Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Replication, Integration, and Packaging of Plasmid DNA following Cotransfection with Baculovirus Viral DNA

Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.     

Cloning and characterization of herpes simplex virus type 1 oriL: comparison of replication and protein-DNA complex formation by oriL and oriS.

The herpes simplex virus type 1 genome contains three origins of DNA replication: two copies of oriS and one copy of oriL. Although oriS has been characterized extensively, characterization of oriL has been severely limited by the inability to amplify oriL sequences in an undeleted form in Escherichia coli. We report the successful cloning of intact oriL sequences in an E. coli strain, SURE, which contains mutations in a series of genes involved in independent DNA repair pathways shown to be important in the rearrangement and deletion of DNA containing irregular structures such as palindromes. The oriL-containing clones propagated in SURE cells contained no deletions, as determined by Southern blot hybridization and DNA sequence analysis, and were replication competent in transient DNA replication assays. Deletion of 400 bp of flanking sequences decreased the replication efficiency of oriL twofold in transient assays, demonstrating a role for flanking sequences in enhancing replication efficiency. Comparison of the replication efficiencies of an 822-bp oriS-containing plasmid and an 833-bp oriL-containing plasmid demonstrated that the kinetics of replication of the two plasmids were similar but that the oriL-containing plasmid replicated 60 to 70% as efficiently as the oriS-containing plasmid at both early and late times after infection with herpes simplex virus type 1. The virus-specified origin-binding protein (OBP) and a cellular factor(s) (OF-1) have been shown in gel mobility shift experiments to bind specific sequences in oriS (C.E. Dabrowski, P. Carmillo, and P.A. Schaffer, Mol. Cell. Biol. 14:2545-2555, 1994; C.E. Dabrowski and P.A. Schaffer, J. Virol. 65:3140-3150, 1991). Although the nucleotides required for the binding of OBP to OBP binding site I in oriL and oriS are the same, a single nucleotide difference distinguishes OBP binding site III in the two origins. The nucleotides adjacent to oriS sites I and III have been shown to be important for the binding of OF-1 to oriS site I. Several nucleotide differences exist in these sequences in oriL and oriS. Despite these minor nucleotide differences, the protein-DNA complexes that formed with oriL and oriS sites I and III were indistinguishable when extracts of infected and uninfected cells were used as the source of protein. Furthermore, the results of competition analysis suggest that the proteins involved in protein-DNA complex formation with sites I and III of the two origins are likely the same.     

Cycled DNA immunoprecipitation procedure to enrich the target sequences for DNA binding proteins with the fold purification monitored.

Using centromere DNA binding protein (CENP-B) expressed as a fusion to beta-galactosidase in Escherichia coli, we established a cycled DNA immunoprecipitation procedure for enriching CENP-B binding sequences and monitoring the enrichment process. Degenerated synthetic oligonucleotides for an authentic CENP-B binding sequence, inserted into a pUC-derived vector, were incubated with the crude CENP-B extract. DNA-protein complexes formed in vitro were immunologically precipitated utilizing the beta-galactosidase moiety as a tagged antigen. The effectiveness of repeating cycles of immunoprecipitation was demonstrated by the color selection method designed for pUC-derived plasmids, after introducing the precipitated plasmids into Escherichia coli. After three cycles of DNA immunoprecipitation, only a few kinds of sequences constituted the majority. By repeating two more cycles, the most predominant sequence was finally enriched until homogeneous, indicating the enrichment of the binding sequences in a hierarchical order. Further application to human genomic DNA showed that two EcoRI DNA fragments, 0.49 and 0.78 kb in size, were exclusively identified. This procedure can be applied to the systematic analysis of binding sequences for any other DNA binding proteins without production of any specific antibodies or further purification.     

Cloning of DNA segments of phage 2C, which allows autonomous plasmid replication in Bacillus subtilis

The chromosome of Bacillus subtilis phage 2C is a 100-MDa double-stranded DNA molecule, containing hydroxymethyluracil in place of thymine and carrying redundant ends each encompassing 10% of the genome. 2C DNA was cleaved with EcoRI and HindIII, and cloned in the shuttle plasmids pSC 540 and pCP 115, both containing segments originating from B. subtilis and Escherichia coli plasmids. These chimaerical plasmids, carrying the chloramphenicol resistance gene, were unable to replicate in B. subtilis; this ability was restored, however, after the insertion of viral DNA segments. Physical maps of the recombinant plasmids were made; a large deletion of the E. coli-derived segment of pSC 540 was observed (which paralleled a loss of replication in this host), whereas addition of 2C DNA segments in pCP 115 was not accompanied by deletion (replication in E. coli was conserved in this case). Cloned viral segments mapped mostly, but not exclusively, within the redundant ends of 2C DNA. It is suggested that the thirteen recombinant clones carried the replication origin region of phage 2C DNA, and that these sequences originated within or close to the redundant extremities of the viral chromosome.     

In vivo resolution of circular plasmids containing concatemer junction fragments from minute virus of mice DNA and their subsequent replication as linear molecules.

