P2 DNA Replication Research Articles

The multifunctional bacteriophage P2 cox protein requires oligomerization for biological activity.

The Cox protein of bacteriophage P2 is a multifunctional protein of 91 amino acids. It is directly involved in the site-specific recombination event leading to excision of P2 DNA out of the host chromosome. In this context, it functions as an architectural protein in the formation of the excisome. Cox is also a transcriptional repressor of the P2 Pc promoter, thereby ensuring lytic growth. Finally it promotes derepression of prophage P4, a nonrelated defective satellite phage, by activating the P4 P(LL) promoter that controls P4 DNA replication. In this case it binds upstream of the P(LL) promoter, which normally is activated by the P4 Delta protein. In this work we have analyzed the native form of the Cox protein in vivo, using a bacteriophage lambda cI-based oligomerization assay system, and in vitro, using gel filtration, cross-linking agents, and gel retardation assays. We found that P2 Cox has a strong oligomerization function in vivo as well as in vitro. The in vitro analysis indicates that its native form is a tetramer that can self-associate to octamers. Furthermore we show that oligomerization is necessary for the biological activity by characterizing different cox mutants and that oligomerization is mediated by the C-terminal region.

Identification of Two Replicons in Phage-Plasmid P4

DNA replication of phage-plasmid P4 proceeds bidirectionally from theori1site (previously namedori), but requires a secondcis-acting region,crr.Replication depends on the product of the P4 α gene, a protein with primase and helicase activity, that binds bothori1andcrr.A negative regulator of P4 DNA replication, the Cnr protein, is required for copy number control of plasmid P4. Using a plasmid complementation test for replication, we found that two replicons, both dependent on the α gene product, coexist in P4. The first replicon is made by thecnrand α genes and theori1andcrrsites. The second is limited to the α andcrrregion. Thus, in the absence of theori1region, replication can initiate at a different site. By deletion mapping, acis-acting region,ori2,essential for replication of the α-crrreplicon was mapped within a 270-bp fragment in the first half of the α gene. Theori2site was found to be dispensable in a replicon that containsori1.A construct that besidescrrand α carries also thecnrgene was unable to replicate, suggesting that Cnr not only controls replication fromori1,but also silencesori2.

Derepression of prophage P2 by satellite phage P4: cloning of the P4 epsilon gene and identification of its product.

Escherichia coli phage P4 lacks all of the genetic information necessary for capsid, tail, and lysis functions. P4 is therefore dependent on a helper phage, such as P2, for lytic propagation. During P4 superinfection of a P2 lysogen, the P2 prophage is derepressed by the action of the P4-encoded epsilon gene. We have cloned the epsilon gene and identified the 10-kDa E protein. The epsilon gene product is the only P4 protein required to derepress prophage P2, which leads to in situ P2 DNA replication. A two-plasmid derepression assay system has been developed to examine the derepression activity of E. The reporter plasmid contains the two face-to-face promoters, Pe and Pc, involved in the lysis-lysogeny transcriptional switch of phage P2 and the immunity repressor C. The Pe promoter is coupled to a cat reporter gene. In the construct, the C repressor is transcribed from the Pc promoter and represses the Pe promoter, which mimics the in situ-repressed P2 prophage. The E protein is supplied in trans from a compatible plasmid in which the epsilon gene is under the control of the T7 promoter. We show here that in the two-plasmid assay system, induction of the E protein derepresses the Pe promoter. The ash9 mutation, which is located upstream of the epsilon gene, enhances the E-mediated derepression of the Pe promoter. The purified E protein shows no specific DNA binding activity, and the implications of this are discussed.

Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha.

Bacteriophage P4 DNA replication depends upon the phage-encoded alpha protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [alpha cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp alpha that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of alpha protein in vitro. The alpha cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp alpha domain that binds specifically to DNA and that gp(alpha)cr mutations impair this interaction. We hypothesize that gp alpha-Cnr interaction is essential for the control of P4 DNA replication.

