Dgp1 and Dfp1 are closely related plasmids in the Dictyostelium Ddp2 plasmid family

Dictyostelium plasmids Dgp1 and Dfp1, two members of the Ddp2 plasmid family, are 86% identical in nucleotide sequence. These small (4481 and 5015 bp), high copy number, nuclear plasmids carry both a gene homologous to the Ddp2 rep gene and a long 0.47- to 0. 48-kb inverted repeat region. Their Rep proteins are 82.8% identical in amino acid sequence and carry all 10 of the conserved peptide sequence motifs found in the Ddp2 family Rep proteins. Unlike other members of this family, Dgp1 carries two copies and Dfp1 carries four copies of a 162- to 166-bp direct repeat element. Both the direct and inverted repeat elements, as well as the promoter of the rep gene, are highly conserved (81 to 90% identical) between Dgp1 and Dfp1. In contrast, these regions are not highly conserved and the Rep proteins are only about 40% identical among the other known members of the plasmid family.     

The sequence and organization of Ddp2, a high-copy-number nuclear plasmid of Dictyostelium discoideum

The complete nucleotide sequence of the plasmid Ddp2 found in the nucleus of the simple eukaryote Dictyostelium discoideum is reported. This 5852-bp plasmid contains a 2661-bp open reading frame (ORF), named the "Rep gene," and 501-bp imperfect inverted repeats. A 1762-bp section of Ddp2, which includes one of the 501-bp repeat sequences, could be deleted without abolishing extrachromosomal replication. Deletion of the second 501-bp repeat, or interruption of the Rep gene, removed the ability to replicate extrachromosomally. We suggest that Ddp2 encodes a protein, "REP," that positively regulates replication initiation, a regulatory mechanism different from that of the yeast 2 mu plasmid which also possesses inverted repeat sequences. Ddp2 has a structure similar to that of plasmid pDG1, found in an unidentified isolate of Dictyostelium, with a similar sized ORF and inverted repeats. A common evolutionary origin is suggested by considerable sequence homology between the ORFs of pDG1 and Ddp2.     

Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418.

A protocol that allows the rapid isolation and growth of large numbers of independent G418-resistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies. Discovery that aggregation of nontransformants was inhibited at a nonselective level of G418 (25 to 35 micrograms/ml) led to the development of a vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddp1 and Ddp2. We conclude that the origin of replication of plasmid Ddp1 does not alone confer stable maintenance and thus, Ddp1 must bear additional sequences required for its own maintenance. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3' end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified.     

Identification of the origin of replication of the eukaryote Dictyostelium discoideum nuclear plasmid Ddp2

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryote Dictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication in D. discoideum, provided that the Rep gene, which acts in trans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp "TGTCATGACA" palindromes separated by a poly(T.A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication.     

Identification of the origin of replication of the eukaryoteDictyostelium discoideum nuclear plasmid Ddp2

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryoteDictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication inD. discoideum, provided that the Rep gene, which acts intrans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp “TGTCATGACA” palindromes separated by a poly(T · A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication.     

Analysis of the Effects of Charge Cluster Mutations in Adeno-Associated Virus Rep68 Protein In Vitro

The Rep78 and Rep68 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which are required for viral replication, regulation of AAV promoters, and preferential integration of the AAV genome into a region of human chromosome 19. These proteins bind the hairpin structures formed by the AAV inverted terminal repeat (ITR) origins of replication, make site- and strand-specific endonuclease cuts within the AAV ITRs, and display nucleoside triphosphate-dependent helicase activities. Additionally, several mutant Rep proteins display negative dominance in helicase and/or endonuclease assays when they are mixed with wild-type Rep78 or Rep68, suggesting that multimerization may be required for the helicase and endonuclease functions. Using overlap extension PCR mutagenesis, we introduced mutations within clusters of charged residues throughout the Rep68 moiety of a maltose binding protein-Rep68 fusion protein (MBP-Rep68Δ) expressed in Escherichia coli cells. Several mutations disrupted the endonuclease and helicase activities; however, only one amino-terminal-charge cluster mutant protein (D40A-D42A-D44A) completely lost AAV hairpin DNA binding activity. Charge cluster mutations within two other regions abolished both endonuclease and helicase activities. One region contains a predicted alpha-helical structure (amino acids 371 to 393), and the other contains a putative 3,4 heptad repeat (coiled-coil) structure (amino acids 441 to 483). The defects displayed by these mutant proteins correlated with a weaker association with wild-type Rep68 protein, as measured in coimmunoprecipitation assays. These experiments suggest that these regions of the Rep molecule are involved in Rep oligomerization events critical for both helicase and endonuclease activities.     

Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli.

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.     

Exonuclease III promotes in vitro binding of the replication initiator protein of plasmid pSC101 to the repeated sequences in the ori region

Purified Rep protein, a replication initiator protein of plasmid pSC101, has less binding affinity for the direct repeats (DR) in the replication origin region (ori) than that for the inverted repeats (IR) in the promoter region of the structure gene of Rep (rep) (Sugiura, S. et al. (1990) J. Biochem. 107, 369-376). We found a protein factor that promotes binding of purified Rep to the DR sequence in the cell extract of Escherichia coli. In the presence of the factor, DNA fragments containing the DR sequence can form a specific DNA-protein complex by the addition of low concentrations of Rep. On the contrary, IR-containing DNA loses its binding activity for Rep by preincubation with the factor. We purified extensively the factor and identified it as exonuclease III (exo III). Enzymatic action of the factor or authentic exo III at 37 degrees C is necessary for binding of Rep to DR-DNA. This binding of Rep to duplex DNA treated with exo III is DR-sequence specific. Since Rep cannot bind to the single stranded DR sequence, the present finding suggests that partial single-stranded regions around the DR sequence are required for binding of Rep.     

