R-loop-dependent rolling-circle replication and a new model for DNA concatemer resolution by mitochondrial plasmid mp1

The mitochondrial (mt) plasmid mp1 of Chenopodium album replicates by a rolling-circle (RC) mechanism initiated at two double-stranded replication origins (dso1 and dso2). Two-dimensional gel electrophoresis and electron microscopy of early mp1 replication intermediates revealed novel spots. Ribonucleotide (R)-loops were identified at dso1, which function as a precursor for the RCs in vivo and in vitro. Bacteriophage T4-like networks of highly branched mp1 concatemers with up to 20 monomer units were mapped and shown to be mainly formed by replicating, invading, recombining and resolving molecules. A new model is proposed in which concatemers were separated into single units by a "snap-back" mechanism and homologous recombination. dso1 is a recombination hotspot, with sequence homology to bacterial Xer recombination cores. mp1 is a unique eukaryotic plasmid that expresses features of phages like T4 and could serve as a model system for replication and maintenance of DNA concatemers.     

Role of individual monomers of a dimeric initiator protein in the initiation and termination of plasmid rolling circle replication

Plasmids of the pT181 family encode initiator proteins that act as dimers during plasmid rolling circle (RC) replication. These initiator proteins bind to the origin of replication through a sequence-specific interaction and generate a nick at the origin that acts as the primer for RC replication. Previous studies have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for plasmid replication. The tyrosine residue at position 191 of the initiator RepC protein of pT181 is known to be involved in nicking at the origin. We have generated heterodimers of RepC that consist of different combinations of wild type, DNA binding, and nicking mutant monomers to identify the role of each of the two monomers in RC replication. One monomer with DNA binding activity was sufficient for the targeting of the initiator to the origin, and the presence of Tyr-191 in one monomer was sufficient for the initiation of replication. On the other hand, a dimer consisting of one monomer defective in DNA binding and the other defective in origin nicking failed to initiate replication. Our results demonstrate that the monomer that promotes sequence-specific binding to the origin must also nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of the initiator was required for nicking at the origin to initiate replication, it was dispensable for termination, suggesting that alternate amino acids in the initiator may promote termination but not initiation.     

Atypical location of double-strand origin of replication (nic site) on the plasmid pGA1 from Corynebacterium glutamicum

The double-strand origin of replication (dso) of the rolling-circle-replicating (RC) plasmid pGA1 from Corynebacterium glutamicum was analyzed using the runoff DNA synthesis assay. The site- and strand-specific breakage of double-stranded plasmid DNA, representing the nic site of dso, was localized precisely within the sequence 5'-CTGG decreases AT-3' in the distal part of the pGA1 rep gene. This location of dso differs from the dso positions found on other RC plasmids and is in agreement with the classification of the plasmid pGA1 into a new group of RC plasmids.     

Rolling-circle replication of mitochondrial DNA in the higher plant Chenopodium album (L.).

The mitochondrial genomes of higher plants are larger and more complex than those of all other groups of organisms. We have studied the in vivo replication of chromosomal and plasmid mitochondrial DNAs prepared from a suspension culture and whole plants of the dicotyledonous higher plant Chenopodium album (L.). Electron microscopic studies revealed sigma-shaped, linear, and open circular molecules (subgenomic circles) of variable size as well as a minicircular plasmid of 1.3 kb (mp1). The distribution of single-stranded mitochondrial DNA in the sigma structures and the detection of entirely single-stranded molecules indicate a rolling-circle type of replication of plasmid mp1 and subgenomic circles. About half of the sigma-like molecules had tails exceeding the lengths of the corresponding circle, suggesting the formation of concatemers. Two replication origins (nicking sites) could be identified on mpl by electron microscopy and by a new approach based on the mapping of restriction fragments representing the identical 5' ends of the tails of sigma-like molecules. These data provide, for the first time, evidence for a rolling-circle mode of replication in the mitochondria of higher plants.     

Effects of temperature-sensitive variants of the Bacillus subtilis dnaB gene on the replication of a low-copy-number plasmid.

