Role of the Dna/Membrane Complex in Prokaryotic Dna Replication

Firshein, William

doi:10.1146/annurev.mi.43.100189.000513

Cited by 88 publications

(58 citation statements)

References 91 publications

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“…Initiation of chromosomal DNA replication involves a number of DNA-membrane interactions (10,11,14). The nature of many of these has been defined by using minichromosomes, episomes that incorporate the DNA base sequence of the chromosomal origin of replication, oriC.pGO46 is one such episome, constructed by insertion of an oriC sequence at the pUC9 cloning site (11).…”

Section: Resultsmentioning

confidence: 99%

Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli.

Rosen

Orman

Rakita

et al. 1990

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

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Neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host defense functions. The mechanisms of action of most of these systems are incompletely understood. The present report indicates that microbicidal activity by a neutrophil-derived antimicrobial system, consisting of myeloperoxidase, enzymatically generated hydrogen peroxide, and chloride ion, is accompanied by prompt cessation of DNA synthesis in Escherichia coli, as determined by markedly reduced incorporation of [3H]thymidine into trichloracetic acid-precipitable material. Simultaneously, the myeloperoxidase system mediates a decline in the ability of E. coli membranes to bind hemimethylated DNA sequences containing the E. coli chromosomal origin of replication (oriC). Binding of oriC to the E. coli membrane is an essential element of orderly chromosomal DNA replication. Comparable early changes in DNA synthesis and DNAmembrane interactions were not observed with alternative oxidant or antibiotic-mediated microbicidal systems. It is proposed that oxidants generated by the myeloperoxidase system modify theE. coli membrane in such a fashion that oriC binding is markedly impaired. As a consequence chromosomal DNA replication is impaired and organisms can no longer replicate.Human neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host-defense functions. Among these is one comprised of myeloperoxidase, hydrogen peroxide, and an oxidizable halide cofactor, usually chloride. The best characterized microbicide of the system is the hypohalous acid, HOCI, which gives rise to additional, derivative microbicidal oxidants, most notably chloramines (1,2). Characterization of the pathways by which loss of viability is effected by these oxidants remains an area of active investigation.Biochemical lesions induced by HOCI-in amounts just sufficient to cause loss of bacterial viability-are largely confined to the cell envelope. Membrane-associated functions that are inactivated in Escherichia coli include loss of FoF1 ATPase activity (3), succinate-dependent respiration (4, 5), nutrient transport (3, 6), and modification of proteins involved in remodeling the bacterial cell wall during growth and division (R.M.R. and H.R., unpublished observations). In contrast, cytosolic proteins with HOCI vulnerable sites, such as the sulfhydryl-containing enzyme aldolase, are oxidized in intact E. coli only by exposure to amounts of HOCI far in excess of those required to produce loss of viability (7,8). Thus, it appears that injuries to cell envelope structures precede those to cytosolic structures and that the surface injuries are sufficient to effect loss of viability.McKenna and Davies recently reported prompt cessation of DNA synthesis in E. coli incubated with low concentrations of HOCI (9). This inhibition was observed under conditions where protein synthesis was relatively spared and where membrane integrity was fully preserved. Among several proposed mechanisms for loss of DN...

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Section: Resultsmentioning

confidence: 99%

Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli.

Rosen

Orman

Rakita

et al. 1990

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

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“…Despite extensive studies on DNA replication, relatively little is known about its in vivo organization, which, in prokaryotes, occurs at the cell membrane (1)(2)(3)(4). The well studied Bacillus subtilis phage 29 (5) is one of the few systems for which this fundamental process has been investigated.…”

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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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“…1). Besides acting as a scaffold and inherently compartmentalizing DNA replication, membranes are also likely to play an important role in the organization and function of the DNA replication complex (for recent reviews see Refs.…”

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confidence: 99%

“…The genome of 29 consists of a linear double-stranded DNA (dsDNA) 1 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 (4 -6).…”

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Phage φ29 DNA Replication Organizer Membrane Protein p16.7 Contains a Coiled Coil and a Dimeric, Homeodomain-related, Functional Domain

Muñoz‐Espín

Mateu

Villar

et al. 2004

Journal of Biological Chemistry

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The Bacillus subtilis phage 29-encoded membrane protein p16.7 is one of the few proteins known to be involved in prokaryotic membrane-associated DNA replication. Protein p16.7 contains an N-terminal transmembrane domain responsible for membrane localization. A soluble variant lacking the N-terminal membrane anchor, p16.7A, forms dimers in solution, binds to DNA, and has affinity for the 29 terminal protein. Here we show that the soluble N-terminal half of p16.7A can form a dimeric coiled coil. However, a second domain, located in the C-terminal half of the protein, has been characterized as being the main domain responsible for p16.7 dimerization. This 70-residue C-terminal domain, named p16.7C, also constitutes the functional part of the protein as it binds to DNA and terminal protein. Sequence alignments, secondary structure predictions, and spectroscopic analyses suggest that p16.7C is evolutionarily related to DNA binding homeodomains, present in many eukaryotic transcriptional regulator proteins. Based on the results, a structural model of p16.7 is presented.In the case of bacteria, replication of the chromosome, plasmids, and infecting phages occurs at the cell membrane (for review see Ref. 1). Besides acting as a scaffold and inherently compartmentalizing DNA replication, membranes are also likely to play an important role in the organization and function of the DNA replication complex (for recent reviews see Refs. 2 and 3). Nevertheless, very little is known about proteins participating in these processes. We started to investigate this fundamental process by using the Bacillus subtilis phage 29, one of the best studied phages (for review see Ref. 4), as a model system. The genome of 29 encodes most, if not all, proteins required for phage DNA replication, and detailed knowledge is available on in vitro 29 DNA replication. This makes 29 an attractive system to study membrane-associated DNA replication.The genome of 29 consists of a linear double-stranded DNA (dsDNA) 1 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 (4 -6). First, the TP-containing DNA ends are recognized by a 29 DNA polymerase/TP 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 completed. Consequently, the DNA replication intermediates can contain extremely long stretches of ssDNA.Ivarie and Pène (7) provided evidence that early expressed 29 protein(s) are required for membrane-associated 29 DNA replication. Gene 16.7, present in an early expressed operon, is conserved in all 29-related phages studied so far (4). Previously, we have shown (4, 8, 9) that p16.7 (130 amino acids) is involved in the organization of membrane-associated 29 DNA replication. Protein p16.7 is a membrane protein, and its Nterminal transmembrane domain is responsible for membrane localization (8...

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Role of the Dna/Membrane Complex in Prokaryotic Dna Replication

Cited by 88 publications

References 91 publications

Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli.

Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli.

Structure of the Functional Domain of φ29 Replication Organizer

Phage φ29 DNA Replication Organizer Membrane Protein p16.7 Contains a Coiled Coil and a Dimeric, Homeodomain-related, Functional Domain

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