Cloning and mapping of the replication origin of Escherichia coli.

The replication origin of Escherichia coli has been cloned on a nonreplicating DNA fragment coding for ampicillin resistance. This recombinant DNA, named pSY211, replicates depending on the presence of the replication origin and can be recovered as a closed circular plasmid DNA of 10.7 megadaltons (Mdal). A restriction map has been constructed. EcoRI cleaves pSY211 into two fragments: one is the ampicillin fragment of 4.5 Mdal and the other is a chromosomal fragment of 6 Mdal and contains the origin. The 6 Mdal EcoRI fragment has four BamHI sites, three HindIII sites, and one Xho I site. A mutant of pSY211 has been isolated which is lacking two BamHI fragments of the chromosomal fragment. In recA hosts, pSY211 is lost at a high frequency. In recA+ hosts, pSY211 is integrated into the chromosome due to nucleotide sequence homology between pSY211 and the replication origin of the E. coli chromosome. The integration site has been mapped. We conclude that the replication origin is located at a site between uncA and rbsK, at about 83 min on the genetic map of E. coli.     

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.     

A DNA fragment containing the origin of replication of the Escherichia coli chromosome.

A 38 kilobase pair region of the Escherichia coli K12 chromosome containing the replication origin has been physically mapped with restriction endonucleases EcoRI and HindIII. Replication starts within or very near a 1.3 kilobase pair HindIII fragment in the middle of this region and proceeds outward in both directions with apparently equal speed. This pattern was observed in both dnaA and dnaC temperature-sensitive (ts) initiation mutants at the start of the synchronous round of replication which occurs after downshift from the nonpermissive to the permissive temperature.     

Isolation of a replication origin complex from Escherichia coli.

A complex consisting of replicative origin DNA and several proteins was isolated from Escherichia coli. Cells of temperature-sensitive mutants were labeled at the origin and fractionated by sucrose gradient centrifugation. A complex highly purified in origin DNA sedimented as a unique band. This complex dissociated at high concentration, above 0.2 M KCl. Upon dialysis, the complex reformed, allowing further purification of its constituents. Three major protein bands were found, corresponding to proteins of the outer membrane. The complex did not sediment with membrane fractions, but adhered to the outer membrane in the presence of magnesium.     

Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13.

The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained within a 507-nucleotide intergenic region of the viral genome. Chimeric plasmids have been constructed by inserting restriction endonuclease fragments of the M13 intergenic region into the plasmid pBR322. Replication of these hybrid plasmids, under conditions not permissive for the plasmid replicon, depends on specific segments of the M13 origin region and on the presence of M13 helper virus. Thus M13-infected polA- Escherichia coli can be transformed to ampicillin resistance by hybrid plasmids that have a functional M13 origin. Cells transformed to drug resistance by plasmids bearing M13 origin sequences contain the duplex chimeric DNA at high copy number but do not accumulate significant amounts of single-stranded plasmid DNA. Rare transducing phages carrying single-stranded chimeric DNA are produced and can be detected by their ability to transduce cells to ampicillin resistance. Plasmids containing a 270-nucleotide fragment from the gene II-proximal half of the intergenic region produce transformants at high frequency under nonpermissive conditions. A central Hae III fragment, Hae III-G, containing the nucleotide sequence coding for the RNA primer for the complementary strand and the nicking site for gene II protein, is sufficient for plasmid replication in M13-infected polA- cells but not for high frequency transformation. Additional sequence information on the gene II side of the Hae III-G fragment is necessary for efficient transformation by the plasmid DNA.     

Enzymatic replication of the origin of the Escherichia coli chromosome.

An enzyme system that replicates plasmids bearing the origin of the Escherichia coli chromosomes (oriC) has the following physiologically relevant features. The system (i) depends completely on low levels of exogenously furnished supercoiled oriC plasmids, (ii) uses only those plasmids that contain the intact oriC region of about 245 base pairs, (iii) initiates replication within or near the oriC sequence and proceeds bidirectionally, (iv) proceeds linearly, after a 5-min lag, for 30-40 min to produce as much as a 40% increase over the input DNA, (v) depends on RNA polymerase and gyrase as indicated by total inhibition by rifampicin and nalidixate, (vi) depends on replication proteins (e.g., dnaB protein and single-stranded DNA binding protein) as judged by specific antibody inhibitions, (vii) operates independently from protein synthesis, and (viii) depends on dnaA activity, as suggested by the inactivity of enzyme fraction from each of two dnaA temperature-sensitive mutant strains, and complementation (with a 15-fold overproduction of complementing activity) by a fraction from a strain containing the dnaA gene cloned in a multicopy plasmid. Resolution and analysis of factors that control the initiation of a chromosome cycle should become accessible through its enzyme system.     

A single-stranded DNA binding protein binds the origin of replication of the duplex kinetoplast DNA.