The dnaB gene of Bacillus subtilis is involved in the initiation of DNA replication and also in the binding of the chromosomal origin to the bacterial membrane. We studied the effect of temperature-sensitive dnaB mutants (dnaB1 and dnaB19) on the replication and on the DNA-membrane binding of the plasmid pKW1, which was derived from the low-copy-number plasmid pBS2. In the dnaB19 mutant, pKW1 was not able to replicate at the restrictive temperature. In the dnaB1 mutant, however, the dimeric form of pKW1 DNA was preferentially produced as the restrictive temperature, but the replication of the monomeric form was totally blocked. We also examined the effects of the dnaB(Ts) gene on the DNA-membrane binding of both the double-stranded and single-stranded DNA from pKW1. The single-stranded DNA from pKW1 was prepared from the DNA of the phage M13 mp19, which contained the origin of replication of pKW1. In the dnaB1 mutant, pKW1 DNA in both the double-stranded and single-stranded form was released from the membrane at the restrictive temperature. On the other hand, in the dnaB19 mutant, only double-stranded DNA, and not single-stranded DNA, was released from the membrane at the restrictive temperature. These results suggest that the product of the dnaB gene has at least two domains which influence the replication of DNA and the binding of DNA to the cell membrane in separate ways.     

Inheritance of the replication complex by one of two daughter copies during lambda plasmid replication in Escherichia coli.

Direct measurement of DNA synthesis confirmed that lambda plasmid replication proceeds for several hours in an amino acid-starved relA mutant of Escherichia coli, leading to plasmid amplification; this replication is lambda cro-independent, but requires the function of lambda O initiator in the absence of its synthesis. This suggests that after the assembly of the replication complex (RC) at ori lambda the lambda O protein remains in this structure and the affinity of lambda O to ori lambda is alleviated in the assembled RC allowing its movement along the DNA. During amino acid starvation the lambda plasmid DNA synthesis per bacterial mass occurs at a constant level, as would be expected if the number of functioning RCs remained constant. This favors the idea that under these conditions the next replication round operates due to the activity of the RC inherited from the preceding round. Density shift experiments reveal indeed that, from two daughter plasmid copies synthesized after the onset of amino acid starvation only one is able to enter into the next round of replication. We infer that this is the plasmid copy that inherits the lambda O-enclosing RC from the previous replication round. Moreover, the same results of density shift experiments were obtained for plasmids synthesized before the onset of amino acid starvation. Therefore, we presume that in lambda plasmid-harboring bacteria growing in nutrient medium, every second plasmid circle bears an RC that originates from the preceding round of replication. This structure has to be assembled de novo only on the daughter plasmid copy that does not inherit the parental RC. In the absence of lambda O initiator synthesis in amino acid-starved relA cells this process cannot occur, leaving as the only replication pathway that driven by the parental RC. Our results are discussed in relation to the model of regulation of lambda plasmid replication.     

Small cryptic plasmids of Streptococcus pneumoniae belong to the pC194/pUB110 family of rolling circle plasmids

The DNA sequences of two related plasmids pPR1 and pPR3 described previously in Streptococcus pneumoniae isolates from Germany and Spain were now determined. Both plasmids belong to a family of rolling circle (RC) plasmids found in a variety of bacteria. Their GC content with 32% is lower than that of the S. pneumoniae chromosomal DNA. The plasmid pPR3 has a molecular size of 3160 bp with four putative open reading frames, whereas pPR1 contained a deletion of 313 bp that included the 5′-part of ORF2 and upstream regions and differed by three bp from pPR3. The predicted protein of ORF1 showed high similarity to replication proteins of RC plasmids with 74% identical amino acids to RepA of Streptococcus thermophilus plasmids. Sequences similar to the plus origin of replication of ssDNA plasmids were present in both plasmids. They also contained a 152-bp region with over 83% identity to the minus origin of replication of the Streptococcus agalacticae plasmid pMV158.     

Characterization of putative rolling-circle plasmids from the Gram-negative bacterium Xylella fastidiosa and their use as shuttle vectors

This study was initiated to characterize a small Xylella fastidiosa (X. fastidiosa) plasmid and attempt to create a X. fastidosa/Escherichia coli shuttle vector that was stable in planta. Restriction enzyme analysis of a 1.3kb plasmid DNA from a grape-infecting strain of X. fastidiosa (UCLA) revealed the presence of three similar, but genetically distinct, plasmids, pUCLAs. Evidence that suggests the pUCLA plasmids replicate via a rolling-circle (RC) mechanism include: (i) the presence of ssDNA in X. fastidiosa cells; (ii) the presence of conserved motifs in the predicted ORF1 that are typical of initiator (Rep) proteins associated with RC replication; (iii) high amino acid identity between the putative Rep proteins of pUCLAs and Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa that replicates by a RC mechanism; and (iv) the presence of a putative origin of replication upstream of ORF1 that has the potential to form secondary hairpin structures. One DNA motif present in pUCLA shared sequence similarity to known nicking sites in the origins of replication of other RC plasmids and phages. The shuttle vector, pXF001, successfully transformed grape X. fastidiosa strains and was found to be present as autonomous, structurally unchanged DNA molecules in X. fastidiosa. However, pXF001 was not stably maintained in X. fastidiosa without antibiotic selection.     

