Characterization of a mini-F plasmid derived from an F mutant expressing incompatibility in the autonomous but not in the integrated state

Plasmid DNA from Escherichia coli F' ser/MA219 harboring an altered F' factor, which expressed incompatibility in the autonomous but not in the integrated state (DeVries and Maas, 1973, J. Bacteriol. 115, 213-220), was digested with the restriction endonuclease EcoRI and ligated to a nonreplicating trpED fragment. A miniplasmid was obtained containing a 5.7-kb EcoRI fragment capable of self-replication. This plasmid, designated pRE300, was incompatible with mini-F as well as with ColE1 derivatives. It represents a cointegrate formed in vivo between a 2.2-kb segment of the F replication region and a ColE1-type replicon of unknown derivation. The F-derived component of pRE300 corresponds to a minimalized F replicon (43.85-46.05 kb F) retaining oriII and the incB locus but missing the incC and incD functions. It is postulated that the Inc- mutation resulted from the insertion of a transposable DNA sequence into the incC locus of the parent F plasmid.     

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.     

Direct repeats of the F plasmid incC region express F incompatibility

The nucleotide sequence of the incompatibility region incC, located at 45.8--46.4 kb on the F plasmid map, was determined. This region consists of 543 bp and contains sufficient information to code for only two small polypeptides of 34 and 30 amino acids each. Deletion of the ATG start codons for these two polypeptides has no effect on expression of incC incompatibility. A prominent feature of this sequence is the presence of five 22 bp direct repeats. A 58 bp segment of the incC region that contains two of these direct repeats was inserted into plasmid pACYC184, which is compatible with the F plasmid. The pACYC184 plasmid containing the direct-repeat sequences now expresses incompatibility with the F'lac plasmid and replication-proficient derivatives of the mini-F plasmid.     

The iteron regions necessary for the RepE-iteron interaction in vivo in mini-F plasmids of Escherichia coli

We have determined the nucleotide positions of an incC iteron essential for RepE binding by analyzing mutated incC iterons defective in exerting incompatibility towards mini-F plasmids. The mutations affecting this incompatibility occurred mostly at two positions within the incC iteron, i.e. an iteron conserved position and a mini-F specific position. Most of the iterons with a base-change at either of these two positions had lost the binding affinity for RepE. This agrees with the crystallographic structure of the RepE-iteron complex which showed that the N and C terminal domains of RepE interact with the two major grooves on one face of the iteron DNA. These grooves contain the iteron conserved and mini-F specific positions necessary for RepE binding. Thus the binding mode may be common to in the case of mini-F like plasmids.     

DNA sequence of an amber replication mutant indicates that a 29 kd protein is the product of the F plasmid replication gene

Summary DNA sequencing shows that the mutational alteration resulting from an amber-suppressible replication-defective mutation of F plasmid is a single base pair change from C:G to T:A which yields an amber codon in the coding frame for a 29 kd polypeptide located in the minimal replication region. We thus identified the gene indispensable for F DNA replication as the coding frame which encodes a 29 kb polypeptide. We will designate this gene repA.Abbreviations kb kilobase pairs - bp base pair - kd kilodaltons     

Isolation and characterization of plasmids carrying a partially defective Escherichia coli replication origin

The replication origin (oriC) of the Escherichia coli chromosome has been cloned and the region essential for chromosomal replication has been delimited to 245 base pairs. In previous studies the ability of recombinants between oriC and ColE1-type vectors, to transform E. coli polA- strains was used to determine which nucleotides in oriC are essential for replication. In this paper we have used a different approach by isolating partial defective replication mutants of a minichromosome (pCM959) that contains oriC as the single replication origin. Our results demonstrate that many mutations are allowed within oriC that do not affect oriC function as measured by the ability to transform E. coli polA- strains. In the minimal oriC region we detected 8 mutations at positions that are conserved in the sequence of six bacterial origins. The implications of these results on previous work will be discussed. Our data also demonstrate that a mutation producing an oriC- phenotype may be suppressed by secondary mutations. An E. coli strain was found that facilitates the isolation of partially defective minichromosomes. The results with this strain indicate a specific function of the sequence surrounding the base pair at position 138.     

Different phenotypes of Walker-like A box mutants of ParA homolog IncC of broad-host-range IncP plasmids

The promiscuous IncPα plasmids RK2 and R995 encode a broad-host-range partition system, whose essential components include the incC and korB genes and a DNA site (O(B)) to which the korB product binds. IncC2, the smaller of the two incC products, is sufficient for stabilization of R995ΔincC. It is a member of the type Ia ParA family of partition ATPases. To better understand the role of ATP in partition, we constructed three alanine-substitution mutants of IncC2. Each mutation changed a different residue of the Walker-like ATP-binding and hydrolysis motif, including a lysine (K10) conserved solely among members of the ParA and MinD families. All three IncC2 mutants were defective in plasmid partition, but they differed from one another in other respects. The IncC2 T16A mutant, predicted to be defective in Mg2? coordination, was severely impaired in all activities tested. IncC2 K10A, predicted to be defective in ATP hydrolysis, mediated enhanced incompatibility with R995 derivatives. IncC2 K15A, predicted to be defective in ATP binding, exhibited two distinct incompatibility properties depending on the genotype of the target plasmid. When in trans to plasmids carrying a complementable incC deletion, IncC2 K15A caused dramatic plasmid loss, even at low levels of expression. In trans to wild-type R995 or to R995ΔincC carrying a functional P1 partition system, IncC2 K15A-mediated incompatibility was significantly less than that caused by wild-type IncC2. All three Walker-like A box mutants were also defective for the host toxicity that normally results from co-overexpression of incC and korB. The phenotypes of the mutants support a model in which nucleotide hydrolysis is required for separation of paired plasmid complexes and possible interaction with a host factor.     

