RepA-WH1 prionoid: Clues from bacteria on factors governing phase transitions in amyloidogenesis

In bacterial plasmids, Rep proteins initiate DNA replication by undergoing a structural transformation coupled to dimer dissociation. Amyloidogenesis of the ‘winged-helix’ N-terminal domain of RepA (WH1) is triggered in vitro upon binding to plasmid-specific DNA sequences, and occurs at the bacterial nucleoid in vivo. Amyloid fibers are made of distorted RepA-WH1 monomers that assemble as single or double intertwined tubular protofilaments. RepA-WH1 causes in E. coli an amyloid proteinopathy, which is transmissible from mother to daughter cells, but not infectious, and enables conformational imprinting in vitro and in vivo; i.e. RepA-WH1 is a ‘prionoid’. Microfluidics allow the assessment of the intracellular dynamics of RepA-WH1: bacterial lineages maintain two types (strains-like) of RepA-WH1 amyloids, either multiple compact cytotoxic particles or a single aggregate with the appearance of a fluidized hydrogel that it is mildly detrimental to growth. The Hsp70 chaperone DnaK governs the phase transition between both types of RepA-WH1 aggregates in vivo, thus modulating the vertical propagation of the prionoid. Engineering chimeras between the Sup35p/[PSI⁺] prion and RepA-WH1 generates [REP-PSI⁺], a synthetic prion exhibiting strong and weak phenotypic variants in yeast. These recent findings on a synthetic, self-contained bacterial prionoid illuminate central issues of protein amyloidogenesis.     

The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response

Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid. Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication. Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid-borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess. We show that delayed cell division occurs by RepA-mediated induction of the SOS response and can be reversed by over-expression of the host DNA primase, DnaG. The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites. The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls--potentially pre-empting plasmid loss in cultures of dividing cells.     

Fluorescence studies of the replication initiator protein RepA in complex with operator and iteron sequences and free in solution

RepA, the replication initiator protein from the Pseudomonas plasmid pPS10, regulates plasmid replication and copy number. It is capable of autorepression, in which case it binds as a dimer to the inverted repeat operator sequence preceding its own gene. RepA initiates plasmid replication by binding as a monomer to a series of four adjacent iterons, which contain the same half-repeat as found in the operator sequence. RepA contains two domains, one of which binds specifically to the half-repeat. The other is the dimerization domain, which is involved in protein-protein interactions in the dimeric RepA-operon complex, but which actually binds DNA in the monomeric RepA-iteron complex. Here, detailed fluorescence studies on RepA and an N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-labeled single-cysteine mutant of RepA (Cys160) are described. Using time-resolved fluorescence depolarization measurements, the global rotational correlation times of RepA free in solution and bound to the operator and to two distinct iteron dsDNA oligonucleotides were determined. These provide indications that, in addition to the monomeric RepA-iteron complex, a stable dimeric RepA-iteron complex can also exist. Further, F?rster resonance energy transfer between Trp94, located in the dimerization domain, and N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-Cys160, located on the DNA-binding domain, is observed and used to estimate the distance between the two fluorophores. This distance may serve as an indicator of the orientation between both domains in the unbound protein and RepA bound to the various cognate DNA sequences. No major change in distance is observed and this is taken as evidence for little to no re-orientation of both domains upon complex formation.     

Protein domains and conformational changes in the activation of RepA, a DNA replication initiator.

RepA is the DNA replication initiator protein of the Pseudomonas plasmid pPS10. RepA has a dual function: as a dimer, it binds to an inversely-repeated sequence acting as a repressor of its own synthesis; as a monomer, RepA binds to four directly-repeated sequences to constitute a specialized nucleoprotein complex responsible for the initiation of DNA replication. We have previously shown that a Leucine Zipper-like motif (LZ) at the N-terminus of RepA is responsible for protein dimerization. In this paper we characterize the existence in RepA of two protein globular domains C-terminal to the LZ. We propose that dissociation of RepA dimers into monomers results in a conformational change from a compact arrangement of both domains, competent for binding to the operator, to an extended species that is suited for iteron binding. This model establishes the structural basis for the activation of DNA replication initiators in plasmids from Gram-negative bacteria.     

