The integral membrane protein p16.7 organizes in vivophi29 DNA replication through interaction with both the terminal protein and ssDNA

Serna-Rico, Alejandro; Muñoz‐Espín, Daniel; Villar, Laurentino; Salas, Margarita; Meijer, Wilfried J. J.

doi:10.1093/emboj/cdg221

Cited by 19 publications

(34 citation statements)

References 31 publications

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“…Structural Basis for p16.7C Oligomerization-Recent studies have shown that p16.7 multimerization is a prominent feature in its DNA binding mode (11). Here we have demonstrated that p16.7C retains the capacity to form multimers, implying that the C-terminal half of p16.7 contains one or more regions responsible for p16.7C dimer-dimer interaction.…”

Section: The Functional Domain Of Protein P167 Can Form Multimers-gelmentioning

confidence: 59%

“…First, it binds nonspecifically to both ss-and dsDNA (10 -12). Second, p16.7 can form multimers, both in vitro and in vivo, and it has been shown that multimerization is crucial for its DNA binding mode (11). Third, it has affinity for the 29 terminal protein.…”

Section: Discussionmentioning

confidence: 99%

“…Labeled fragments were either used directly (dsDNA) or after heat-denaturation (ssDNA). Micrococcal nuclease digestions of the ssDNA nucleoprotein complexes were performed as described before (11). DNase I reactions contained, in 20 l, besides the end-labeled dsDNA fragment and the indicated amount of protein, 25 mM Tris-HCl (pH 7.5) and 10 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

“…After complex formation, samples were treated with micrococcus nuclease (B) or DNase I (C) as described under "Experimental Procedures," and the DNA products fractionated through polyacrylamide gels under denaturing conditions. The digestion pattern observed in B is the consequence of preferential cleavage by micrococcal nuclease at AT-rich regions (11,39). Digestion of the ssDNA fragment without protein during a shorter period of time or using lower micrococcus nuclease concentrations gave patterns highly similar to those obtained in the presence of limited amounts of p16.7Cb.…”

Section: The Functional Domain Of Protein P167 Can Form Multimers-gelmentioning

confidence: 99%

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Structure of the Functional Domain of φ29 Replication Organizer

Asensio

Albert

Muñoz‐Espín

et al. 2005

Journal of Biological Chemistry

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The Bacillus subtilis phage 29-encoded membrane protein p16.7 is one of the few proteins involved in prokaryotic membrane-associated DNA replication that has been characterized at a functional and biochemical level. In this work we have determined both the solution and crystal structures of its dimeric functional domain, p16.7C. Although the secondary structure of p16.7C is remarkably similar to that of the DNA binding homeodomain, present in proteins belonging to a large family of eukaryotic transcription factors, the tertiary structures of p16.7C and homeodomains are fundamentally different. In fact, p16.7C defines a novel dimeric six-helical fold. We also show that p16.7C can form multimers in solution and that this feature is a key factor for efficient DNA binding. Moreover, a combination of NMR and xray approaches, combined with functional analyses of mutants, revealed that multimerization of p16.7C dimers is mediated by a large protein surface that is characterized by a striking self-complementarity. Finally, the structural analyses of the p16.7C dimer and oligomers provide important clues about how protein multimerization and DNA binding are coupled.Despite extensive studies on DNA replication, relatively little is known about its in vivo organization, which, in prokaryotes, occurs at the cell membrane (1-4). The well studied Bacillus subtilis phage 29 (5) is one of the few systems for which this fundamental process has been investigated. The genome of 29 consists of a linear double-stranded DNA (dsDNA) 1 that contains a terminal protein covalently linked at each 5Ј-end. Initiation of 29 DNA replication, as well as that of most other linear genomes containing a terminal protein attached to its DNA ends, occurs via a so-called protein-primed mechanism (5-7). The 29 genome encodes most, if not all, proteins required for phage DNA replication making 29 an attractive system to study membrane-associated DNA replication.The early-expressed 29 gene 16.7, which is conserved in all 29-related phages studied so far, encodes a well characterized membrane protein, p16.7 (130 amino acids), that plays an important role in membrane-associated 29 DNA replication (5,8,9). It contains an N-terminal transmembrane domain which is responsible for membrane localization (9) (a schematic organization of protein p16.7 is shown in Fig. 1A). Analyses of a soluble variant lacking the N-terminal membrane anchor, p16.7A, revealed that it has affinity for both single-stranded (ss) and dsDNA, as well as for the 29 terminal protein. Moreover, p16.7A, which is a dimer in solution, can form multimers, especially upon DNA binding, and multimerization is important for the mode by which it binds DNA (9 -11). Recently, it was shown that the dimerization and DNA-binding activities of p16.7 are confined to the C-terminal half of the protein, p16.7C (12).Here, we show that p16.7C is able to form multimers in solution and that this process is enhanced in the presence of DNA. To gain insight into the multiple features and functions of p16.7C we ...

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Section: The Functional Domain Of Protein P167 Can Form Multimers-gelmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: The Functional Domain Of Protein P167 Can Form Multimers-gelmentioning

confidence: 99%

See 2 more Smart Citations

Structure of the Functional Domain of φ29 Replication Organizer

Asensio

Albert

Muñoz‐Espín

et al. 2005

Journal of Biological Chemistry

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“…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).…”

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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

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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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Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low‐Affinity Ligands and Enzymes

2020

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Directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology. That progress is heavily dependent on the robustness and flexibility of the available selection platforms, given the significant cost to (re)develop a given platform to target a new desired function. Bacterial cell display has a significant track record as a viable strategy for the engineering of mesophilic enzymes, as enzyme activity can be probed directly and free from interference from the cellular milieu, but its adoption has lagged behind other display-based methods. Herein, we report the development of SNAP as a quantitative reporter for bacterial cell display, which enables fast troubleshooting and the systematic development of the display-based selection platform, thus improving its robustness. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed for the specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes, thus making it a highly flexible platform for these biochemical functions. Together, this establishes bacterial display as a robust and flexible platform, ideally suited for the systematic engineering of ligands and enzymes needed for XNA molecular biology.

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The integral membrane protein p16.7 organizes in vivophi29 DNA replication through interaction with both the terminal protein and ssDNA

Cited by 19 publications

References 31 publications

Structure of the Functional Domain of φ29 Replication Organizer

Structure of the Functional Domain of φ29 Replication Organizer

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

Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low‐Affinity Ligands and Enzymes

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