During replication of their linear, single-stranded DNA genomes, parvoviruses generate a series of concatemeric duplex intermediates. We have cloned, into Escherichia coli plasmids, junction fragments from these palindromic concatemers of minute virus of mice DNA spanning both the right end-to-right end (viral 5' to 5') and left end-to-left end (viral 3' to 3') fusions. When mouse cells were transfected with these circular plasmids and superinfected with minute virus of mice, the viral junctions were resolved and the plasmids replicated as linear chromosomes with vector DNA in their centers and viral DNA at their termini. Resolution did not occur when the concatemer joint was replaced by a different palindromic sequence or when the transfected cells were not superinfected, indicating the presence of latent origins of replication which could only be activated by a viral trans-acting factor(s). Moreover, the products of resolution and replication from the two termini were characteristically different. Analysis of individual terminal fragments showed that viral 5' (right-end) sequences were resolved predominantly into "extended" structures with covalently associated copies of the virally encoded NS-1 polypeptide, while bridges derived from the 3' (left) end resolved into both NS-1-associated extended termini and lower-molecular-weight "turn-around" forms in which the two DNA strands were covalently continuous. This pattern of resolution exactly coincides with that seen at the two termini of replicative-form intermediates in normal virus infections. These results demonstrate that the bridge structures are authentic substrates for resolution and indicate that the frequency with which extended versus turn-around forms of each terminus are generated is an intrinsic property of the telomere.     

DnaA Protein of Escherichia coli: oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids

Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non-permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC-containing plasmid, suggesting that the structure of the DnaA-oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.     

Human origins of DNA replication selected from a library of nascent DNA

The identification of metazoan origins of DNA replication has so far been hampered by the lack of a suitable genetic screening and by the cumbersomeness of the currently available mapping procedures. Here we describe the construction of a library of nascent DNA, representative of all cellular origin sequences, and its utilization as a screening probe for origin identification in large genomic regions. The procedure developed was successfully applied to the human 5q31.1 locus, encoding for the IL-3 and GM-CSF genes. Two novel origins were identified and subsequently characterized by competitive PCR mapping, located approximately 3.5 kb downstream of the GM-CSF gene. The two origins (GM-CSF Ori1 and Ori2) were shown to interact with different members of the DNA prereplication complex. This observation reinforces the universal paradigm that initiation of DNA replication takes place at, or in close proximity to, the binding sites of the trans-acting initiator proteins.     

Requirements for species-specific papovavirus DNA replication.

Replication of papovavirus DNA requires a functional replication origin, a virus-encoded protein, large T antigen, and species-specific permissive factors. How these components interact to initiate and sustain viral DNA replication is not known. Toward that end, we have attempted to identify the viral target(s) of permissive factors. The functionally defined replication origins of polyomavirus and simian virus 40, two papovaviruses that replicate in different species (mice and monkeys, respectively), are composed of two functionally distinct domains: a core domain and an auxiliary domain. The origin cores of the two viruses are remarkably similar in primary structure and have common binding sites for large T antigen. By contrast, their auxiliary domains share few sequences and serve as binding sites for cellular proteins. It seemed plausible, therefore, that if cellular permissive factors interacted with the replication origin, their targets were likely to be in the auxiliary domain. To test this hypothesis we constructed hybrid origins for DNA replication that were composed of the auxiliary domain of one virus and the origin core of the other and assessed their capacity to replicate in a number of mouse and monkey cell lines, which express the large T antigen of one or the other virus. The results of this analysis showed that the auxiliary domains of the viral replication origins could substitute for one another in DNA replication, provided that the viral origin core and its cognate large T antigen were present in a permissive cellular milieu. Surprisingly, the large T antigens of the viruses could not substitute for one another, regardless of the species of origin of the host cell, even though the two large T antigens bind to the same sequence motif in vitro. These results suggest that species-specific permissive factors do not interact with the origin-auxiliary domains but, rather, with either the origin core or the large T antigen or with both components to effect DNA replication.     

Herpes simplex virus induces the replication of foreign DNA.

Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.     

Specificity of RepC protein in plasmid pT181 DNA replication

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 DNA replication specifically requires the plasmid-encoded initiator protein, RepC. The initiator protein binds specifically to a 32-base pair sequence within the pT181 origin of replication. RepC protein also has a nicking-closing activity that is specific for the pT181 origin. Replication of pT181 initiates by covalent extension of the nick and proceeds by a rolling circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural organization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross-reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC221 and pS194 replicated efficiently in a RepC-dependent in vitro system. However, replication of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.     

T-antigen binding to site I facilitates initiation of SV40 DNA replication but does not affect bidirectionality.