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

Interaction of initiator proteins with the origin of replication of an IncL/M plasmid

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.     

Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA

The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.     

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators

DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.     

RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in Xenopus egg extracts

Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase α and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.     

Excess intracellular concentration of the pSC101 RepA protein interferes with both plasmid DNA replication and partitioning.

RepA, a plasmid-encoded gene product required for pSC101 replication in Escherichia coli, is shown here to inhibit the replication of pSC101 in vivo when overproduced 4- to 20-fold in trans. Unlike plasmids whose replication is prevented by mutations in the repA gene, plasmids prevented from replicating by overproduction of the RepA protein were lost rapidly from the cell population instead of being partitioned evenly between daughter cells. Removal of the partition (par) locus increased the inhibitory effect of excess RepA on replication, while host and plasmid mutations that compensate for the absence of par, or overproduction of the E. coli DnaA protein, diminished it. A repA mutation (repA46) that elevates pSC101 copy number almost entirely eliminated the inhibitory effect of RepA at high concentration and stimulated replication when the protein was moderately overproduced. As the RepA protein can exist in both monomer and dimer forms, we suggest that overproduction promotes RepA dimerization, reducing the formation of replication initiation complexes that require the RepA monomer and DnaA; we propose that the repA46 mutation alters the ability of the mutant protein to dimerize. Our discovery that an elevated intracellular concentration of RepA specifically impedes plasmid partitioning implies that the RepA-containing complexes initiating pSC101 DNA replication participate also in the distribution of plasmids at cell division.     

Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1.

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.     

Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a ∼1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21–24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20–50 nucleotides was ∼3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.MCM10 is a ubiquitous, conserved gene essential for DNA replication in eukaryotes. It was first discovered in yeast genetic screens designed to detect mutants defective in DNA synthesis and minichromosome maintenance (1, 2). In vivo, Mcm10 associates with chromatin and chromosomal replication origins in human cells (hMcm10), Xenopus laevis (XMcm10), Schizosaccharomyces pombe (SpMcm10), and Saccharomyces cerevisiae (ScMcm10) (3–6). In S. cerevisiae, initiation of chromosomal replication occurs at multiple specific sites known as autonomously replicating sequences (ARSs)² (7). Mutations in MCM10 enhance the loss rate of plasmids bearing specific ARSs (8), suggesting a function for ScMcm10 in initiation.In eukaryotic systems replication initiation is a cell cycle-regulated process characterized by a multistep sequential loading of ORC, Cdc6, Cdt1, and the Mcm2–7 complex onto the origin in G₁ to form the pre-RC complex. Binding of ORC, Cdc6p, and Cdt1p to chromatin is a prerequisite for the recruitment of Mcm2–7 (9, 10). The next step in the assembly of the initiation replication apparatus in S. cerevisiae involves protein kinases (Cdc28 and Cdc7/Dbf4), and recruitment of Mcm10, Cdc45, and the GINS complex. The mechanism for targeting Mcm10 to replications origins is unknown. However, recent studies in S. cerevisiae have shown that Mcm10 and Mcm2–7 physically interact (6, 11). It is now believed that in late G₁, chromatin-bound Mcm2–7 is responsible for the recruitment of Mcm10 presumably via protein-protein interactions (12). Prior studies in the Xenopus laevis system reached similar conclusions (4). Additional interactions of Mcm10 with other components of the pre-RC cannot be excluded (13).A key step in the initiation of replication is the local melting of an origin DNA sequence, which occurs at the G₁/S transition and throughout the S phase. The mechanism of this essential DNA-melting process is not understood. There is no evidence in S. cerevisiae that the assembled pre-RC complex leads to the melting of an origin DNA sequence. This unwinding may occur only following the recruitment of Mcm10, raising the possibility that Mcm10 is a key component of the initiation machinery responsible for this process. Results of a study in the Xenopus egg extract system (4), which showed that omission of XMcm10 blocks unwinding of plasmid DNA and initiation of DNA replication, are consistent with this notion. An additional function of Mcm10 in initiation is in the recruitment of Cdc45 to replication origins, presumably via Mcm10-Cdc45 physical interactions (5, 14). Cdc45 is believed to be important for the activation of replication origins and the assembly of the replication elongation complex (15). Upon initiation of DNA replication, ScMcm10 moves from the origin to origin-proximal sequences suggesting that ScMcm10 associates with moving replication forks (12) and is consistent with the observation that elevated temperatures cause pausing of replication forks in a mcm10-1 ts mutant (8). Both ScMcm10 and SpMcm10 interact with DNA polymerase α supporting the notion that replication fork movement requires Mcm10. ScMcm10 and polymerase α form a complex on and off the DNA in vivo (12). In S. pombe, SpMcm10 stimulates the activity of polymerase α in vitro and associates with a primase activity (16, 17) that has not been reported in other eukaryotes (18).Previous studies with Mcm10 in other systems showed that Mcm10 binds DNA. Using a filter binding assay Fien and Hurwitz (16) reported that SpMcm10 from S. pombe binds well to ssDNA but barely interacts (20-fold lower affinity) with dsDNA. It has been suggested that binding of SpMcm10 to ssDNA is important for the recruitment of polymerase α (16). Recently, it has been reported that a DNA binding activity is also associated with XMcm10 protein from X. laevis. Measurements of fluorescence anisotropy were used to show binding of XMcm10 to short, 25-nucleotide-long oligonucleotides (18). These studies have shown that XMcm10 has similar affinities for ssDNA and dsDNA. Unlike SpMcm10, which harbors a single DNA-binding domain in the N-terminal half of the protein, XMcm10 seems to contain two distinct domains for binding DNA. The biological implication of having two DNA-binding domains is not clear.It appears that there are differences in the quaternary structure of Mcm10 from different organisms. Although SpMcm10 and XMcm10 may be a homodimer in solution (17, 18), a recent electron microscopy study suggested that human hMcm10 has a hexameric ring structure (19). The same study reported that hMcm10 interacts with ssDNA but failed to bind dsDNA. The differences in structure and DNA binding properties may reflect differences in the function of Mcm10 in various organisms as well as in the protein preparations.Here we report, for the first time, the characterization of the DNA binding properties of purified Mcm10 from S. cerevisiae. We show that ScMcm10 forms a stable complex with dsDNA and ssDNA. In addition, we demonstrate that dsDNA longer than 50 bp sustains oligomerization of ScMcm10. The number of ScMcm10 molecules bound is directly proportional to the size of the dsDNA, suggesting that ScMcm10 is tightly packed on the dsDNA, perhaps in a head to tail oligomeric structure. In contrast to a 25-bp-long dsDNA, which supports the binding of a single copy of ScMcm10, ssDNA containing only 20 nucleotides may sustain binding of as many as three copies of ScMcm10, suggesting that a ScMcm10 complex composed of at least 3 subunits assembles on ssDNA. We believe that these distinct binding properties to dsDNA and ssDNA are important for the ScMcm10 functions in initiation, formation of replication forks, and the maintenance of replication fork progression during chromosomal DNA replication.     

Missing-base and ethylation interference footprinting of P1 plasmid replication initiator.

RepA, the replication initiator protein of plasmid P1, binds to specific 19 bp sequences on the plasmid DNA. Earlier footprinting studies with dimethylsulfate identified the guanines that contact RepA through the major groove of DNA. In this study, base elimination was used to identify the contribution of all four bases to the binding reaction. Depurination and depyrimidation of any base in the neighborhood of the contacting guanines was found to decrease RepA binding. These results are consistent with the notion that RepA contacts bases of two consecutive major grooves on the same face of DNA. We also observed that depurination but not methylation of three guanines (G3, G8 and G9) affected binding. We identified the DNA phosphate groups (3 in the top strand, one of which mapped between G8 and G9, and 4 in the bottom strand, one of which was adjacent to C3) that strongly interfered with RepA binding upon ethylation. These results indicate that certain bases (e.g. G3, G8 and G9) may not contact RepA directly but contribute to base and backbone contacts by maintaining proper structure of the binding site.