SOS induction by P1 Km miniplasmids.

We have constructed (in vitro) a set of P1 miniplasmids. The smallest of these that could function as an independent replicon contained the right side of EcoRI-5 plus all of EcoRI-8. Those miniplasmids that lack EcoRI-6 induce the SOS pathway of the cell as shown by (i) increased expression of the recA operon, (ii) excision of the cryptic genetic element e14, (iii) spontaneous induction of lambda, and (iv) dependence of e14 excision on recA+ function. This induction was contingent upon the replication of the P1 Km miniplasmids from their P1 origin and, thus, was apparently caused by an aberrant initiation of DNA replication. When P1 EcoRI-6 was present in cis or trans with a P1 Km miniplasmid, neither e14 nor lambda was excised, but the expression of the recA operon was still induced. These results suggest that P1 EcoRI fragments 5 and 8 are insufficient for normal replication, and thus our P1 Km miniplasmids induced SOS functions. A product of EcoRI-6 may partially restore normal replication.     

In organello replication and viral affinity of linear, extrachromosomal DNA of the ascomycete Ascobolus immersus

Summary Linear, extrachromosomal DNA's of the filamentous fungus Ascobolus immersus are localized within the mitochondria. These linear plasmids have no homology to the high molecular weight mtDNA (hmw mtDNA). For analysis of plasmid replication an in organello DNA synthesis system was developed, in which radionucleotides were incorporated into intact mitochondria. Plasmid DNA is labelled preferentially in this system. From replication analysis of a specific plasmid there is evidence of a virus-like protein-primed replication. Sequence analysis of this plasmid reveals that a viral DNA polymerase is encoded. Thus, these genetic elements presumably are viral remnants rather than true plasmids.     

Replication of plasmid chimera pBR-mtB-A containing rat liver mtDNA fragment in Escherichia coli polA cells

Escherichia coli K-12 strains p108 (polA6), p3478 (polA1), and KS55 (polA12, ts) deficient in DNA polymerase I were transformed by recombinant pBR-mtB-A plasmid containing BamHI-A fragment of rat liver mtDNA and pBR322 plasmid. The physical map of the pBR-mtB-A, containing the recognition sites for SalI, EcoRI and HinIII endonucleases, was constructed and the orientation of mtDNA fragment joined to pBR322 plasmid was studied. The phenotypic selection using ampicillin containing medium at permissive and nonpermissive temperature (KS55 strain), or at 37 °C (polA6 and polA1 strains) revealed that only the cells transformed with the hybrid plasmid are able to grow under these conditions. The presence of mtDNA insertions in chimeric DNA molecules of pBR-mtB-A in polA strains was proved by electrophoretic and hybridization analysis. Thus the results obtained demonstrate the replication of the vehicle containing both plasmid replicon and mitochondrial origin in the conditions nonpermissive for the stable reproduction of the plasmid DNA alone.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Localization of an origin of DNA replication within the TRS/IRS repeated region of the herpes simplex virus type 1 genome

An assay has been developed and used to locate an origin of DNA replication on the herpes simplex virus type 1 (HSV-1) genome. Baby hamster kidney cells were transfected with circular plasmid molecules containing cloned copies of HSV-1 DNA fragments, and helper functions were provided by superinfection with wild-type HSV-1. The presence of an HSV-1 origin of replication within a plasmid enabled amplification of the vector DNA sequences, which was detected by the incorporation of [32P]orthophosphate. By screening various HSV-1 DNA fragments it was possible to identify a 995-bp fragment that maps entirely within the reiterated sequences flanking the short unique region of the viral genome and contains all the cis-acting signals necessary to function as an origin of viral DNA replication. The products of plasmid replication were shown to be high mol. wt. DNA molecules consisting of tandem duplications of the complete plasmid, suggesting that replication was occurring by a rolling-circle mechanism.     

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.     

Low doses of ultraviolet radiation and oxidative damage induce dramatic accumulation of mitochondrial DNA replication intermediates,fork regression,and replication initiation shift

Mitochondrial DNA is prone to damage by various intrinsic as well as environmental stressors. DNA damage can in turn cause problems for replication, resulting in replication stalling and double-strand breaks, which are suspected to be the leading cause of pathological mtDNA rearrangements. In this study, we exposed cells to subtle levels of oxidative stress or UV radiation and followed their effects on mtDNA maintenance. Although the damage did not influence mtDNA copy number, we detected a massive accumulation of RNA:DNA hybrid–containing replication intermediates, followed by an increase in cruciform DNA molecules, as well as in bidirectional replication initiation outside of the main replication origin, O_H. Our results suggest that mitochondria maintain two different types of replication as an adaptation to different cellular environments; the RNA:DNA hybrid–involving replication mode maintains mtDNA integrity in tissues with low oxidative stress, and the potentially more error tolerant conventional strand-coupled replication operates when stress is high.     

Template requirements for in vivo replication of adenovirus DNA.

The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.     