Replication of the kinetoplast DNA (kDNA) minicircle of trypanosomatids initiates at a conserved 12-nt sequence, 5'-GGGGTTGGTGTA-3', termed the universal minicircle sequence (UMS). A sequence-specific single-stranded DNA-binding protein from Crithidia fasciculata binds the heavy strand of the 12-mer UMS. Whereas this UMS-binding protein (UMSBP) does not bind a duplex UMS dodecamer, it binds the double-stranded kDNA minicircle as well as a duplex minicircle fragment containing the origin-associated UMS. Binding of the minicircle origin region by the single-stranded DNA binding protein suggested the local unwinding of the DNA double helix at this site. Modification of thymine residues at this site by KMnO4 revealed that the UMS resides within an unwound or otherwise sharply distorted DNA at the minicircle origin region. Computer analysis predicts the sequence-directed curving of the minicircle origin region. Electrophoresis of a minicircle fragment containing the origin region in polyacrylamide gels revealed a significantly lower electrophoretic mobility than expected from its length. The fragment anomalous electrophoretic mobility is displayed only in its native conformation and is dependent on temperature and gel porosity, indicating the local curving of the DNA double helix. We suggest that binding of UMSBP at the minicircle origin of replication is possible through local unwinding of the DNA double helix at the UMS site. It is hypothesized here that this local melting is initiated through the untwisting of unstacked dinucleotide sequences at the bent origin site.     

Cloning of the uvrC gene of Escherichia coli: expression of a DNA repair gene.

We have cloned the uvrC gene of Escherichia coli, using an F' plasmid carrying the uvrC region as a source of DNA. Two plasmids, pSC101 and pBR322, were used as cloning vectors. The recombinant plasmids were selected for their ability to complement the uvrC defect of E. coli strains AB1884 and N177. We conclude that the uvrC structural gene is contained in a 1.9-kilobase DNA fragment. The protein encoded by the uvrC gene appears to have a monomer molecular weight of 64,500 as analyzed by denaturing polyacrylamide gel electrophoresis. Strains containing multicopy uvrC+ plasmids overproduce a factor that is missing in lysates of uvrC- mutants and required for an in vitro model repair reaction. The expression of uvrC+ hybrid plasmids suggests that the structural gene is separated by at least 0.8 kilobase from the regulatory region.     

Nucleotide sequence of Escherichia coli K-12 replication origin.

From subfragments of an EcoRI fragment (9 kilobase pairs) that contained the replication origin of the Escherichia coli chromosome and had been cloned as a recombinant with a nonreplicating DNA fragment coding for ampicillin resistance, small derivative plasmids were constructed. The smallest of these, pTSO151, contained a segment of 463 base pairs as the chromosomal component. Another plasmid, pSY134, constructed from BamHI digests of the EcoRI fragment and mini-F(pMF21), contained a region of 422 base pairs identical with a corresponding region in pTSO151. We conclude that the replication origin of E. coli chromosome is located within this 422-base-pair segment. The nucleotide sequence of this segment is presented.     

