Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli

Summary Increased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible pL promoter, stimulated initiation of replication from oriC about threefold. The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA. The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content. DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC. Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC. The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC.     

In vitro roles of Escbericbia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids

Summary Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins, DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.     

Mechanisms of primer RNA synthesis and D-loop/R-loop-dependent DNA replication in Escherichia coli

In DNA replication, DNA chains are generally initiated from small pieces of ribonucleotides attached to DNA templates. These ‘primers’ are synthesized by various enzymatic mechanisms in Escherichia coli. Studies on primer RNA synthesis on single-stranded DNA templates containing specific ‘priming signals’ revealed the presence of two distinct modes, ie immobile and mobile priming. The former includes primer RNA synthesis by primase encoded by dnaG and by RNA polymerase containing a σ⁷⁰ subunit. Priming is initiated at a specific site in immobile priming. Novel immobile priming signals were identified from various plasmid replicaons, some of which function in initiation of the leading strand synthesis. The latter, on the other hand, involves a protein complex, primosome, which contains DnaB, the replicative helicase for E coli chromosomal replication. Utilizing the energy fueled by ATP hydrolysis of DnaB protein, primosomes are able to translocate on a template DNA and primase synthesizes primer RNAs at multiple sites. Two distinct primosomes. DnaA-dependent primosome supports normal chromosomal identified, which are differentially utilized for E coli chromosomal replication. Whereas DnaA-dependent primosome supports normal chromosomal replication from oriC, the PriA-dependent primosome functions in oriC-independent chromosomal replication observed in DNA-damaged cells or cells lacking RNaseH activity. In oriC-independent replication, PriA protein may recognize the D- or R-loop structure, respectively, to initiate assembly of a primosome which mediates primer RNA synthesis and replication fork progression.     

DNA lesions that block DNA replication are responsible for the dnaA induction caused by DNA damage

Summary The initiation protein DnaA of Escherichia coli regulates its own expression autogenously by binding to a 9 by consensus sequence, the dnaA box, between the promoters dnaAP1 and dnaAP2. In this study, we analysed dnaA regulation in relation to DNA damage and found dnaA expression to be inducible by DNA lesions that inhibit DNA replication. On the other hand, coding DNA lesions were not able to induce dnaA expression. These results suggest that an additional regulatory mechanism is involved in dnaA gene expression and that DnaA protein may play a role in cellular responses to DNA damage. Furthermore, they strongly suggest that in response to DNA replication inhibition by DNA damage, and enhanced (re)initiation capacity is induced by oriC.     

DNA gyrase activity regulates DnaA‐dependent replication initiation in Bacillus subtilis

In bacteria, initiation of DNA replication requires the DnaA protein. Regulation of DnaA association and activity at the origin of replication, oriC, is the predominant mechanism of replication initiation control. One key feature known to be generally important for replication is DNA topology. Although there have been some suggestions that topology may impact replication initiation, whether this mechanism regulates DnaA‐mediated replication initiation is unclear. We found that the essential topoisomerase, DNA gyrase, is required for both proper binding of DnaA to oriC as well as control of initiation frequency in Bacillus subtilis. Furthermore, we found that the regulatory activity of gyrase in initiation is specific to DnaA and oriC. Cells initiating replication from a DnaA‐independent origin, oriN, are largely resistant to gyrase inhibition by novobiocin, even at concentrations that compromise survival by up to four orders of magnitude in oriC cells. Furthermore, inhibition of gyrase does not impact initiation frequency in oriN cells. Additionally, deletion or overexpression of the DnaA regulator, YabA, significantly modulates sensitivity to gyrase inhibition, but only in oriC and not oriN cells. We propose that gyrase is a negative regulator of DnaA‐dependent replication initiation from oriC, and that this regulatory mechanism is required for cell survival.     

Dam methylated and hemimethylatedoriC plasmids are replicated symmetrically; a novel and general test of replication symmetry

Summary Deoxyadenosine methylation (dam) of the numerous GATC sequences present in theEscherichia coli origin of chromosomal replication (oriC) has been shown to be important both in vivo and in vitro for efficient initiation of DNA synthesis. Recent in vivo data suggest that initiation is only inefficient when these sequences are hemimethylated. This raises the interesting possibility that initiation may be inefficient because it only takes place on one strand of the template, i.e., replication is asymmetric on hemimethylated DNA. We tested this possibility by a novel and rapid approach which relies on the specificities of the restriction endonucleasesMboI,MboII andDpnI. Although we show that replication takes place equally well on both strands of methylated and hemimethylatedoriC DNA templates, the method should be applicable to the analysis of replication symmetry on most DNA templates which contain methylated deoxyadenosine GATC sequences as part ofMboII restriction sites.     

