The integral membrane protein p16.7 organizes in vivo phi29 DNA replication through interaction with both the terminal protein and ssDNA

Remarkably little is known about the in vivo organization of membrane-associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage phi29 as a model system. Previously, we demonstrated that the phi29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated phi29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the phi29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C-terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA-binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo phi29 DNA replication is presented.     

The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 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 phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.     

Structural and functional analysis of phi29 p16.7C dimerization mutants: identification of a novel aromatic cage dimerization motif

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage varphi29-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.     

Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA replication

An early expressed operon, located at the right end of the linear bacteriophage phi29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in phi29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a phi29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo phi29 DNA replication is discussed.     

The phage phi29 membrane protein p16.7, involved in DNA replication, is required for efficient ejection of the viral genome

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 phi29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step approximately 65% 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.     

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.     

In vivo assembly of phage phi 29 replication protein p1 into membrane-associated multimeric structures

The mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown. In the case of the Bacillus subtilis phage 29, the viral protein p1 enhances the rate of in vivo viral DNA replication. Previous work showed that p1 generates highly ordered structures in vitro. We now show that protein p1, like integral membrane proteins, has an amphiphilic nature. Furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location. By combining in vivo chemical cross-linking and cell fractionation techniques, we also demonstrate that p1 assembles in infected cells into multimeric structures that are associated with the bacterial membrane. These structures exist both during viral DNA replication and when 29 DNA synthesis is blocked due to the lack of viral replisome components. In addition, protein p1 encoded by plasmid generates membrane-associated multimers and supports DNA replication of a p1-lacking mutant phage, suggesting that the pre-assembled structures are functional. We propose that a phage structure assembled on the cell membrane provides a specific site for 29 DNA replication.     

Initiation of phage phi 29 DNA replication by the terminal protein modified at the carboxyl end

A mutant at the carboxyl end of the terminal protein, p3, of phage phi 29 DNA has been constructed by inserting an containing the stop translation codon TGA in the three possible reading frames, immediately downstream of a phage phi 29 DNA fragment coding for all but the last five amino acids of protein p3. The activity in the formation of the p3-dAMP initiation complex in vitro of this mutant as well as another one previously isolated, also mutated at the carboxyl end, have been tested. The results obtained suggest that an intact carboxyl end in the phage phi 29 terminal protein is essential for its normal primer function in DNA replication.     

A temperature sensitive mutant of Escherichia coli which does not allow replication of RNA phage at a high temperature.

A conditional mutant, referred to as RepR43, was isolated from Escherichia coli W2252 by N-methyl-N'-nitro-N-nitroso-guanidine mutagenesis. Although RepR43 does not permit growth of RNA phage beta at the restrictive temperature, 43 degrees C, cell growth and synthesis of macromolecules such as RNA and protein continue at a somewhat reduced rate. Several lines of evidence indicate that a RepR43 function is indispensable for normal phage RNA replication. In addition, this function appears to be involved in the maintenance of the perpetuated phage genome. The addition of 10% sucrose to the medium at the restrictive temperature resulted in the production of the phage, suggesting that the mutant cell might have an altered membrane organization which interferes with normal viral replication.     

Studies with Bacteriophage φII. Events Following Infection of Male and Female Derivatives of Escherichia coli K-12

We studied the course of infection of the female-specific bacteriophage phiII in male and female cells isogenic except for the presence of the substituted sex factor, F'lac. Both male and female cells are killed by phiII; however, only limited phage replication occurs in male cells. Host macromolecular synthesis stops abruptly at 4 to 6 min after infection of male cells, and synthesis of phage components cannot be detected. Experiments with chloramphenicol indicate that phage deoxyribonucleic acid (DNA) penetrates into male cells, since protein synthesis after infection is required to stop synthesis of DNA in males. Phage DNA becomes membrane-associated in both female and male cells. In male cells, parental phage DNA does not dissociate from the membrane during the latent period as is the case with females, indicating a block in phage DNA replication. Isolation of nonrestricting F'lac mutations indicates involvement of a specific episome product in phiII restriction.     

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ø29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su ^?) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.     

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.     

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ?29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su ^–) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.     

Bacteriophage f1 Infection: Fate of the Parental Major Coat Protein

The major coat protein of infecting f1 phage is incorporated into the inner membrane of the host cell, even in the absence of phage f1DNA penetration and replication. The major coat protein monomers are reutilized in the assembly of new phage. They are not conserved as a single unit but behave as independent units which are slowly incorporated into newly manufactured phage.     

Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5

Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.