An enhancer of DNA replication.

cmp, a nucleotide sequence element in the plasmid pT181 of Staphylococcus aureus, acts as an enhancer of DNA replication. When cmp is present on an unrelated vector along with the pT181 origin of replication, it increases the ability of the linked pT181 origin to compete with a coresident pT181 plasmid for the initiator protein RepC. cmp is contained within a 156-base-pair segment, and its deletion from pT181 reduces by twofold the frequency of plasmid replication under derepressed conditions. The enhancer sequence contains a locus of DNA bending, and enhancer activity decreases with distance from the replication origin.     

Mechanism of plasmid pT181 DNA replication

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. This nick presumably serves as the start-site of pT181 replication by extension synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. In vitro replication of a recombinant plasmid carrying two pT181 origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading-strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two PT181 origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.     

Processing of plasmid DNA with ColE1-like replication origin

With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.     

Synthesis of single-stranded plasmid pT181 DNA in vitro. Initiation and termination of DNA replication

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.     

Behavior of the [Mi-3] Mutation and Conversion of Polymorphic Mtdna Markers in Heterokaryons of Neurospora Crassa

We have examined the behavior of the [mi-3] mitochondrial mutation and two physical mtDNA markers in heterokaryotic cultures of Neurospora crassa. Previous workers showed that a 1.2-kilobase insertion in the larger polymorphic form of EcoRI-5 restriction fragment is a site of high frequency and rapid unidirectional gene conversion. We have confirmed this observation and determined by DNA sequence analysis that the insertion in the EcoRI-5 fragment corresponds precisely to an optional intron that contains a long open reading frame in the ND1 gene. Thus, the conversion of the short, intron-lacking, form of EcoRI-5 to the longer, intron-containing, form may be analogous to the unidirectional gene conversion events catalyzed by intron-encoded proteins in other organisms. The resolution of two polymorphic forms of the mtDNA EcoRI-9 restriction fragment in our heterokaryons differs from that observed previously and suggests that this locus is not a site of gene conversion in our heterokaryon pair. The size polymorphism of the EcoRI-9 fragments is due to a tandemly reiterated 78-base-pair sequence which occurs two times in the short form and three times in the long form. One copy of the repeat unit and 66 base pairs following it have been duplicated from the ND2 gene which is located about 30 kilobases distant on the mtDNA. In contrast to the [poky] mitochondrial mutant, which was completely dominant over wild-type mitochondria in heterokaryons, the [mi-3] mutant was recovered in only seven of twenty heterokaryons after ten cycles of conidiation and subculturing. The resolution of the [mi-3] or wild-type phenotype in heterokaryons may depend solely on random factors such as allele input frequency, drift, and segregation rather than specific dominant or suppressive effects.     

Replication initiates at multiple locations on an autonomously replicating plasmid in human cells.

We have used a two-dimensional gel electrophoresis mapping technique to determine where DNA replication initiates on a plasmid which utilizes a fragment of human DNA to replicate autonomously in human cells. Replication was found to initiate at multiple locations on the plasmid carrying the human sequence, in contrast to the pattern seen for an Epstein-Barr virus vector which served as a control with a fixed origin. The family of repeats, a portion of the Epstein-Barr virus origin of replication which is present our plasmid, was shown to function as a replication fork barrier. The nature of the stalled replicative intermediates on the human DNA-based plasmid further indicated that replication did not initiate at a single fixed position each time the plasmid replicated. The results suggest that the replication apparatus used to duplicate DNA in human cells may not have precise sequence requirements which target initiation to specific locations.     

Klebsiella pneumoniae origin of replication (oriC) is not active in Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides.

A DNA fragment carrying genes encoding the conjugal transfer system of the broad host range plasmid RK2 was inserted into a plasmid carrying the chromosomal origin of replication (oriC) from Klebsiella pneumoniae. The resulting plasmid, pEON1, was readily transferred between gram-negative bacteria and carried two potential origins of replication: oriC and the replication origin from pBR322 (oriPBR). Although pEON1 could be transferred to Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides, pEON1 was not maintained in these strains. However, an oriC-containing plasmid was maintained in these nonenteric bacteria when an RK2 origin of replication was present on the plasmid. Thus, the inability of pEON1 to be established in a nonenteric bacterium represents a failure of oriC to function as an origin of replication rather than a toxic effect of oriC. The initiation potential of the chromosomal origin of replication from K. pneumoniae appears to be realized only in enteric bacteria.     

Fate of polyoma origin of replication after its direct introduction into mice

Recently we have developed a method for direct introduction of calcium phosphate-precipitated DNA into newborn rats. To examine whether the foreign DNA can replicate, a plasmid containing a polyoma origin of replication was injected into newborn mice. The plasmid was found intact in liver and spleen and able to transform bacteria. The foreign DNA had disappeared by the seventh day after injection. Yet, the plasmid DNA containing the polyoma origin of replication had undergone replication in both the liver and the spleen.     

A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.