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.     

Inhibition of DNA replication in vitro by pefloxacin

Pefloxacin (a novel quinolone antibiotic) is demonstrated to be a drug inhibiting DNA replication 10-times more efficiently than oxolinic acid measured either in toluene-treated E. coli or in an in vitro replication system for oriC plasmids [6]. DNA repair synthesis is not inhibited by the drug.     

Random initiation of replication of plasmids P1 and F (oriS) when integrated into the Escherichia coli chromosome

We have constructed intP1 and intFs strains of Escherichia coli in which the basic replicons of either plasmid P1 or plasmid F (oriS) were integrated into an inactivated oriC, such that chromosome replication is controlled by the integrated plasmid replicon. In this study, we have further analysed these strains, and density-shift experiments revealed that chromosome replication occurred randomly during the cell cycle. Flow-cytometry analyses of exponentially growing populations supported this conclusion, and also showed that the DNA/mass ratio of the strains decreased with increasing growth rate. Flow cytometry of exponentially growing cultures treated with rifampicin demonstrated that initiation of replication was uncoordinated in cells containing multiple replication origins.     

OriC plasmids do not affect timing of chromosome replication in Escherichia coli K12

Summary The variability of the time interval between successive rounds of chromosome replication was estimated by density-shift experiments, by measuring the conversion of heavy DNA to hybrid density and light DNAs upon transfer of a steady-state culture growing in medium with [¹³C]glucose and ¹⁵NH₄Cl to medium with light isotopes. The coefficient of variation (CV%) for the interreplication time of the Escherichia coli K12 chromosome was found to be 17%, i.e. similar to that for interdivision time. The presence of additional copies of oriC in the cell on a high copy number plasmid did not increase the CV of interreplication time. It is concluded that a single rate-limiting event is unlikely to time the initiation of chromosome replication. The regulation of initiation at oriC and the coordination with cell division is discussed.     

Increased expression of a hemimethylated oriC binding protein, SeqA, in an aphA mutant

In Escherichia coli, the origin of DNA replication, oriC, becomes transiently hemimethylated at the GATC sequences immediately after initiation of replication and this hemimethylated state is prolonged because of its sequestration by a fraction of outer membrane. This sequestration is dependent on a hemimethylated oriC binding protein such as SeqA. We previously isolated a clone of phage λgtll called hobH, producing a LacZ fusion protein which recognizes hemimethylated oriC DNA. Very recently, Thaller et al. (FEMS Microbiol. Lett. 146 (1997)191–198)found that the same DNA segment encodes a non-specific acid phosphatase, and named the gene aphA. We show here that the interruption of the aphA reading frame by kanamycin resistance gene insertion, abolishes acid phosphatase (NAP) activity. Interestingly, in the membrane of the null mutant, the amount of SeqA protein is about six times higher than that in the parental strain, suggesting the existence of a regulatory mechanism between SeqA and NAP expression.     

The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis

Summary The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and an approximate copy number of 20–50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S. lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.     

Interdependence and nodule specificity of cis-acting regulatory elements in the soybean leghemoglobin lbc3 and N23 gene promoters

Summary A composite plasmid comprising the mini-F and pBR322 replicons was found to inhibit cell growth of a host with conditional mutations in dnaA and rnh under restrictive conditions, where the normal initiation of replication from oriC was inactivated, but the alternative replication initiation from oriK was active. It was further shown that the composite plasrnid inhibited stable DNA replication (SDR) which occurs constitutively in cells mutant for rnh. Neither pBR322 nor mini-F alone produced these inhibitory effects. Deletion analyses revealed that the mini-F segment responsible for the inhibition of both processes was the promoter region of the sopA gene which had been cloned into a site upstream of the bla gene on pBR322 in such an orientation as to cause overexpression of bla. Inserting the promoter of the Escherichia coli lac gene into the same site had the same effect. Introduction of a deletion and a frameshift mutation into bla abolished the inhibition. Thus, the inhibition of growth and SDR appear to be due to overproduction of the bla gene product, -lactamase.     