Multiple regulatory mechanisms controlling phage-plasmid P4 propagation

Bacteriophage P4 autonomous replication may result in the lytic cycle or in plasmid maintenance, depending, respectively, on the presence or absence of the helper phage P2 genome in the Escherichia coli host cell. Alternatively, P4 may lysogenize the bacterial host and be maintained in an immune-integrated condition. A key step in the choice between the lytic / plasmid vs. the lysogenic condition is the regulation of P4 α operon. This operon may be transcribed from two promoters, P LE and P LL, and encodes both immunity (promoter proximal) and replication (promoter distal) functions. P LE is a constitutive promoter and transcription of the downstream replication genes is regulated by transcription termination. The trans-acting immunity factor that controls premature transcription termination is a short RNA encoded in the P LE proximal part of the operon. Expression of the replication functions in the lytic/plasmid condition is achieved by activation of the P LL promoter. Transcription from P LL is insensitive to the termination mechanism that acts on transcription starting from P LE. P LL is also negatively regulated by P4 orf88, the first gene downstream of P LL. An additional control on P4 DNA replication is exerted by the P4 cnr gene product.

Multiple regulatory mechanisms controlling phage-plasmid P4 propagation.

Bacteriophage P4 autonomous replication may result in the lytic cycle or in plasmid maintenance, depending, respectively, on the presence or absence of the helper phage P2 genome in the Escherichia coli host cell. Alternatively, P4 may lysogenize the bacterial host and be maintained in an immune-integrated condition. A key step in the choice between the lytic/plasmid vs. the lysogenic condition is the regulation of P4 alpha operon. This operon may be transcribed from two promoters, PLE and PLL, and encodes both immunity (promoter proximal) and replication (promoter distal) functions. PLE is a constitutive promoter and transcription of the downstream replication genes is regulated by transcription termination. The trans-acting immunity factor that controls premature transcription termination is a short RNA encoded in the PLE proximal part of the operon. Expression of the replication functions in the lytic/plasmid condition is achieved by activation of the PLL promoter. Transcription from PLL is insensitive to the termination mechanism that acts on transcription starting from PLE.PLL is also negatively regulated by P4 orf88, the first gene downstream of PLL. An additional control on P4 DNA replication is exerted by the P4 cnr gene product.

Domain structure of phage P4 alpha protein deduced by mutational analysis

Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.

Bacteriophage P4 DNA replication

Replication of satellite phage P4 of Escherichia coli is dependent on three phage-encoded elements: the origin ( ori), a cis replication element ( crr), and the product of the α gene, gpα. In vitro P4 replication is origin-specific resulting in monomeric form I DNA. DNA synthesis requires chromosomally encoded proteins DNA polymerase III holoenzyme, SSB, DNA gyrase and probably topoisomerase I; host-encoded initiation and priming functions are dispensable. The α protein is multifunctional in P4 replication, combining three activities in a single polypeptide chain. First, the protein complexes specifically with type I repeats at ori and crr. Second, the helicase activity associated with gpα unwinds DNA with 3′ → 5′ polarity. Third, the primase activity results in the synthesis of RNA primers. Defined sequence motifs in gpα correlate with the helicase and primase activities which are arranged in distinct, separable domains. Primase activity is associated with the N-terminal half of the protein, ori / crr binding with the C-terminal portion. A model for the initiation mechanism of P4 replication which resembles that of mammalian simian virus 40 is discussed.

Bacteriophage P4 DNA replication.

Replication of satellite phage P4 of Escherichia coli is dependent on three phage-encoded elements: the origin (ori), a cis replication element (crr), and the product of the alpha gene, gp alpha. In P4 replication is origin-specific resulting in monomeric form I DNA. DNA synthesis requires chromosomally encoded proteins DNA polymerase III holoenzyme, SSB, DNA gyrase and probably topoisomerase I; host-encoded initiation and priming functions are dispensable. The alpha protein is multifunctional in P4 replication, combining three activities in a single polypeptide chain. First, the protein complexes specifically with type I repeats at ori and crr. Second, the helicase activity associated with gp alpha unwinds DNA with 3'--> 5' polarity. Third, the primase activity results in the synthesis of RNA primers. Defined sequence motifs in gp alpha correlate with the helicase and primase activities which are arranged in distinct, separable domains. Primase activity is associated with the N-terminal half of the protein, ori/crr binding with the C-terminal portion. A model for the initiation mechanism of P4 replication which resembles that of mammalian simian virus 40 is discussed.