SV40 origin auxiliary sequence 1 (aux-1) encompasses T-antigen (T-ag) binding site I and facilitates origin core (ori-core) activity in whole cells or cell extracts. Aux-1 activity depended completely upon its sequence, orientation and spacing relative to ori-core. Aux-1 activity was lost either by inserting 10 base pairs between aux-1 and ori-core or by placing either orientation of aux-1 on the opposite side of ori-core. Reversing the orientation of aux-1 in its normal position actually inhibited replication. Easily unwound DNA sequences that stimulate yeast or E. coli origins of replication could not replace aux-1. Aux-1 did not affect bidirectional replication. Replication remained bidirectional even when aux-1 was inactivated, and deletion of aux-1 did not affect selection of RNA-primed DNA synthesis initiation sites in the origin region: the transition from discontinuous to continuous DNA synthesis that marks the origin of bidirectional replication occurred at the same nucleotide locations in both wild-type and aux-1 deleted origins. These results support a model for initiation of SV40 DNA replication in which T-ag binding to aux-1 (T-ag binding site I) facilitates the efficiency with which T-ag initiates replication at ori-core (T-ag binding site II) without affecting the mechanism by which initiation of DNA replication occurs.     

Plasmid rolling-circle replication: highlights of two decades of research

This review provides a historical perspective of the major findings that contributed to our current understanding of plasmid rolling-circle (RC) replication. Rolling-circle-replicating (RCR) plasmids were discovered approximately 20 years ago. The first of the RCR plasmids to be identified were native to Gram-positive bacteria, but later such plasmids were also identified in Gram-negative bacteria and in archaea. Further studies revealed mechanistic similarities in the replication of RCR plasmids and the single-stranded DNA bacteriophages of Escherichia coli, although there were important differences as well. Three important elements, a gene encoding the initiator protein, the double strand origin, and the single strand origin, are contained in all RCR plasmids. The initiator proteins typically contain a domain involved in their sequence-specific binding to the double strand origin and a domain that nicks within the double strand origin and generates the primer for DNA replication. The double strand origins include the start-site of leading strand synthesis and contain sequences that are bound and nicked by the initiator proteins. The single strand origins are required for synthesis of the lagging strand of RCR plasmids. The single strand origins are non-coding regions that are strand-specific, and contain extensive secondary structures. This minireview will highlight the major findings in the study of plasmid RC replication over the past twenty years. Regulation of replication of RCR plasmids will not be included since it is the subject of another review.     

In vivo selection of randomly mutated retroviral genomes.

Darwinian evolution, that is the outgrowth of the fittest variants in a population, usually applies to living organisms over long periods of time. Recently, in vitro selection/amplification techniques have been developed that allow for the rapid evolution of functionally active nucleic acids from a pool of randomized sequences. We now describe a modification of the nucleic acid-evolution protocol in which selection and amplification take place inside living cells by means of a retroviral-based replication system. We have generated a library of HIV-1 DNA genomes with random sequences in particular domains of the TAR element, which is the binding site for the Tat trans-activator protein. This mixture of HIV genomes was transfected into T cells and outgrowth of the fittest viruses was observed within two weeks of viral replication. The results of this in vivo selection analysis are consistent with the notion that primary sequence elements in both TAR bulge and loop domains are critical for Tat-mediated trans-activation and viral replication.     

DNA bending in the mycobacterial plasmid pAL5000 origin-RepB complex

Plasmid pAL5000 represents a family of relatively newly discovered cryptic plasmids in gram-positive Actinomycetes bacteria. The replication regions of these plasmids comprise a bicistronic operon, repA-repB, encoding two replication proteins. Located upstream is a cis-acting element that functions as the origin of replication. It comprises an ~200-bp segment spanning two binding sites for the replication protein RepB, a low-affinity (L) site and a high-affinity (H) site separated by an ~40-bp spacer sequence. The trajectory of the DNA in the RepB-origin complex has been investigated, and it has been found that the origin undergoes significant bending movements upon RepB binding. RepB binding not only led to local bending effects but also caused a long-range polar curvature which affected the DNA sequences 3′ to the H site. These movements appear to be essential for the in-phase alignment of the L and H sites that leads to the formation of a looped structure. A novel property of RepB unearthed in this study is its ability to form multimers. This property may be an important factor that determines the overall trajectory of the DNA in the RepB-origin complex. The results presented in this study suggest that the origins of replication of pAL5000 and related plasmids are highly flexible and that multimeric, RepB-like initiator proteins bind the origin and induce local deformations and long-range curvatures which are probably necessary for the proper functioning of the origin.     

Repetitive plasmid sequences generate DNA fingerprinting patterns in mammals

Bacterial plasmids with stringently regulated copy numbers have directly repeated DNA sequences, termed iterons, in the vicinity of their replication origins. These sequences bind a specific protein exerting a key role in the initiation of plasmid replication. Plasmids P1, pSC101 and RFS1010 have different iteron sequences and belong to three different incompatibility groups. Used as DNA probes each of these plasmids generates specific patterns in mammals similar to those obtained by the DNA fingerprinting technique. The iteron-containing regions were identified as the part of the plasmids responsible for those patterns by using polymerase chain reaction (PCR) amplified DNA segments that contained the iteron regions as probes.     

Hemimethylation prevents DNA replication in E. coli

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.