Isolation and characterization of herC, a mutation of Escherichia coli affecting maintenance of ColE1

Summary Two modes of ColE1 DNA replication are known, one dependent on RNase H, and the other RNase H independent. The cer114 mutant of the ColE1 replicon is defective in both modes and carries a single base pair alteration 95 by upstream of the replication origin. An Escherichia coli mutant which restored maintenance of the cer114 replicon was isolated. This host suppressor mutant is defective in RNase H and carries a herC, mutation located at 62 min of the E. coli chromosome. The herC, mutation is recessive to its wild-type allele and supports maintenance of the mutant replicon in the absence of RNase H. The herC, mutation alone conferred cold-sensitive growth, suggesting that the herC, gene product is essential for cell growth. The 1832 by E. coli DNA fragment, containing the wild-type allele of the herC, mutation, was cloned and an open reading frame for the HerC protein was determined.     

dam methylation and the initiation of DNA replication on oriC plasmids

Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.     

DnaA structure, function, and dynamics in the initiation at the chromosomal origin

Escherichia coli DnaA is the initiator of chromosomal replication. Multiple ATP-DnaA molecules assemble at the oriC replication origin in a highly regulated manner, and the resultant initiation complexes promote local duplex unwinding within oriC, resulting in open complexes. DnaB helicase is loaded onto the unwound single-stranded region within oriC via interaction with the DnaA multimers. The tertiary structure of the functional domains of DnaA has been determined and several crucial residues in the initiation process, as well as their unique functions, have been identified. These include specific DNA binding, inter-DnaA interaction, specific and regulatory interactions with ATP and with the unwound single-stranded oriC DNA, and functional interaction with DnaB helicase. An overall structure of the initiation complex is also proposed. These are important for deepening our understanding of the molecular mechanisms that underlie DnaA assembly, oriC duplex unwinding, regulation of the initiation reaction, and DnaB helicase loading. In this review, we summarize recent progress on the molecular mechanisms of the functions of DnaA on oriC. In addition, some members of the AAA+ protein family related to the initiation of replication and its regulation (e.g., DnaA) are briefly discussed.     

Identification and autonomous replication capability of a chromosomal replication origin from the archaeon <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

Here, we describe the identification of a chromosomal DNA replication origin (oriC) from the hyperthermophilic archaeon Sulfolobus solfataricus (subdomain of Crenarchaeota). By means of a cumulative GC-skew analysis of the Sulfolobus genome sequence, a candidate oriC was mapped within a 1.12-kb region located between the two divergently transcribed MCM- and cdc6-like genes. We demonstrated that plasmids containing the Sulfolobus oriC sequence and a hygromycin-resistance selectable marker were maintained in an episomal state in transformed S. solfataricus cells under selective pressure. The proposed location of the origin was confirmed by 2-D gel electrophoresis experiments. This is the first report on the functional cloning of a chromosomal oriC from an archaeon and represents an important step toward the reconstitution of an archaeal in vitro DNA replication system.     

The role of Helicobacter pylori DnaA domain I in orisome assembly on a bipartite origin of chromosome replication

The main roles of the DnaA protein are to bind the origin of chromosome replication (oriC), to unwind DNA and to provide a hub for the step-wise assembly of a replisome. DnaA is composed of four domains, with each playing a distinct functional role in the orisome assembly. Out of the four domains, the role of domain I is the least understood and appears to be the most species-specific. To better characterise Helicobacter pylori DnaA domain I, we have constructed a series of DnaA variants and studied their interactions with H. pylori bipartite oriC. We show that domain I is responsible for the stabilisation and organisation of DnaA-oriC complexes and provides cooperativity in DnaA–DNA interactions. Domain I mediates cross-interactions between oriC subcomplexes, which indicates that domain I is important for long-distance DnaA interactions and is essential for orisosme assembly on bipartite origins. HobA, which interacts with domain I, increases the DnaA binding to bipartite oriC; however, it does not stimulate but rather inhibits DNA unwinding. This suggests that HobA helps DnaA to bind oriC, but an unknown factor triggers DNA unwinding. Together, our results indicate that domain I self-interaction is important for the DnaA assembly on bipartite H. pylori oriC.     

Mutations in the DnaA binding sites of the replication origin of Escherichia coli.

Summary Mutations (base changes) were introduced into the four DnaA binding sites (DnaA boxes) of theEscherichia coli replication origin,oriC. Mutations in a single DnaA box did not impair the ability of these origins to replicate in vivo and in vitro. A combination of mutations in two DnaA boxes, R1 and R4, resulted in slower growth of theoriC plasmid-bearing host cells. DnaA protein interaction with mutant and wild-type DnaA boxes was analyzed by DNase I footprinting. Binding of DnaA protein to a mutated DnaA box R1 was not affected by a mutation in DnaA box R4 and vice versa. Mutations in DnaA boxes R1 and R4 did not modify the ability of the DnaA protein to bind to other DnaA boxes inoriC.