Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids.

Based on the ability to complement the poor growth of an M13 phage derivative lacking the complementary strand origin, eleven single-strand initiation sequences (ssi) for DNA replication are identified in the F, R6K, R100 and ColE2 plasmids. Six of them were from F, two from near the gamma and alpha origins (ori) of R6K, two from the vicinity of the basic replicon of R100 and one from near the ori of ColE2. They can be classified into two groups based on the morphology of the plaques and the length of nucleotide (nt) sequences required for ssi activity; one group that gives rise to larger and clearer plaques and can be reduced to nearly 100 nt (seven out of eleven), and another that generates smaller and less clear plaques and requires more than 200 nt for full activity (four out of eleven). Sequence homology is detected among some members from both groups. The possible biological roles of the ssi are discussed.     

Biochemical investigations of control of replication initiation of plasmid R6K

The mechanistic basis of control of replication initiation of plasmid R6K was investigated by addressing the following questions. What are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? Did the primary control involve only initiator protein-ori DNA interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? Mutations at two different regions of the pi-encoding sequence individually caused some loss of negative control as indicated by a relatively modest increase in copy number. However, combinations of the mutation P42L, which caused loss of DNA looping, with those located in the region between the residues 106 and 113 induced a robust enhancement of copy number. These mutant forms promoted higher levels of replication in vitro in a reconstituted system consisting of 22 purified proteins. The mutant forms of pi were susceptible to pronounced iteron-induced monomerization in comparison with the WT protein. As contrasted with the changes in DNA-protein interaction, we found no detectable differences in protein-protein interaction between wild type pi with DnaA, DnaB helicase, and DnaG primase on one hand and between the high copy mutant forms and the same host proteins on the other. The DnaG-pi interaction reported here is novel. Taken together, the results suggest that both loss of negative control due to iteron-induced monomerization of the initiator and enhanced iteron-initiator interaction appear to be the principal causes of enhanced copy number.     

Molecular cloning of replication and incompatibility regions from the R-plasmid R6K.

The construction of a physical map of R6K DNA on the basis of specific cleavage of R6K DNA by HindIII and EcoRI restriction endonucleases allowed us to determine the location of the R6K replication and drug resistance regions. Molecular cloning techniques were used to dissect the replication and incompatibility functions of R6K. This R-plasmid possesses two origins of replication, α and β, separated by a stretch of 3900 nucleotides. A region close to ori α. controls the copy number of the composite replicon. Inverted duplications which are 100 to 200 nucleotides long are found at the positions of ori α and ori β, respectively. A 1400-nucleotide long sequence within the region bounded by the inverted duplications and separate from the origins and the control region is involved in the R6K self-replication and replication under conditions of polymerase I deprivation. This region also contains some of the incompatibility genes of R6K. The sequentially asymmetrically bidirectional mode of R6K replication is due to the existence of a replication termination site. This terminator is located outside the sequences bounded by the inverted duplications and is not essential for plasmid DNA replication.     

Incompatibility and replication of plasmids]

The identified basic replicons rep1 and rep2 of plasmid pAP42 belong to different groups of incompatibility (inc FIX and inc FVIII). The replicons are partly incompatibile with other inc F-groups too. The results indicate connection between plasmid incompatibility and their replication.     

A comparison of the origin of replication of pSa with R6K

Summary The plasmid pOri3 is a derivative of the origin of replication of pSa. Replication is defective as a result of a truncated repA gene, the product of which is required for plasmid replication. The defective replication is complemented by the presence of the intact repA gene of pSa, or by the presence of the plasmid R6K. The basis of this complementation has been examined by comparing the nucleotide sequence of the origin of pSa with that of R6K. A 13 base pair sequence present twice in the origin of pSa has homology with a 13 base pair sequence that is present fourteen times in the origin of R6K. These sequences may be the binding sites for the initiator proteins of these two plasmids. The location of these binding sites relative to the genes for the initiator proteins suggests that an autoregulatory control mechanism for the synthesis of the initiator proteins may also play a role in the control of plasmid copy number.     

