Biochemical investigations of control of replication initiation of plasmid R6K

The mechanistic basis of control of replication initiation of plasmid R6K was investigated by addressing the following questions. What are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? Did the primary control involve only initiator protein-ori DNA interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? Mutations at two different regions of the pi-encoding sequence individually caused some loss of negative control as indicated by a relatively modest increase in copy number. However, combinations of the mutation P42L, which caused loss of DNA looping, with those located in the region between the residues 106 and 113 induced a robust enhancement of copy number. These mutant forms promoted higher levels of replication in vitro in a reconstituted system consisting of 22 purified proteins. The mutant forms of pi were susceptible to pronounced iteron-induced monomerization in comparison with the WT protein. As contrasted with the changes in DNA-protein interaction, we found no detectable differences in protein-protein interaction between wild type pi with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other. The DnaG-pi interaction reported here is novel. Taken together, the results suggest that both loss of negative control due to iteron-induced monomerization of the initiator and enhanced iteron-initiator interaction appear to be the principal causes of enhanced copy number.     

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.     

An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control

Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.     

The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.

Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30 degrees C and at 42 degrees C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

Replication of antibiotic resistance plasmid R6K DNA in vitro.

A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.     

RNA polymerase is required for DNA initiation in vitro

Summary We have previously reported in vitro complementation assays for chromosome initiation that enable dnaA and dnaC mutant extracts to synthesize DNA. To examine the role of RNA polymerase in chromosome initiation, inhibitors of the enzyme and anti-RNA polymerase antibody were used. Though rifampicin failed to efficiently inhibit ribonucleoside triphosphate polymerization under the assay conditions, both streptolydigin and anti-RNA plymerase antibody abolished ribonucleic acid synthesis completely. Antibody effectively inhibited chromosome initiation in the dnoA mutant based reaction but streptolydigin did not. Neither streptolydigin nor antibody affected the dnaC-dependent assay. It was concluded that RNA polymerase is required for initiation but not necessarily to polymerize a polyribonucleotide. A scheme for the sequence of initiation events is presented.     

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins

Summary The functional R6K origin is composed of two DNA elements, one of 580 bp carrying the origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previosly identified as also required for and origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for origin activity. The function of the origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the origin depends on the R6K replication initiation protein .Within the 580 bp of the origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the replicon. Deletion of the 96 bp or 98 bp results in inactivation of the and the origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the and the origins of R6K. DNA homology analysis performed on , and origin sequences, also reveals 10–23 bp sequences in the and the origins that are related to the family of 22 bp direct repeats in the origin which were shown previously to be binding sites for the protein.     

DNA sequences required for the initiation of adenovirus type 4 DNA replication in vitro

In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid containing 140 base-pairs of the right terminus of adenovirus type 4 supported initiation of DNA replication in vitro, provided that the plasmid was linearized in such a way as to locate the viral terminal sequences at the molecular ends of the DNA. Initiation by adenovirus type 4-infected cell extracts was also supported by a plasmid containing the complete adenovirus type 2 inverted terminal repeat (ITR). Deletion analysis of both adenovirus types 2 and 4 ITRs revealed that only the terminal 18 base-pairs of the genomes (perfectly conserved between the 2 viruses) were required for initiation in vitro. Thus, initiation was not enhanced by the presence of either the NFI site, the NFIII site or both sites together. Fractionation of a HeLa cell nuclear extract, by ion-exchange chromatography, identified a nuclear factor that stimulated the initiation reaction four- to fivefold. The stimulatory factor did not correspond to either of the cellular proteins NFI or NFIII which stimulate adenovirus type 2 DNA replication in vitro. Initiation in vitro was also supported by single-stranded DNA templates, albeit at a lower efficiency. Studies with synthetic oligonucleotides indicated a surprising specificity for initiation: whereas the strand used as template during initiation in vivo was active as a template for initiation in vitro, the complementary strand was inactive.     