Completion of DNA replication in Escherichia coli

The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans. However, this process remains poorly understood. Here we show that, in Escherichia coli, the completion of replication involves an enzymatic system that effectively counts pairs and limits cellular replication to its doubling point by allowing converging replication forks to transiently continue through the doubling point before the excess, over-replicated regions are incised, resected, and joined. Completion requires RecBCD and involves several proteins associated with repairing double-strand breaks including, ExoI, SbcDC, and RecG. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. In some bacterial viruses, the completion mechanism is specifically targeted for inactivation to allow over-replication to occur during lytic replication. The results suggest that a primary cause of genomic instabilities in many double-strand-break-repair mutants arises from an impaired ability to complete replication, independent from DNA damage.During chromosomal replication, cells tightly regulate the processes of initiation, elongation, and completion to ensure that each daughter cell inherits an identical copy of the genetic information. Although the mechanisms regulating initiation and elongation have been well characterized (reviewed in refs. 1, 2), the process of how cells recognize replicated regions and complete replication at the precise doubling point remains a fundamental question yet to be addressed. Whether this event occurs once per generation as in Escherichia coli or thousands of times per generation as in human cells, the failure to efficiently carry out this function would be expected to result in a loss of genomic stability. Considering the large number of proteins that cells devote to ensuring the fidelity of replication initiation and elongation, it seems highly probable that the final critical step in this process will be also be tightly regulated and controlled enzymatically.In some aspects, one could argue that the efficiency of completion is likely to be more critical to the faithful duplication of the genome than that of initiation. When replication origins fail to initiate efficiently, elongation of replication forks from neighboring origins is often able to compensate (3, 4), and both prokaryotic and eukaryotic cells are able to tolerate variations in their origin number without severe phenotypic consequences (5–7). However, a failure to accurately limit or join any event where forks converge would be expected to result in duplications, deletions, rearrangements, or a loss of viability depending upon how the DNA ends are resolved at segregation.A number of studies suggest that an ability to sense when all sequences in the genome have doubled is critical to genomic replication. In vitro, converging replisomes continue through their meeting point as one replisome displaces the other, resulting in over-replication, or a third copy, of the region where the forks meet (8). Complicating the process of genomic doubling even further, several studies have suggested that illegitimate initiations of replication frequently occur at single-strand nicks, gaps, D-loops, and R-loops throughout the genomes of both prokaryotes and eukaryotes (9–14). Similar to when replication forks continue through a previously replicated template, each of these events would generate a third copy of the chromosomal region where the event occurs. Thus, over-replication may be inherent and promiscuous during the duplication of genomes. If true, then to ensure that each sequence of the genome replicates once, and only once, per generation, cells must encode an enzymatic system that is essentially able to count in pairs and efficiently degrade odd or over-replicated regions until the two nascent end pairs of replication events can be joined.The model organism E. coli is particularly well-suited to dissect how this fundamental process occurs. In E. coli, the completion of replication occurs at a defined region on the genome, opposite to the bidirectional origin of replication (15). Most completion events can be further localized to one of six termination (ter) sequences within the 400-kb terminus region due to the action of Tus, which binds to ter and inhibits replication fork progression in an orientation-dependent manner, in effect stalling the replication fork at this site until the second arrives (16, 17). Although Tus confines converging replication forks to a specific region, it does not appear to be directly involved in the completion reaction because tus mutants have no phenotype and complete replication normally (18). Furthermore, plasmids and bacteriophage lacking ter sequences are maintained stably (19).Many mutants impaired for either replication initiation or elongation were initially isolated based on their growth defects or an impaired ability to maintain plasmids (20–22). We reasoned that mutants impaired for the ability to complete replication might be expected to exhibit similar phenotypes and initially focused our attention on the properties of recBC and recD mutants. RecB-C-D forms a helicase–nuclease complex that is required for homologous repair of double-strand breaks in E. coli (23, 24). The enzyme uses specific DNA sequences, termed “Chi sites,” to initiate recombination between pairs of molecules. Loss of RecB or C inactivates the enzyme complex, whereas loss of RecD inactivates the nuclease and Chi recognition, but retains helicase activity (23, 24). Here, we show that inactivation of RecBCD leads to a failure to recognize and join replicating molecules at their doubling point. Although the completion process requires RecBCD, it is distinct from double-strand break repair and does not involve a double-strand break intermediate, homologous recombination, or RecA.     

Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication.

Soluble protein fractions from Escherichia coli dnaA+ cells but not dnaA temperature-sensitive cells replicate plasmids containing the E. coli chromosomal origin of replication (oriC). Complementation of these mutant fractions provided an assay for dnaA protein activity in initiation of replication at oriC. From a strain (constructed in vitro) that overproduces the dnaA protein more than 200-fold, the 52,000-dalton polypeptide was purified to near homogeneity. Although the protein tends to aggregate, monomer-sized protein purified by high-performance liquid chromatography is fully active for replication. It binds specifically and tightly to oriC in a supercoiled plasmid as judged by a Millipore filter-binding assay and by protection of the unique HindIII site within the oriC sequence. In the oriC replication reaction, dnaA protein acts at an early step preceding DNA synthesis.     

Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units.

Electron microscopy shows that complexes of the single-strand DNA binding protein (SSB) of Escherichia coli and phage fd DNA appear as beaded fiber loops containing an average of 38 beads, 1 per 170 bases of DNA. Extensive digestion of native unfixed SSB-fd DNA complexes with micrococcal nuclease reveals a protected DNA fragment of 145 bases, while shorter digestion periods result in a sequence of fragments in multiples of 160 +/- 25 bases. Digestion of these complexes with DNase I produces a repeating pattern of bands, multiples of approximately 15 bases with strong bands at 60, 105, 118, 130, 145, 150, and 210 bases. Isopycnic banding in CsCl solution yields densities of 1.272 and 1.700 g/ml, respectively, for SSB alone and for fd DNA and, after fixation, of 1.388 g/ml for fd DNA-SSB beaded fibers and 1.373 g/ml for the individual protein-DNA beads. Based on these data and the molecular weights of SSB and fd DNA, we suggest that the nucleoprotein chain consists of eight molecules of SSB bound to 145 bases of DNA, with these units linked by roughly 30 bases of protein-free DNA. The excellent concord between results obtained by enzyme digestion of unfixed native samples and, after fixation, by electron microscopy and density banding supports the conclusion that SSB organizes single-stranded DNA in a manner similar to the organization of duplex DNA by histones.     