A new gene of bacteriophage P4 that controls DNA replication

Bacteriophage P4 replication may result in either a lytic cycle or plasmid maintenance, depending on the presence or absence, respectively, of helper phase P2 genome. Bacteriophage P4 DNA replication depends on the product of gene alpha, which has origin recognition, primase, and helicase activities. An open reading frame with the coding capacity for a protein of 106 amino acids (orf106) is located upstream of the alpha gene. Genes orf106 and alpha are transcriptionally coregulated. Three amber mutations and an internal deletion (del51) were introduced into orf106. All of the amber mutations exhibited a polar effect on transcription of the downstream alpha gene. The P4 del51 mutant was slightly defective in lytic growth and could not be propagated in the plasmid state. In this latter condition, P4 DNA overreplication was observed. Overexpression of Orf106 severely inhibited P4 DNA replication, preventing P4 lytic growth and plasmid maintenance. The inhibitory effect of Orf106 on P4 replication was not observed when both orf106 and alpha were overexpressed. We suggest that orf106 is involved in P4 replication and that a balanced expression of orf106 relative to alpha may be necessary for proper P4 DNA replication. In particular, orf106 appears to be essential for the control of P4 genome replication in the plasmid state. We propose that orf106 be named cnr, for copy number regulation.

Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein.

Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an initiator of the replication by inducing a single-stranded cut at the origin of replication (ori). The exact location of the cut induced by the A protein in vivo is determined in this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (iii) primer extensions. It is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, in a region devoid of secondary structures. The A gene has been cloned into an expression vector, and the A protein has been purified. The purified A protein does not bind to double-stranded ori containing DNA, but it cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein.

Studies of Bacteriophage P2 DNA Replication: The DNA Sequence of the Cis-acting gene A and ori Region and Construction of a P2 Mini-chromosome

A self-replicating plasmid was constructed from the 76·7 to 91·6% region of bacteriophage P2, which contains the P2 origin of replication ( ori) and the genetically defined replication genes B and A. The sequence of the 76·7 to 80·2% has been determined previously, and the sequence of the 80·2 to 91·6% region is now reported. The sequenced region contained gene A, which predicts a 761 amino acid polypeptide known to induce a single-strand cut at or near ori , and ori, which has been located by electron microscopy to about 89% from the left end of the phage genome. Analysis of plasmid-encoded proteins in minicells indicated that the A gene product was about 78 kilodaltons. Five previously unknown open reading frames ( orf-80, orf-81, orf-82, orf-83 and orf-91) were discovered. They have been cloned and their respective products were identified. The products of orf-80, orf-82 and gene A were found to be lethal to the host when overexpressed. The predicted amino acid sequences of the orf-82 and orf-83 gene products were similar to two early gene products of phage 186; the orf-91 product resembled the hypothetical protein of orfd of retron Ec67. Similarities between the products of P2 gene A , 186 gene A and orf2 and orf3 of Ec67 were also found. A P2 mini-chromosome has been constructed that contains only the P2 A gene and the β-lactamase gene as a marker.

Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene

The bacteriophage P2 ogr gene encodes an 8.3-kDa protein that is a positive effector of P2 late gene transcription. The ogr gene is preceded by a promoter sequence (Pogr) resembling a normal Escherichia coli promoter and is located just downstream of a late transcription unit. We analyzed the kinetics and regulation of ogr gene transcription by using an ogr-specific antisense RNA probe in an S1 mapping assay. During a normal P2 infection, ogr gene transcription starts from Pogr at an intermediate time between the onset of early and late transcription. At late times after infection the ogr gene is cotranscribed with the late FETUD operon; the ogr gene product thus positively regulates its own synthesis from the P2 late promoter PF. Expression of the P2 late genes also requires P2 DNA replication. Complementation experiments and transcriptional analysis show that a nonreplicating P2 phage expresses the ogr gene from Pogr but is unable to transcribe the late genes. A P2 ogr-defective phage makes an increased level of ogr mRNA, consistent with autogenous control from Pogr. Transcription of the ogr gene in the prophage of a P2 heteroimmune lysogen is stimulated after infection with P2, suggesting that Pogr is under indirect immunity control and is activated by a yet-unidentified P2 early gene product during infection.

