in the function of many proteins involved in these processes (for review, see Ref. 1). Despite this, little is known about the in vivo organization of DNA replication. To gain a better insight in this fundamental process, we studied the in vivo DNA replication of the Bacillus subtilis bacteriophage 29 (2). The detailed knowledge of its in vitro mechanism of DNA replication (for reviews, see Refs. 3 and 4) made 29 an attractive system for this study.The genome of 29 is a linear double-stranded DNA (dsDNA) 1 of 19,285 base pairs that contains a terminal protein (TP) covalently linked at each 5Ј end. Fig. 1A shows a schematic representation of the genetic and transcriptional organization of the 29 genome. Regulation of 29 DNA transcription, which can be divided into an early and a late stage, has been studied extensively in vivo as well as in vitro (for reviews, see Refs. 3 and 5). The late expressed genes, all transcribed from a single operon present in the central part of the genome, encode the structural proteins of the phage, proteins involved in morphogenesis, and those required for lysis of the infected cell. The early expressed genes are present in two operons that flank the late operon. The early operon located at the left side of the 29 genome encodes the transcriptional regulator protein p4 and various proteins that are directly involved in phage DNA replication, such as the DNA polymerase, TP, singlestranded DNA-binding protein (SSB), double-stranded DNAbinding protein, and protein p1. The operon located at the right side of the 29 genome encodes, in addition to proteins p17 and p16.7, four putative proteins of unknown function.A schematic overview of the in vitro 29 DNA replication mechanism is shown in Fig. 1B. Initiation of 29 DNA replication occurs via a so-called protein-primed mechanism (reviewed in Refs. 3, 4, and 6). The TP-containing DNA ends constitute the origins of replication. Initiation of DNA replication starts by recognition of the origin by a heterodimer formed by the 29 DNA polymerase and the primer TP. The DNA polymerase then catalyzes the addition of the first dAMP to the primer TP. Next, after a transition step, these two proteins dissociate, and the DNA polymerase continues processive elongation until replication of the nascent DNA strand is completed. Replication, which starts at both DNA ends, is coupled to strand displacement. This results in the generation of socalled type I replication intermediates consisting of full-length double-stranded 29 DNA molecules with one or more singlestranded DNA (ssDNA) branches of varying lengths. When the two converging DNA...

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The Putative Coiled Coil Domain of the φ29 Terminal Protein Is a Major Determinant Involved in Recognition of the Origin of Replication

Cited by 24 publications

References 35 publications

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

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

Functional linkages between replication proteins of genes 1, 3 and 5 of <i>Bacillus subtilis</i> phage <b>φ</b>29

The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

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