Negative control of plasmid pSC101 replication by increased concentrations of both initiator protein and iterons

Increased intracellular concentrations of the initiator protein Rep (or RepA) interfere with pSC101 DNA replication, and mutated Rep proteins that result in an increase in plasmid copy numbers do not inhibit the replication. A rep mutant (rep(inh)) defective in the inhibitory activity was isolated and found to be a new high copy number mutant. The inhibitory function of Rep was enhanced by the coexistence of directly repeated sequences (DR; iterons) in the replication origin region (ori), but not by the inverted repeat sequences (IR) in ori and the rep promoter. This synergistic effect of Rep and DR sequences for the replication inhibition was dependent on their intracellular concentrations. Considering that DR sequences are the specific binding sites of the Rep monomer form, the Rep monomer-DR complex might be responsible for the inhibition of the plasmid replication. Furthermore, the Rep monomer in the crude cell extracts facilitated dimerization of DR DNA fragments by DNA ligase. Neither synergistic inhibitory function with DR nor Rep mediated dimerization of DR DNA was observed in high copy number mutant Rep proteins. The role of the Rep-iteron complex in the copy number control of pSC101 is discussed.     

Distinct functions of the two specificity determinants in replication initiation of plasmids ColE2-P9 and ColE3-CA38

The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, alpha and beta, in the origins as specificity determinants and showed that when each component of the region A-site alpha pair and the region B-site beta pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site alpha. By using an electrophoretic mobility shift assay, we demonstrated that region B and site beta play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site alpha were involved in the efficient unwinding of several nucleotide residues around site alpha, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.     

Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.     

A single-stranded region located downstream of the repeated sequence in the ori region of pSC101 is required for binding of the Rep protein in vitro.

Purified replication initiator protein (Rep) of plasmid pSC101 binds preferentially to two inverted repeats (IR) overlapping the promoter of its own structure gene, rep. However, the protein has much lower binding affinity for directly repeated (DR) sequences in the replication origin (ori) that are similar to the symmetric sequences. Exonuclease III (exo III) promotes in vitro binding of Rep to the origin repeats. In the present studies, DNA containing the DR sequences was degraded unidirectionally by exo III and then formed a complex with Rep. Analyses of DNA from the complex revealed that Rep bound to the DR sequences only when the degradation proceeded from the 3' end proximal to IR to the DR sequences, resulting in conversion of the duplex structure in a specific downstream region of DR into the single-stranded form. The degradation in the opposite direction had no effect on binding of Rep. These results suggest that a localized structural change of DNA adjacent to DR is required for Rep binding to double-stranded DR sequences. By contrast, exo III strikingly inhibited binding of Rep to DNA containing the IR sequences by introducing a single-stranded moiety into duplex IR sequences.     

Activation of the ATPase activity of adeno-associated virus Rep68 and Rep78

Rep68 and Rep78 DNA helicases, encoded by adeno-associated virus 2 (AAV2), are required for replication of AAV viral DNA in infected cells. They bind to imperfect palindromic elements in the inverted terminal repeat structures at the 3'- and 5'-ends of virion DNA. The ATPase activity of Rep68 and Rep78 is stimulated up to 10-fold by DNA containing the target sequence derived from the inverted terminal repeat; nontarget DNA stimulates ATPase activity at 50-fold higher concentrations. Activation of ATPase activity of Rep68 by DNA is cooperative with a Hill coefficient of 1.8 +/- 0.2. When examined by gel filtration at 0.5 M NaCl in the absence of DNA, Rep68 self-associates in a concentration-dependent manner. In the presence of DNA containing the binding element, Rep68 (and Rep78) forms protein-DNA complexes that exhibit concentration-dependent self-association in gel filtration analysis. The ATPase activity of the isolated Rep68-DNA and Rep78-DNA complexes is not activated by additional target DNA. Results of sedimentation velocity experiments in the presence of saturating target DNA are consistent with Rep68 forming a hexamer of the protein with two copies of the DNA element. Activation of the ATPase activity of Rep68 is associated with the formation of a protein-DNA oligomer.     

Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.     

The ColE2-P9 Rep protein binds to the origin DNA as a monomer

The Rep proteins of some plasmid replicons have two functions. Dimers bind to the operator sequences acting as auto-repressors, whereas monomers bind to the iterons to initiate replication of DNA. The ColE2 Rep proteins are present mostly in a dimeric form with some multimers larger than dimers in solution, while the form of Rep binding to Ori is not known. We used an EMSA-based method to determine the molecular weight of Rep in the Rep-Ori complex. The result suggested that Rep binds to Ori as a monomer. In addition, the result of EMSA using the Rep protein fused with the maltose binding protein and the His6-tag also supported this conclusion. We proposed that dimerization of Rep might probably be involved in keeping the copy number of the ColE2 plasmid at the normal low level by limiting the amount of active monomeric forms of Rep in the host cell.