INTERACTIONS BETWEEN SATELLITE BACTERIOPHAGE P4 AND ITS HELPERS

The helper dependence of satellite phage P4 superimposes an additional set of regulatory interactions on those required for the independent maintenance of P4 or its helpers. These interactions allow P4 to exploit a helper phage under a variety of circumstances and can affect expression of the immunity functions and late genes of both phages. The phage P2 lysis/lysogeny decision involves two competing repressors regulating mutually exclusive promoters in the early control region. In the absence of a helper phage, the P4 immunity function plays a role in the choice between lysogeny or the multicopy plasmid state. No evidence exists for a P4-encoded immunity repressor; in P4-lysogenic cells, expression of the P4 DNA replication gene alpha appears to be prevented by premature termination of transcription. Immunity-independent expression of alpha in the multicopy plasmid state involves initiation of transcription at an alternative upstream promoter that is positively regulated by P4 delta protein; the same promoter is activated by P2 Cox protein during derepression of P4 by P2. The mechanism of derepression of P2 by P4 remains to be determined, and the relationship between the P4 immunity and derepression functions and the mutations that allow P4 to grow with a P3 prophage helper is an intriguing area for further exploration. Expression of P2 and P4 late genes is regulated by phage-encoded, zinc-binding transcriptional activators that appear to interact directly with the alpha subunit of RNA polymerase of E. coli. Stimulation of P2 late transcription by P2 Ogr protein depends upon phage DNA replication, whereas activation of transcription from the same promoters by the related P4 delta gene product is replication-independent. Elucidation of the mechanisms underlying these interactions promises to provide new insights into strategies for control of gene expression.

Bacteriophage P2 ogr and P4 delta genes act independently and are essential for P4 multiplication

Satellite bacteriophage P4 requires the products of the late genes of a helper phage such as P2 for lytic growth. Expression of the P2 late genes is positively regulated by the P2 ogr gene in a process requiring P2 DNA replication. Transactivation of P2 late gene expression by P4 requires the P4 delta gene product and works even in the absence of P2 DNA replication. We have made null mutants of the P2 ogr and P4 delta genes. In the absence of the P4 delta gene product, P4 multiplication required both the P2 ogr protein and P2 DNA replication. In the absence of the P2 ogr gene product, P4 multiplication required the P4 delta protein. In complementation experiments, we found that the P2 ogr protein was made in the absence of P2 DNA replication but could not function unless P2 DNA replicated. We produced P4 delta protein from a plasmid and found that it complemented the null P4 delta and P2 ogr mutants.

Regulation of the plasmid state of the genetic element P4.

After infection of sensitive cells in the absence of a helper phage, the satellite bacteriophage P4 enters a temporary phase of uncommitted replication followed by commitment to either the repressed-integrated condition or the derepressed-high copy number mode of replication. The transient phase and the stable plasmid condition differ from each other in the pattern of protein synthesis, in the rate of P4 DNA replication and in the expression of some gene functions. The regulatory condition characteristic of the P4 plasmid state affects a superinfecting genome, preventing the establishment of the P4 immune condition.

The replication of bacteriophage P4 DNA in vitro: Partial purification of the P4 α gene product

A soluble enzyme system has been prepared from a phage P4-infected Escherichia coli strain that supports the replication of exogenous, supercoiled P4 DNA. This DNA synthesis in vitro depends upon the four deoxyribonucleotides and ATP, but is enhanced about four- to fivefold by the presence of other ribonucleotides. E. coli DNA polymerase III holoenzyme, the E. coli single-strand DNA binding protein, and the partially purified P4 α gene product are required for replication in vitro. Rifamycin does not inhibit P4 replication in vitro. Since the P4 α gene codes for a rifamycin-resistant RNA polymerase (Barrett et al., 1983), and since P4 DNA replication is independent of the host primase (Bowden et al., 1975), we believe the α gene product is functioning as a P4-specific DNA primase.