A family of cosmid vectors with the multi-copy R6K replication origin

A family of cosmid vectors was constructed which contain replication origins (ori) derived from the multicopy plasmid R6K, a kanamycin resistance gene and two cos sites, permitting efficient library construction. Additional features of later constructs are (i) the presence of NotI sites flanking the site of insertion to allow intact excision of inserts, (ii) the facility for selective cloning of the ends of inserts for rapid chromosome walking, and (iii) the use of a mutated R6K ori leading to an increased copy number.     

Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K

The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature-sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined.     

Construction of plasmid R6K derivatives in vitro: Characterization of the R6K replication region

Low molecular weight derivatives of the antibiotic resistance plasmid R6K have been constructed in vitro using the restriction endonucleases HindIII and HaeII. Common to all of the derivatives that replicate autonomously in Escherichia coli is a 2.1-kb segment of the DNA at the region of the origin of replication. This 2.1-kb region does not contain the asymmetric terminus of replication present in the parent plasmid. The derivatives replicate under relaxed control and are incompatible with intact R6K. A restriction endonuclease cleavage map of the plasmid is presented.     

Plasmid-encoded initiation protein is required for activity at all three origins of plasmid R6K DNA replication in vitro

DNA replication of plasmid R6K initiates at three unique sites, ori alpha, ori beta, and ori gamma. Replicating DNA molecules of a deletion derivative of R6K were synthesized in an in vitro system containing pi protein fraction from cells carrying a mini-R6K derivative that produced only this initiation protein as an R6K-encoded protein and analyzed by electron miscroscopy. Requirement of pi protein for the activity of all these three replication origins in vitro was verified. Frequencies of initiation at the three origins were almost equal.     

植物乳杆菌编码复制起始蛋白天然质粒的系统进化分析

【目的】探究复制起始蛋白(Replication initiation protein,Rep)是否可以作为天然质粒系统进化关系研究的分子标记。【方法】以植物乳杆菌天然质粒编码的Rep为研究对象,通过构建Rep系统进化树详细分析和讨论这些质粒的系统进化关系。【结果】植物乳杆菌45个编码Rep天然质粒可以划分为5个进化关系紧密的家族和1个独立进化质粒p G6302,家族1-4质粒可以进一步划分为10个进化关系更近的亚家族类群,因此这些质粒可能起源于6个祖先质粒。【结论】Rep氨基酸序列显示了适度的保守性和变异性,是植物乳杆菌编码Rep质粒进化研究理想的分子标记,为植物乳杆菌天然质粒系统进化研究提供了一种简单、有效的分析方法和标准,并为植物乳杆菌或其他乳酸菌天然质粒系统进化研究提供了分子水平的佐证和依据。     

Structural properties of the beta origin of replication of plasmid R6K

The beta origin of replication of plasmid R6K, one of three active R6K origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. The nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (Stalker, D., Kolter, R., and Helinski, D. (1982) J. Mol. Biol. 161, 33-43). In this work, the sequence of the remaining portion of this 1964-bp segment was obtained. In addition to its activity as an origin of replication, this sequence also contains sufficient information for autonomous replication in Escherichia coli. A 277-bp region containing seven 22-bp direct repeats is present at one end of the beta origin segment (Stalker, D., Kolter, R., and Helinski, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1150-1154) while the other end contains a 140-bp sequence that includes a relaxation complex site. The 277-bp direct repeat region is required for activity of the beta origin. The start of the beta origin of replication as mapped by electron microscopy (Crosa, J. (1980) J. Biol. Chem. 255, 11075-11077) lies approximately 1000 bp away from the 277-bp region. The pi structural gene, which makes up most of the sequence between the direct repeats and the beta origin, is required in cis for beta origin activity. The pi protein also is required for beta origin activity but can be provided in trans. The nucleotide sequence just beyond the pi structural gene and within or near the start of beta origin of replication contains an open reading frame for a 151-amino acid protein. Deletions ranging from 94 bp to 1590 bp were obtained within the 1964-bp beta origin region. In every case, the deletion results in loss of origin activity even when the deleted sequence plus adjacent regions are provided in trans. These observations suggest a requirement for a specific secondary structure over an extensive region for beta origin activity.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

