The replication termination signal terB of the Escherichia coli chromosome is a deletion hot spot.

Hybrids composed of phage M13, plasmid pBR322 and the termination signal of Escherichia coli chromosome replication terB were used to show that arrest of DNA synthesis creates a very efficient deletion hot spot. Up to 80% of deletions occurring in these hybrids had one deletion end-point at terB provided that (i) terB was oriented to arrest M13 and pBR322 leading strand synthesis; and (ii) the host cells contained the Tus protein necessary for arresting DNA synthesis at terB. The position of terB and the flanking sequences had little effect on deletion hot spot activity. About 90% of the deletions at terB ended 5-6 nucleotides in front of the major replication arrest site. We propose two models to account for deletion formation and speculate that many genome rearrangements may be due to the pausing of DNA replication.     

Deletion analysis of the cloned replication origin region from bacteriophage M13.

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.     

Blocking rolling circle replication with a UV lesion creates a deletion hotspot

UV light irradiation increases genetic instability by causing mutations and deletions. The mechanism of UV-induced rearrangements was investigated making use of deletion-prone plasmids. Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide. A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed. In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots. These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate. They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion. Furthermore, UV-induced deletions occurred independently of pBR322 replication. We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone. We propose that the deletion-prone properties of a UV-arrested polymerase depend on the associated helicase.     

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.     

Deletion hot spots in chimeric Escherichia coli plasmids.

Deletions form frequently in chimeric plasmids composed of M13mp2, pBR322, and pC194 (B. Michel and S. D. Ehrlich, Proc. Natl. Acad. Sci. USA 83:3386-3390, 1986). They are generated by joining of the nucleotide neighboring the nick site in the M13 replication origin to a nonadjacent nucleotide. This nucleotide is most often located within particular short plasmid regions, named deletion hot spots. Three natural hot spots were present in the chimeric plasmids. Two were active only when the DNA replication initiated at the M13 origin was allowed to progress; the third was active only in the presence of wild-type amounts of DNA ligase. Three artificial hot spots were generated by creating palindromic sequences in the plasmids.     

Generation of deletions through a cis-acting mutation in plasmid pC194

Summary When plasmid pC194-1 is ligated to pBR322 to generate plasmid pHV15-1, deletions occur with high frequency within the joined pBR322 DNA. Generation of deletions is recE4 independent, and occurs in B. subtilis with a 1,000-fold higher frequency than in Escherichia coli. In the hybrid plasmid pVH15-1, deletion end-points are not at random, but at defined locations within pBR322. We propose that the base alteration, characterizing pC194-1, has stabilized within the plasmid a stem/loop structure, which acts as a deletion generator.     

Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.     

Recovery of recombinant bacterial plasmids from E. coli transformed with DNA from microinjected mouse cells

We have previously described the isolation of thymidine kinase positive (TK+), human beta-globin gene-containing colonies following co-microinjection of mouse TK- L cells with two recombinant pBR322 plasmids, one containing the TK gene of herpes simplex virus type I (plasmid pXl), and the second containing a human genomic DNA fragment within which is the human beta-globin gene (plasmid pRKl). DNA isolated from one such clone was used in bacterial transformation experiments with a selection for tetracycline-resistant colonies (that is, for cells containing pRKl). A total of forty-two tetracycline-resistant colonies were isolated, thirty of which contained circular pRK1 molecules identical to those originally injected. The remaining twelve colonies contained unique plasmids that were grouped into five different classes of recombinant molecules. All five of these unique recombinant classes appear to contain a common deletion endpoint occurring at a specific region of the pBR322 segment of pRKl. Four of the unique recombinant classes appear to have arisen from the deletion of a segment of a pRKl trimer or dimer molecule, while the fifth class appears to have resulted from recombination between pRKl and pXl followed by a deletion event within this recombinant. It is uncertain whether these deletions are occurring within the eukaryotic cell or upon subsequent transformation of the bacterial cell. If the latter, then the passage of the plasmid DNA through the eukaryotic cell alters a specific site of the pBR322 DNA in such a way that deletions can occur at a high frequency in this region when the plasmid DNA is introduced back into a bacterial cell. Thus, we have established a prokaryote-eukaryote-prokaryote DNA transfer and recovery system which should be useful in studies on DNA replication and the regulation of gene expression in higher eukaryotes.     