Reconstitution of R6K DNA replication in vitro using 22 purified proteins

We have reconstituted a multiprotein system consisting of 22 purified proteins that catalyzed the initiation of replication specifically at ori gamma of R6K, elongation of the forks, and their termination at specific replication terminators. The initiation was strictly dependent on the plasmid-encoded initiator protein pi and on the host-encoded initiator DnaA. The wild type pi was almost inert, whereas a mutant form containing 3 amino acid substitutions that tended to monomerize the protein was effective in initiating replication. The replication in vitro was primed by DnaG primase, whereas in a crude extract system that had not been fractionated, it was dependent on RNA polymerase. The DNA-bending protein IHF was needed for optimal replication and its substitution by HU, unlike in the oriC system, was less effective in promoting optimal replication. In contrast, wild type pi-mediated replication in vivo requires IHF. Using a template that contained ori gamma flanked by two asymmetrically placed Ter sites in the blocking orientation, replication proceeded in the Cairns type mode and generated the expected types of termination products. A majority of the molecules progressed counterclockwise from the ori, in the same direction that has been observed in vivo. Many features of replication in the reconstituted system appeared to mimic those of in vivo replication. The system developed here is an important milestone in continuing biochemical analysis of this interesting replicon.     

Purification of pT181-encoded repC protein required for the initiation of plasmid replication

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 replication specifically requires the plasmid-encoded repC protein. An in vitro system has been shown to carry out semiconservative replication of pT181 and its derivative plasmids (Khan, S A., Carleton, S. M., and Novick, R. P. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4902-4906). We have used this replication assay to isolate repC protein, which was purified to near homogeneity. The repC gene was cloned into the pKJB825 plasmid that contains the phage lambda temperature-sensitive repressor gene, cI857, and the rightward promoter, PR. Upon temperature induction, Escherichia coli clones containing the recombinant plasmid overproduced repC protein, which was purified in significant quantities. The molecular weight of repC protein under denaturing conditions is 38,000, which is consistent with the size predicted from the DNA sequence data. Presence of repC protein was absolutely essential for the initiation of replication of pT181 and its derivatives in vitro.     

Structure-based functional analysis of the replication protein of plasmid R6K: key amino acids at the pi/DNA interface

In previous work, we characterized the bases in an iteron of plasmid R6K that are important for the binding of pi protein monomers and dimers. Here we investigate the following six amino acids of pi, encoded by pir, hypothesized to be important for DNA contact: Ser71, Try74, Gly131, Gly211, Arg225, and Arg254.     

A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division.     

Three novel plasmid R6K proteins act in concert to distort DNA within the α and β origins of DNA replication

Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified. The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (αLIR and βLIR), which are essential elements for the two distally located R6K α and β DNA replication origins. DDP1 and DDP2 are encoded by two tandem genes located at the 5' end of αLIR, whereas a gene coding for DDP3 is located at the 3' end of βLIR. DDP1 and DDP2 are required for primary DNA distortion within αLIR or βLIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences. Creation of DNA distortion within αLIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA-binding site. The possible relevance of these findings to R6K replication is discussed.     

Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication.

Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.     

Activity in vitro of three replication origins of the antibiotic resistance plasmid RSF1040

Replicating molecules of plasmid RSF1040, a deletion mutant of R6K, were synthesized in vitro and analyzed by electron microscopy. Initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma, within a 3900-base pair segment of the R6K genome. These sites are indistinguishable from the origins that are active in vivo. Frequencies of initiation at these three origins, however, are different from those observed in vivo. Replication proceeds unidirectionally in either direction from ori beta and ori gamma and in one direction from ori alpha. The replication terminus of the R6K genome is inactive in the in vitro system.     

The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding

We have tagged the replication initiator protein of the plasmid R6K near the C-terminal end by fusion, in the correct reading frame, with the 89 amino acid long N-terminal alpha-donor polypeptide of beta-galactosidase of E. coli. This fusion was carried out with recombinant DNA methods. The protein chimera thus generated retained the activities of both initiation of DNA replication in vivo at the replication origin gamma of R6K and hydrolysis of beta-galactopyranoside when complemented in vivo with the alpha-acceptor polypeptide coded by the lac Z gene containing the M15 deletion. Using the simple and convenient assay for detecting beta-galactosidase, we have partially purified the tagged replication initiator, and have demonstrated that the protein binds to specific DNA sequences of the R6K chromosome. The protein bound to DNA sequences located at two places in the 5' untranslated leader region of the initiator protein cistron.