Cloning of an origin of DNA replication of Xenopus laevis.

DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Escherichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the [3H]-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of the segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. The DNA synthesis in pSW14 was shown to be replication rather than repair synthesis, based on a buoyant density shift of the product when iododeoxyuridine was used for labeling. To determine the number of replications of pSW14, a novel method was employed. Because pSW14 is a head-to-head dimer of the vector with the Xenopus fragment inserted at an EcoRI site, the plasmid has three methylatable sites--two bracketing the Xenopus fragment and one opposite the fragment. By cotransformation of E. coli with pSW14 and pBR322 containing the EcoRI methylase gene, supercoiled pSW14 was methylated and injected into eggs with [3H]thymidine. Disappearance of modified EcoRI sites by semiconservative replication was followed by measuring the sensitivity to EcoRI endonuclease over time. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated [3H]thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude that pSW14 has a functional origin in the Xenopus DNA segment.     

Protein HU in the enzymatic replication of the chromosomal origin of Escherichia coli.

A protein that stimulates the enzymatic replication of duplex DNAs of recombinant phages and plasmids bearing the Escherichia coli origin of replication (oriC) has been isolated from an extract of E. coli. The isolated protein and the well-known protein HU, a histone-like DNA-binding protein, have identical polypeptide molecular weights and saturate the oriC replication assay at less than 40 dimers per template DNA circle. This level is one-tenth that needed to coat the template. Protein HU from the blue-green alga Anabaena is similarly active. Antibody specific for protein HU from E. coli inhibits replication promoted both by the reconstituted system and by a crude enzyme extract; in both assays, activity is restored by excess of the isolated protein. Cells lysed in 1 M KCl yield 32,000 dimers of the protein per cell, a number consistent with the reported abundance of HU. These data establish the identity of the isolated factor and protein HU and provide an indication of a function for HU in replication.     

Incompatibility of plasmids containing the replication origin of the Escherichia coli chromosome.

Plasmids containing the replication origin of the Escherichia coli chromosome (oriC plasmids) are unstable in certain recA strains of E. coli. However, they can be maintained more stably in other recA strains. This stable maintenance has allowed us to study the incompatibility properties of oriC plasmids. We have found that two oriC plasmids are incompatible: they cannot be stably coinherited in individual dividing cells. An oriC plasmid is excluded from growing bacteria at a much faster rate in the presence of a hybrid plasmid made from an oriC plasmid and a high-copy-number vector plasmid than in the presence of another oriC plasmid. By inserting various segments around the oriC region into high-copy-number vectors, we have shown that two different regions in the vicinity of the oriC region determine incompatibility. One region, which we named incA, includes the region essential for autonomous replication of the oriC plasmid. The other, incB, is adjacent to incA but is not required for autonomous replication.     

Bacteriophage lambda carrying the Escherichia coli chromosomal region of the replication origin.

A transducing phage lambdaasn was isolated. The late gene region of its genome was found to have been substituted by an Escherichia coli chromosomal segment containing the genes bgIR, bgIC, glmS, uncA, and asn. Restriction endonuclease cleavage mapping and electron microscopic analysis of the lambdaasn DNA revealed that the size of the bacterial segment is approximately 1.75 X 10(7) daltons, corresponding to about 26.4 kilobases. The circular DNA of lambdaasn was digested with restriction endonuclease EcoRI, diluted, and sealed with DNA ligase. When the reaction mixture was used to transform a recipient E. coli strain, a small plasmid of about 1 X 10(7) daltons (named pMCR115) was obtained. Restriction endonuclease cleavage mapping of pMCR115 and other evidence suggested that it contained the replication origin (oriC) of the E. coli chromosome.     

Cloning and expression of human erythropoietin cDNA in Escherichia coli.

Human erythropoietin (Ep) cDNA has been cloned in Escherichia coli by using pBR322 as a vector. Polyadenylylated RNA was isolated from selected human renal carcinomas with elevated Ep titers. The presence of Ep mRNA was detected by immunoprecipitation of in vitro translation products with monoclonal antibody to human Ep. Double-stranded cDNA was synthesized and inserted into the Pst I site of pBR322 by homopolymeric dG . dC tailing. The cDNA library was initially screened by colony hybridization with 32P-labeled cDNA synthesized from size-fractionated mRNA enriched in Ep message. Positive colonies were further screened immunologically by in situ radioimmunoassay with monoclonal antibody to human Ep. Three positive clones were identified that express the Ep gene sequences as a beta-lactamase fusion protein. These clones contain inserts of approximately 1400, 600, and 200 base pairs. Human renal Ep mRNA, of which the translation products immunoreact with anti-Ep on immunoblots, was hybrid-selected by plasmid DNA from these recombinants. Purified human Ep competes with 35S-labeled hybrid-selected translation products for antibody binding.