Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K.

A dnaA 'null' strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (alpha, gamma, beta). DnaA binds in vitro to sites in two functionally distinct segments of the central gamma origin. The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication. Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1. When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt pi protein are decreased or if copy-up pi mutant proteins are provided in trans. DnaA does not effect expression of R6K replication initiator protein pi, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes pi. Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the gamma origin and might be sufficient for initiation from the gamma origin and might be sufficient and required for the activity of the alpha and beta origins as well. Implications of the DnaA protein binding to two domains of the gamma origin and the role of the 106-bp origin enhancer in replication are discussed.     

Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein.

Integration host factor (IHF) protein is the only host-encoded protein known to bind and to affect replication of the gamma origin of Escherichia coli plasmid R6K. We examined the ability of R6K origins to replicate in cells lacking either of the two subunits of IHF. As shown previously, the gamma origin cannot replicate in IHF-deficient cells. However, this inability to replicate was relieved under the following conditions: underproduction of the wild-type pi replication protein of R6K or production of normal levels of mutant pi proteins which exhibit relaxed replication control. The copy number of plasmids containing the primary R6K origins (alpha and beta) is substantially reduced in IHF-deficient bacteria. Furthermore, replication of these plasmids is completely inhibited if the IHF-deficient strains contain a helper plasmid producing additional wild-type pi protein. IHF protein has previously been shown to bind to two sites within the gamma origin. These sites flank a central repeat segment which binds pi protein. We propose a model in which IHF binding to its sites reduces the replication inhibitor activity of pi protein at all three R6K origins.     

Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K

The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined.     

A DNA segment conferring stable maintenance on R6K gamma-origin core replicons.

The plasmid R6K gamma origin consists of two adjacent modules, the enhancer and the core, and requires R6K initiator protein pi for replication. While the core alone can replicate at a low level of wild-type pi protein, we show here that host cells do not stably maintain core plasmids. The presence of the enhancer segment confers stable inheritance on core plasmids without a significant change in average plasmid copy number. Deletions and site-directed mutagenesis indicated that the stability of core plasmids is not mediated by binding sites or consensus sequences in the enhancer for DnaA, pi protein, gyrase, Fis, or Dcm methylase. Proper segregation of core plasmids requires only the R6K stb or stability-related region, which includes the 20-bp segment of the 100-bp enhancer adjacent to the core. The use of the pi 116 mutant protein, which increases plasmid copy number fourfold, does not stabilize core plasmids lacking the enhancer. We also show that at an elevated level of wild-type pi, the gamma-origin plasmid is unstable, even in the presence of the enhancer. We discuss the differences and similarities between the R6K stability system and those found in other plasmids.     

Spacing is crucial for coordination of domain functions within the simian virus 40 core origin of replication.

The simian virus 40 core origin of replication is composed of distinct domains that are bracketed by DNA spacers. We created a matched set of insertion mutations in spacer sites to study the spatial relationships among origin domains. Insertions larger than a single base pair severely inhibit replication regardless of the helical phasing between domains. Replication-defective mutations reduce T-antigen binding and T-antigen-induced KMnO4 modifications of DNA to various extents. Mutations in the early half of the origin reduce T-antigen functions in the entire origin, whereas mutations in the late half reduce functions only in that half. Surprisingly, some mutations that severely inhibit DNA replication reduce T-antigen-induced melting and other structural changes within origin DNA to only a limited extent. In contrast, all replication-defective origin mutations prevent T antigen from extending the primary replication bubble beyond the limits of the core origin of replication. We conclude, therefore, that T-antigen-induced events within the core origin must be spatially coordinated for conversion of T-antigen hexamers bound to the core origin into mobile helicase units.     

Small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity

The emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. To address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. In the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. Our focus was on the pir-encoded pi protein of plasmid R6K that possesses both positive and negative functions in controlling gamma origin-based replication. We observed that three of four pir mutations examined, including two in-frame deletions, severely impaired negative plasmid-replication control. The resulting over-replication phenotype was particularly strong when a pir mutant was placed in cis to gamma origin. In conjugative mating experiments with several representatives of the family Enterobacteriaceae, the plasmids expressed postconjugational antimicrobial activity. The potential utility of a conjugation-based antimicrobial approach is discussed. Additionally, we describe the replication inhibitory function of a novel and useful Rep protein variant, pi*M36A;M38A, which binds iteron DNA exclusively as dimers.     

Binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid R6K

Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the gamma origin of plasmid R6K, the Rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. In this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by pi monomers and dimers. We also isolated iteron mutants that affected the binding of pi monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, pi monomers interact with nucleotides spanning the entire length of the iteron. In contrast, pi dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.     

Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance.

We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein. Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.     

Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution.

The replication initiator protein pi of plasmid R6K binds seven 22 bp direct repeats (DR) in the gamma origin. The pi protein also binds to an inverted repeat (IR) in the operator of its own gene, pir, which lies outside the gamma origin sequences. A genetic system was devised to select for pi protein mutants which discriminate between IR and DR (York et al., Gene (Amst.) 116, 7-12, 1992; York and Filutowicz, J. Biol. Chem. 268, 21854-21861, 1993). From this selection the mutant pi S87N protein was isolated which is deficient in repressing the pir gene's expression because it cannot bind to IR at the pir gene operator. Remarkably, we discovered that pi S87N binds to DR cooperatively under conditions where wt pi binds independently. Moreover, the pi S87N is more active as a replication initiator in vivo when supplied at the same level as wt pi. Quantitative binding assays showed that both wt pi and pi S87N bind a DNA fragment containing a single DR unit with a similar affinity (Kd = 0.3 x 10(-12) M). Thus, cooperativity of pi S87N is most likely achieved through altered interactions between promoters bound at adjacent DR units.     

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein.

Replication of the gamma origin of Escherichia coli plasmid R6K requires pi protein, encoded by the R6K pir gene, and many host factors, including DnaA protein. Pi has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function of pi is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using gamma origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type pi protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.     

Plasmid-encoded initiation protein is required for activity at all three origins of plasmid R6K DNA replication in vitro

DNA replication of plasmid R6K initiates at three unique sites, ori alpha, ori beta, and ori gamma. Replicating DNA molecules of a deletion derivative of R6K were synthesized in an in vitro system containing pi protein fraction from cells carrying a mini-R6K derivative that produced only this initiation protein as an R6K-encoded protein and analyzed by electron miscroscopy. Requirement of pi protein for the activity of all these three replication origins in vitro was verified. Frequencies of initiation at the three origins were almost equal.     

Monomer/dimer ratios of replication protein modulate the DNA strand-opening in a replication origin

DNA opening is an essential step in the initiation of replication via the Cairns mode of replication. The opening reaction was investigated in a gamma ori system by using hyperactive variants of plasmid R6K-encoded initiator protein, pi. Reactivity to KMnO4 (indicative of opening) within gamma ori DNA occurred in both strands of a superhelical template upon the combined addition of wt pi, DnaA and integration host factor (IHF), each protein known to specifically bind gamma ori. IHF, examined singly, enhanced reactivity to KMnO4. The IHF-dependent reactive residues, however, are distinct from those dependent on pi (wt and hyperactive variants). Remarkably, the DNA helix opening does not require IHF and/or DnaA when hyperactive variants of pi were used instead of wt protein. We present three lines of evidence consistent with the hypothesis that DNA strand separation is facilitated by pi monomers despite the fact that both monomers and dimers of the protein can bind to iterons (pi binding sites). Taken together, our data suggest that pi elicits its ability to modulate plasmid copy number at the DNA helix-opening step.     

Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers.

Mutated forms of trfA, the replication protein gene of plasmid RK2, that support a minimal RK2 origin plasmid in Escherichia coli at copy numbers up to 23-fold higher than normal have been isolated. Six such high-copy-number (copy-up) mutations were mapped and sequenced. In each case, a single base transition led to an amino acid substitution in the TrfA protein primary sequence. The six mutations affected different residues of the protein and were located within a 69-base-pair region encoding 24 amino acids. Dominance tests showed that each of the mutants can be suppressed by wild-type trfA in trans, but suppression is highly dependent on the amount of wild-type protein produced. Excess mutant TrfA protein provided in trans significantly increased the copy number of RK2 and other self-replicating derivatives of RK2 that contain a wild-type trfA gene. These observations suggest that the mutations affect a regulatory activity of the TrfA replication protein that is a key factor in the control of initiation of RK2 replication.     

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.