The biological equilibrium of base pairs

Summary An inherent feature of double-stranded DNA is the possible replacement of any base pair by another one upon replication. A replication-dependent substitution mutation of a matched base pair requires the temporary formation of a mismatched base pair (mispair). A functionally complementary pair of mispairs is ascribed to each of the four types of substitution mutations. Provided that all types of mispairs can be formed, a dynamic biological equilibrium between the four matched base pairs must exist in all DNA, which is directly related to the formation and stability of the corresponding eight mispairs in vivo. Each nucleotide position in a genome can therefore be described as a system of six dynamic equilibria between the four matched base pairs. After a sufficient number of replications, these equilibrium states will express an overall mutation-selection balance for each individual base pair. In a thermodynamic context, the mispairs represent intermediate states on the transformation pathway between the matched base pairs. Catalysts change the stability and probability of formation of intermediate states. Mutagenic proteins are proposed as hypothetical substitution mutation catalysts in vivo. Functionally, they would be capable of recognizing a particular DNA sequence, tautomerizing a nucleotide base thereof, and hence efficiently inducing a specific misincorporation. Phenomenologically such catalysts would accelerate the rates of substitution mutations and provide pathways for directional mutation pressure. Present address until March 31 1990: University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, Great Britain     

A replication region of the IncHI plasmid, R27, is highly homologous with the RepFIA replicon of F

A region of the IncHI plasmid R27 has been found to share very close nucleotide sequence homology with the RepFIA replicon of F. This region has been located on a 1.6 kb segment of R27 plasmid DNA, and corresponds to ori-2 and the E gene of F. The incC repeat sequence region shows reduced homology, with the F repeats being an imperfect subset of a larger repeated sequence found in R27. The E gene homologue of R27 is able to initiate replication from the F ori-2 sequence and to repress the E gene promoter of F. The results are consistent with the observed incompatibility behaviour of R27, and have a bearing on the specificity of interaction of E protein with its DNA-binding sites.     

Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication.

We recently described a protocol for determination of retrovirus mutation rates, that is, the mutation frequency in a single cycle of retrovirus replication (J.P. Dougherty and H.M. Temin, Mol. Cell. Biol. 6:4378-4395, 1987; J.P. Dougherty and H.M. Temin, p. 18-23, in J. H. Miller and M. P. Calos, ed., Gene Transfer Vectors for Mammalian Cells, 1987). We used this protocol to determine the mutation rates for defined mutations in a replicating retrovirus by using a spleen necrosis virus-based vector. We determined that the mutation rate for a single base pair substitution during replication of this avian retrovirus is 2 x 10(-5) per base pair per replication cycle and the insertion rate is 10(-7) per base pair per replication cycle. It will be possible to use this protocol to determine mutation rates for other retroviruses.     

Trans- and cis-acting elements for the replication of P1 miniplasmids

Replication-deficient mutants of the unit-copy miniplasmid lambda-P1:5R were isolated after hydroxylamine mutagenesis. Complementation tests showed that the majority of these mutants are defective in the production of the repA protein product. Two of these mutants have suppressible nonsense (amber) mutations. The DNA sequence of one of these, repA103, has been determined. The lesion lies within the repA open reading frame, showing that the repA product is essential for plasmid replication. Complementation of deletion mutants of lambda-P1:5R by repA protein showed that the origin of replication lies to the left of repA and that this 300-base-pair origin region is the only portion of the DNA essential for plasmid replication if repA protein is supplied in trans. Six of the 21 hydroxylamine-induced mutants were not complemented by repA. Replication of three of these could be restored by introduction into the plasmid of a wild-type origin region, suggesting that they were origin-defective. The DNA sequence of two mutants was determined. Mutant rep-11 has a 43-base-pair deletion within the incC sequence (incC is a series of five direct repeats of a 19-base-pair sequence known to be involved in the regulation of plasmid replication). The deletion appears to have been generated by homologous recombination between two repeats. Mutant rep-30 has a single base substitution in a region just to the left of incC that destroys one of five G-A-T-C (dam methylation) sites in this region. As lambda-P1:5R is unable to establish itself as a plasmid in a methylase-defective (dam-) strain, it seems probable that methylation of the G-A-T-C sequences is important for origin function. The incC region and the sequences to its left appear to constitute an essential part of the origin of replication.     

DNA strand specificity for UV-induced mutations in mammalian cells.

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.     

Mutator mutations in bacteriophage T4 gene 32 (DNA unwinding protein).

Bacteriophage T4 gene 32 encodes a DNA unwinding protein required for DNA replication, repair, and recombination. Gene 32 temperature-sensitive mutations enhance virtually all base pair substitution mutation rates.     

Highly efficient random mutagenesis in transcription-reverse-transcription cycles by a hydrogen bond ambivalent nucleoside 5'-triphosphate analogue: potential candidates for a selective anti-retroviral therapy

The nucleoside P can base pair with both A and G. We evaluated the mutation frequency induced by the 5'-triphospbate of the ribonucleoside P (PTP) in an in vitro retroviral replication model. After 4 cycles of replication in the presence of PTP, the mutation frequency was raised to 3.8 x 10(-2) per nucleotide and C-to-U and U-to-C mutations were dominantly observed. These results suggest that ambivalent NTP analogues, like PTP, could induce mutations beyond the error threshold of retroviruses.