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.     

Replication enhancer-independent mutation increases the co-operativity with which an initiator protein binds its origin

The plus-strand replication origin of bacteriophage fl has a bipartite structure consisting of a required core origin region and an adjacent A + T-rich enhancer sequence that potentiates replication approximately 100-fold. The core origin binds the initiator protein (gpII) and the enhancer binds the Escherichia coli integration host factor (IHF). gpII binds the core origin in two steps, forming a binding intermediate (complex I) and a functional complex for nicking (complex II). We have used a double-label gel binding assay to determine the stoichiometry of the gpII-origin interaction. The results indicate that complex I contains two gpII molecules per origin, and complex II contains four gpII molecules per origin. Enhancer-independent mutations in gpII allow wild-type levels of replication in the absence of either the enhancer or IHF. We have examined the binding of an enhancer-independent gpII mutant (mp1) protein to the replication origin. The mp1 mutation in gpII (Met40----Ile) increases the co-operativity with which the protein binds to form complex II. In addition, the mutant gpII forms both complexes with a DNA fragment containing only two (beta-gamma) of the three repeats from the core origin sequence, while the wild-type protein forms only complex I with this fragment. We discuss how a mutation that increases the co-operativity of binding of an initiator protein might stimulate DNA replication.     

ORC binding to TRF2 stimulates OriP replication

In higher eukaryotes, the origin recognition complex (ORC) lacks sequence-specific DNA binding, and it remains unclear what other factors specify an origin of DNA replication. The Epstein-Barr virus origin of plasmid replication (OriP) recruits ORC, but the precise mechanism of ORC recruitment and origin activation is not clear. We now show that ORC is recruited selectively to the dyad symmetry (DS) region of OriP as a consequence of direct interactions with telomere repeat factor 2 (TRF2) and ORC1. TRF-binding sites within DS stimulate replication initiation and facilitate ORC recruitment in vitro and in vivo. TRF2, but not TRF1 or hRap1, recruits ORC from nuclear extracts. The amino-terminal domain of TRF2 associated with a specific region of ORC1 and was necessary for stimulation of DNA replication. These results support a model in which TRF2 stimulates OriP replication activity by direct binding with ORC subunits.     

Evidence for a rolling-circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXphi

The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single-stranded DNA genome of CTXphi. In toxigenic V. cholerae strains, the CTXphi genome is typically found in integrated arrays of tandemly arranged CTX prophages. Infected cells that lack a chromosomal integration site harbour the CTXphi genome as a plasmid (pCTX). We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling-circle (RC) mechanism. The initiation and termination sites for pCTX plus-strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus-strand origins of several RC replicons. Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin. Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages. We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus-strand pCTX DNA is initiated and terminated. These data support the model that plus-strand CTXphi DNA is generated from chromosomal prophages via a novel process analogous to RC replication.     

The affinity of EBNA1 for its origin of DNA synthesis is a determinant of the origin's replicative efficiency

Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research.     

DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.     

Characterization of a novel plasmid-like element in Neurospora crassa derived mostly from the mitochondrial DNA.

We have identified a plasmid-like element within mitochondria of Neurospora crassa strain stp-B1. It is derived from the EcoRI-4 and EcoRI-6 regions of the mitochondrial DNA, and an additional 124 bp DNA segment of unknown origin. The plasmid DNA consists of an oligomeric series of circular molecules of monomer length 2.2 kbp. The abundance of the plasmid suggests its autonomous replication and the presence of an efficient origin of replication. An unusually large number of palindromes capable of forming secondary structures are present in the plasmid. Such a palindrome, located near sequences reminiscent of mammalian and fungal mtDNA origins of replication, may define the replication origin of the plasmid. This putative origin might also represent the replication origin of the wild-type mtDNA.     

Replication of a plasmid containing two origins of bacteriophage f1

A chimeric plasmid was constructed that contains a tandem duplication of the bacteriophage f1 origin of DNA replication. This plasmid replicates stably in the absence of phage. When cells carrying this plasmid are infected with f1, two new plasmid-derived DNA species are generated: a smaller, chimeric plasmid containing only one f1 origin of replication, and a miniphage, the genome of which consists of the f1 fragment that was located between the two f1 origins of the original plasmid. These data support the hypothesis (Horiuchi, 1980) that the nucleotide sequence recognized for initiation of plus strand synthesis in f1 DNA replication is also the signal for its termination.     

Rolling-circle replication of bacterial plasmids.

Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication.     

Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing

We have constructed chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII ( pZ145 ) and AhaIII ( pZ150 , pZ152 ) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145 , plasmid pZ146 , that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.