Bacteriophage P4 DNA replication: Location of the P4 origin

An electron microscopic examination of replicating bacteriophage P4 DNA molecules has revealed theta-type structures that replicate bidirectionally from a single origin. Many replicating P4 DNA molecules also contain long (2000 bases) single-strand DNA regions at the growing fork that are deployed in a trans configuration, which supports the concept of continuous leading strand and discontinuous lagging strand syntheses. The position of the P4 origin was localized by the use of a plasmid complementation test for replication in vivo, as well as by labeling of DNA replicating in vitro in the presence of a chain-terminating inhibitor. During this study we discovered a second site on the P4 genome which is essential for replication, and we have named it crr ( cis region required for replication). This site is located at least 3300 bases from the origin but appears to be required for the initiation of DNA replication in vivo as well as in vitro.

Bacteriophage P2 late promoters: II. Comparison of the four late promoter sequences

The late genes of bacteriophage P2 are clustered into four transcription units. We have reported the transcription initiation sites for two of the late messenger RNAs, encoding genes QP and ONMLKRS. We have now located the 5′ ends of the two remaining late mRNAs. The first gene in the VJHG transcription unit has been located by DNA sequence determination of the single nucleotide change in a V amber mutant. Location of the first gene in the FETUD transcription unit has been inferred from the DNA sequence. The 5′ ends of the mRNAs for these two transcription units were located by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S 1. Similar protection of hybrids using RNA that had been 5′ end-labeled with [ α- 32P]GTP and guanylyl transferase confirmed that these 5′ termini resulted from initiation of transcription. The DNA sequences preceding the P2 late transcription starts are different from the Escherichia coli promoter consensus sequences at −10 and −35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression. The four P2 late promoters do share sequence homologies in the −10 and −35 regions, however, and several additional homologies further upstream. P2 late gene expression also appears to involve negative regulation by a product of the ONMLKRS gene cluster. When cells are infected with P2 polar O amber mutants, a marked increase in the levels of proteins encoded by the other three gene clusters is observed. This increase is reflected in the amounts of late mRNAs, suggesting that RNA synthesis is normally repressed or that late mRNAs are more labile in the presence of a gene product from the ONMLKRS transcription unit. Satellite phage P4 induces P2 late gene expression without the usual requirement for P2 DNA replication. The 5′ ends of the P2 late mRNAs are the same during P4 transactivation as during normal P2 late gene expression. Thus, the regulation of P2 late gene expression by P4 does not involve altered promoter selection.

Bacteriophage P2 late promoters: Transcription initiation sites for two late mRNAs

Divergent transcription of two of the bacteriophage P2 late mRNAs, encoding genes QP and ONMLKRS, is initiated from opposite strands of the DNA in a region near the left end of the P2 genome. The first gene in each of these transcription units (P and O) has been located in the nucleotide sequence by amino-terminal sequence analysis of the P gene product and by DNA sequence determination of the single nucleotide changes in two O amber mutants. The 5' ends of the P and O gene mRNAs are separated by 109 nucleotide pairs in the DNA template. The locations of these 5' termini were determined by protection of end-labeled restriction fragments in RNA-DNA hybrids from digestion with nuclease S1. Sequence analysis of mRNA that had been labeled at the 5' end with [alpha-32P]GTP and guanylyl transferase confirmed that these termini resulted from initiation of transcription. The DNA sequences preceding the O and P transcription starts have poor homologies to the bacterial promoter consensus sequences at -10 and -35, consistent with the apparent requirement for phage-encoded proteins in the regulation of P2 late gene expression. The O and P promoter regions also have no detectable homology to each other in the -10 or -35 regions, and are unusually G + C-rich. There are, however, blocks of sequence homology within the transcribed region of each of these two late operons near the 5' end. Satellite phage P4 induces P2 late gene expression without the usual requirement for P2 DNA replication. The 5' ends of the P2 P and O gene transcripts are the same during P4 "transactivation" as during normal P2 late gene expression. Thus the regulation of P2 late gene expression by P4 does not involve a change in the site for initiation of transcription.