n' Protein activator sites of plasmid pBR322 are not essential for its DNA replication

The lagging strand DNA synthesis of the Escherichia coli bacterial chromosome and plasmids is thought to be initiated by the mobile promotor, the primosome. This primosome is assembled at a specific site on single-stranded DNA. This process is initiated by the interaction of one of the at least seven components, the n' protein, with this site. Indeed n' protein activator sites are found in the plasmids Col E1 and pBR322. To investigate the in vivo function of these n' protein sites, deletion derivates of pBR322 were constructed in which the n' protein sites are removed. The deletion plasmids show no change in stability and only threefold reduction in copy number compared to pBR322. Using a transduction system for single-stranded plasmid DNA it was shown that no other specific initiation signals for lagging strand DNA synthesis were present in the deletion plasmids. It was concluded that the n' protein activator sites in pBR322 are not essential for its DNA replication in vivo.     

Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage.

Escherichia coli plasmids pBR313 and pBR322 were transduced by phage M13 with low efficiency (10(-8) transductants/phage). Hybrid plasmids pHV12 or pHV33, composed of Staphylococcus aureus plasmid pC194 and pBR313 or pBR322, respectively, were transduced much more efficiently (10(-4) transductants/phage). Inactivation of either of the two zones necessary for pC194 replication, one coding for a protein, the other not, reduced the transforming efficiency of hybrids to the level of pBR322. Activity of the pC194 replication region was not necessary for the formation of chimeras between M13 and the transduced plasmid in the donor cells, but rather for the establishment of the plasmid in the recipient cells.     

Mechanism of intramolecular recyclization and deletion formation following transformation of Escherichia coli with linearized plasmid DNA.

The deletion end-points of a number of type I (less than monomeric) plasmid deletants obtained by transforming recA+ or recA- E. coli with linear pBR322 DNA were determined by DNA sequencing. In both monodirectional and bidirectional deletions the recyclization point was normally characterized by recombination between directly repeated sequences of between 4 and 10 bp present on each arm of the linearized pBR322 molecule. Frequently, short tracts of uninterrupted homology involved in recombinational recircularization were embedded in regions of relative non-homology. A model predicting the probability of matching sequences in either end of a linear plasmid molecule is presented. It is proposed that exonucleolytic processing of the exposed termini of linear plasmid molecules generates substrates for subsequent recombinational recyclization and deletion. The activity of host recombination and repair functions in recircularizing linear DNA molecules explains the generation of many of the aberrant recombinant DNA constructs obtained during gene cloning procedures.     

Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin

The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome. The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA. Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site. This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome. We refer to this part of the intergenic region as a "replication enhancer" sequence. We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication. Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein.     

Illegitimate recombination occurs between the replication origin of the plasmid pC194 and a progressing replication fork.

Hybrids between plasmids pC194, pBR322 and the bacteriophage f1 undergo deletions in Escherichia coli. The deletions end most often between nucleotides 1445 and 1446 of pC194. That site probably corresponds to a nick in the replication origin of this plasmid. The localization of the other deletion end appears to be determined by the position of the f1 replication fork. Two models accounting for these data are discussed.     

Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by a cell membrane fraction.

The replication of both ColE1-type plasmids and plasmids bearing the origin of replication of the Escherichia coli chromosome (oriC) has been shown to be inhibited by hemimethylation of adenine residues within GATC sequences. In the case of oriC plasmids, this inhibition was previously shown to be mediated by the specific affinity of the hemimethylated origin DNA for an outer cell membrane fraction. Here, we suggest that a similar mechanism is operating in the case of the ColE1-like plasmid pBR322 as (i) a hemimethylated DNA fragment carrying the promoter for the RNA which primes DNA synthesis (RNAII) is specifically bound by the same membrane fraction and, (ii) the addition of the membrane fraction to a soluble assay of pBR322 replication results in preferential inhibition of initiation on the hemimethylated template. We suggest that membrane sequestration of hemimethylated origin DNA and/or associated replication genes following replication may be a common element restricting DNA replication to precise moments in the cell cycle.     

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.