Genetic heteroduplex analysis of the R6K plasmid]

The results of genetic studies of R6K Tra1- and R6Kdelta[Sm1] mutants of R6K plasmid and those of heteroduplex analysis of DNAs have shown that DNA of this drug-resistant factor contains three loops flanked by the inverted repeats. The latter are designated as IR1, IR2 and IR3 and are of 50, 100 and 120 nucleotides in size respectively. IR1 is inserted into the loop flanked by IR2. Loops with these two repeats are located in major EcoR1 fragment, IR3 having been found in minor EcoRI fragment of the plasmid. The evidence obtained from the analysis of heteroduplex R6K/RSF2124 has shown that the loop with IR1 is corresponding to transposon Tn3. The extent of the deletion deltaSm1 indicates that IR2 may be a part of a transposon bearing the resistance to streptomycin. By comparing present data with those obtaine from the analysis of the RSF1040 factor of DNA replication initiation sites (Grosa et al., 1976), it has been suggested that the loop with IR3 represents a transposon with replicative functions (TnRep). The deletion of the mutant plasmid R6Kdelta[Sm1] (7.2 . 10(6) daltons in size) which affected one of the EcoRI sites not only confers the sensitivity to streptomycin but enhances also the efficiency of conjugational transfer and results in the loss of the R6K ability to bring about integrative suppression and to inhibit the fertility of the plasmids from IncP and IncN groups. The deletion mutant proved to have lost the property of incompatibility with the initial plasmid R6K and with itself.     

Roles of long and short replication initiation proteins in the fate of IncP-1 plasmids

Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1β plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-β replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3- to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts.     

The nucleotide sequence of the replication origin beta of the plasmid R6K

We h ave identified by molecular cloning a region of 283 base pairs of the HindIII 2 fragment of R6K which corresponds to the region of the replication origin beta. This 283 base-pair DNA fragment, when present contiguously with the structural gene for the replication initiation protein of R6K, encoded in the HindIII 9-15 and part of HindIII 2 restriction fragments, will support the replication of a plasmid chimera containing the pBR322 replicon in a pol Ats host at the nonpermissive temperature. The nucleotide sequence of the region of replication origin beta has been determined. The nucleotide sequence has some homology with the ori gamma region of R6K; it has a 15-base-pair homology with the replication origin of Escherichia coli.     

The DnaK-DnaJ-GrpE chaperone system activates inert wild type pi initiator protein of R6K into a form active in replication initiation

The plasmid R6K is an interesting model system for investigating initiation of DNA replication, not only near the primary binding sites of the initiator protein pi but also at a distance, caused by pi -mediated DNA looping. An important milestone in the mechanistic analysis of this replicon was the development of a reconstituted replication system consisting of 22 different highly purified proteins (Abhyankar, M. A., Zzaman, S., and Bastia, D. (2003) J. Biol. Chem. 278, 45476-45484). Although the in vitro reconstituted system promotes ori gamma-specific initiation of replication by a mutant form of the initiator called pi*, the wild type (WT) pi is functionally inert in this system. Here we show that the chaperone DnaK along with its co-chaperone DnaJ and the nucleotide exchange factor GrpE were needed to activate WT pi and caused it to initiate replication in vitro at the correct origin. We show further that the reaction was relatively chaperone-specific and that other chaperones, such as ClpB and ClpX, were incapable of activating WT pi. The molecular mechanism of activation appeared to be a chaperone-catalyzed facilitation of dimeric inert WT pi into iteron-bound monomers. Protein-protein interaction analysis by enzyme-linked immunosorbent assay revealed that, in the absence of ATP, DnaJ directly interacted with pi but its binary interactions with DnaK and GrpE and with ClpB and ClpX were at background levels, suggesting that pi is recruited by protein-protein interaction with DnaJ and then fed into the DnaK chaperone machine to promote initiator activation.