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

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

doi:10.1074/jbc.m109312200

Cited by 19 publications

(25 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Gp16.7, which has the potential to bind to both ssDNA and dsDNA, is an integral membrane protein consisting of an N-terminal membrane anchor and a C-terminal cytoplasmic domain. It localizes ϕ29 DNA replication to the membrane (Meijer et al ., 2000, 2001b; Serna-Rico et al ., 2002, 2003). The C-terminal domain of gp16.7 dimerizes in solution but forms larger filaments in the presence of ssDNA (Asensio et al ., 2005; Serna-Rico et al ., 2003).…”

Section: Protein–protein Interactions Of Bacteriophagesmentioning

confidence: 99%

Bacteriophage Protein–Protein Interactions

Häuser¹,

Blasche²,

Dokland³

et al. 2012

Advances in Virus Research

Self Cite

View full text Add to dashboard Cite

Section: Protein–protein Interactions Of Bacteriophagesmentioning

confidence: 99%

Bacteriophage Protein–Protein Interactions

Häuser¹,

Blasche²,

Dokland³

et al. 2012

Advances in Virus Research

Self Cite

View full text Add to dashboard Cite

“…Immunofluorescence studies showed that p16.7 is required for efficient spreading of 29 DNA replication from its initial to additional sites at the membrane of the infected cell (27). 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).…”

mentioning

confidence: 99%

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

et al. 2007

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…Functional studies showed that p16.7 is involved in the organization of membraneassociated 29 DNA replication and that the efficiency of in vivo 29 DNA replication is affected in its absence (9,13,14). Biochemical studies using a soluble variant lacking the N-terminal membrane anchor, p16.7A, revealed (i) that it has singlestranded DNA (ssDNA) and dsDNA binding activity, (ii) that it is a dimer in solution, and (iii) that it can form oligomers, especially upon DNA binding (13)(14)(15). The p16.7 region following the transmembrane domain (approximately residues 30 -60) forms a low affinity coiled-coil (16).…”

mentioning

confidence: 99%

“…The early expressed 29 gene 16.7 is conserved in all 29-related phages studied so far (9). Protein p16.7 (130 amino acids) is a membrane protein, and its N-terminal transmembrane domain (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] is responsible for membrane localization (13). Functional studies showed that p16.7 is involved in the organization of membraneassociated 29 DNA replication and that the efficiency of in vivo 29 DNA replication is affected in its absence (9,13,14).…”

mentioning

confidence: 99%

Structural and Functional Analysis of ϕ29 p16.7C Dimerization Mutants

Muñoz‐Espín

Fuertes

Jiménez

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage 29-encoded membrane protein p16.7 is one of the few proteins known to be involved in the organization of prokaryotic membrane-associated DNA replication. The functional DNA binding domain of p16.7 is constituted by its C-terminal half, p16.7C, which forms high affinity dimers in solution and which can form higher order oligomers. Recently, the solution and crystal structures of p16.7C and the crystal structure of the p16.7C-DNA complex have been solved. Here, we have studied the p16.7C dimerization process and the structural and functional roles of p16.7 residues Trp-116 and Asn-120 and its last nine C-terminal amino acids, which form an extended tail. The results obtained show that transition of folded dimers into unfolded monomers occurs without stable intermediates and that both Trp-116 and the C-terminal tail are important for dimerization and functionality of p16.7C. Residue Trp-116 is involved in formation of a novel aromatic cage dimerization motif, which we call "Pro cage." Finally, whereas residue Asn-120 plays a minor role in p16.7C dimerization, we show that it is critical for both oligomerization and DNA binding, providing further evidence that DNA binding and oligomerization of p16.7C are coupled processes.Compelling evidence has accumulated that DNA replication of eukaryotic and prokaryotic genomes occurs in so-called replication factories, which probably contain, besides replicative DNA polymerases, all other enzymes involved in DNA replication and related processes (1-3). Imaging techniques demonstrated that replication factories are located at rather static positions, implying that they are attached to subcellular structure(s) (reviewed in Ref. 4), which in bacteria is the cytosolic membrane (for reviews, see Refs. 5 and 6). Besides functioning as a scaffold, membrane association inherently compartmentalizes replicative complexes, allowing surface catalysis, which increases the efficiency of the DNA replication process (7,8). Little is known, however, about proteins involved in the organization of membrane-associated replication of genomes. An exception is the well studied Bacillus subtilis phage 29 (for a review, see Ref. 9). The genome of 29 consists of a linear double-stranded DNA (dsDNA) 3 of 19,285 bp that contains a terminal protein (TP) covalently linked at each 5Ј-end. Initiation of 29 DNA replication occurs via a so-called protein-primed mechanism (reviewed in Refs. 10 and 11). The first step involves recognition of the origins of replication, constituted by the TPcontaining DNA ends, by a 29-encoded DNA polymerase/TP heterodimer. After origin recognition, the DNA polymerase catalyzes the covalent attachment of the first nucleotide to the TP molecule present in the heterodimer. Then, after a transition step, these two proteins dissociate, and the DNA polymerase continues processive elongation, which is coupled to strand displacement, until replication of the nascent DNA strand is compl...

show abstract

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

Cited by 19 publications

References 30 publications

Bacteriophage Protein–Protein Interactions

Bacteriophage Protein–Protein Interactions

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

Structural and Functional Analysis of ϕ29 p16.7C Dimerization Mutants

Contact Info

Product

Resources

About