Similarities in control of mini-F plasmid and chromosomal replication in Escherichia coli.

In Escherichia coli, concentrations of a mini-F plasmid with two origins of replication (oriV and oriS) were 50% lower in fast-growing cells than in slow-growing cells. By contrast, a mini-F plasmid deleted for oriV maintained a uniform concentration in both fast- and slow-growing cells, and in this behavior the plasmid mimicked the control by the host of chromosomal origin (oriC) concentration.     

Timing of chromosomal replication in Escherichia coli

We have previously shown that certain mutations in the dnaA and recA genes of Escherichia coli perturb initiation of chromosomal replication so that all origins present are not initiated simultaneously. In this work, several genes whose protein products are involved in initiation of replication have been investigated for their effects on the synchrony of initiation. Some of the mutants (dnaC2, rpoC907, dam3) were found to have the asynchrony phenotype. Also, dnaA(Ts) mutations were shown to be dominant over dnaA+ in terms of initiation synchrony. The mechanism leading to the asynchronous phenotype is discussed.     

Perturbed chromosomal replication in recA mutants of Escherichia coli.

When initiation of DNA replication is inhibited in wild-type Escherichia coli cells by rifampin or chloramphenicol, completion of ongoing rounds of replication (runout of replication) leads to cells containing two, four, or eight fully replicated chromosomes, as measured by flow cytometry. In recombination-deficient recA strains, a high frequency of cells with three, five, six, or seven fully replicated chromosomes was observed in addition to cells with two, four, or eight chromosomes. recA mutants affected only in the protease-stimulating function behaved like wild-type cells. Thus, in the absence of the recombinase function of RecA protein, the frequency of productive initiations was significantly reduced compared with that in its presence. DNA degradation during runout of replication in the presence of rifampin was about 15%. The DNA degradation necessary to account for the whole effect described above was in this range or even lower. However, a model involving selective and complete degradation of partially replicated chromosomes is considered unlikely. It is suggested that the lack of RecA protein causes initiations or newly formed replication forks to stall but remain reactivatable for a period of time by functional RecA protein.     

Intermediates of chromosomal DNA replication in Escherichia coli

The product of bacteriophage T4 gene 63 has two activities, one which catalyzes the attachment of tail fibers to base plates during morphogenesis (TFA) and one which catalyzes the joining of single-stranded polynucleotides (RNA ligase). The only phenotype attributed to mutations in gene 63 is a defect in attachment of tail fibers leading to fiberless T4 particles. However, it is suspected that TFA and RNA ligase are unrelated activities of the same protein since they have very different requirements in vitro.We have isolated new mutants which have lost the RNA ligase but have retained the TFA activity of the product of gene 63. These mutants exhibit defects in T4 DNA replication and late gene expression in some strains of Escherichia coli. This work allows us to draw three conclusions: (1) the TFA and RNA ligase activities are unrelated functions of the gene 63 product making this the prototype for a protein which has more than one unrelated function; (2) the RNA ligase is probably involved in DNA metabolism rather than RNA processing as has been proposed: (3) the RNA ligase and polynucleotide 5′ kinase 3′ phosphatase of T4 perform intimately related functions.     

Suppressors of a UGG missense mutation in Escherichia coli

As part of our investigation of tRNA structure-function relationships, we isolated and preliminarily characterized translational suppressors of the tryptophan codon UGG in a trpA missense mutant of Escherichia coli. the parent strain also contained two other mutant alleles relevant to the suppressor search; these were supD, which codes for a serine-inserting amber suppressor tRNA, and gly V55, the gene for a GGA/G-reading mutationally altered glycine tRNA. On the basis of map location, reversed-phase (RPC-5) column chromatography of glycyl-tRNA, and codon response, several classes have been distinguished so far. The number of suppressors in each class, their codon responses, and their apparent genic identities, respectively, are as follows: class 1--4 suppressors, UGG, supD; class 2--12 suppressors, UGG, glyU; class 3--9 suppressors, UGA and UGG, glyT; class 4--2 suppressors, UGG, glyT; class 5--7 suppressors, UGG, gly V55. Besides these, one suppressor retains supD activity, but so far its map location has not been distinguished from that of supD. Another suppressor clearly does not map near supD or any of the glycine tRNA genes mentioned. These last two suppressors may represent novel missense suppressors such as misacylated tRNA's or mutationally altered aminoacyl-tRNA synthetases, tRNA modification enzymes, or ribosomes. Finally, three other suppressors were obtained from a strain containing glyT56, the gene for an AGA/G-reading form of glyT tRNA. All three occurred at the expense of glyT56 activity and exhibited the the transductional linkage to argH that is characteristic of glyT.     

Cyanobacterial RNA polymerase genes rpoC1 and rpoC2 correspond to rpoC of Escherichia coli.

The DNA-dependent RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of cyanobacteria contains a unique core component, gamma, which is absent from the RNA polymerases of other eubacteria (G. J. Schneider, N. E. Tumer, C. Richaud, G. Borbely, and R. Haselkorn, J. Biol. Chem. 262:14633-14639, 1987). We present the complete nucleotide sequence of rpoC1, the gene encoding the gamma subunit, from the heterocystous cyanobacterium Nostoc commune UTEX 584. The derived amino acid sequence of gamma (621 residues) corresponds with the amino-terminal portion of the beta' polypeptide of Escherichia coli RNA polymerase. A second gene in N. commune UTEX 584, rpoC2, encodes a protein which shows correspondence with the carboxy-terminal portion of the E. coli beta' subunit. The rpoBC1C2 genes of N. commune UTEX 584 are present in single copies and are arranged in the order rpoBC1C2, and the coding regions are separated by short AT-rich spacer regions which have the potential to form very stable secondary structures. Our data indicate the occurrence of divergent evolution of structure in the eubacterial DNA-dependent RNA polymerase.     

The G157C mutation in the Escherichia coli sliding clamp specifically affects initiation of replication

Escherichia coli cells with a point mutation in the dnaN gene causing the amino acid change Gly157 to Cys, were found to underinitiate replication and grow with a reduced origin and DNA concentration. The mutant β clamp also caused excessive conversion of ATP-DnaA to ADP-DnaA. The DnaA protein was, however, not the element limiting initiation of replication. Overproduction of DnaA protein, which in wild-type cells leads to over-replication, had no effect in the dnaN(G157C) mutant. Origins already opened by DnaA seemed to remain open for a prolonged period, with a stage of initiation involving β clamp loading, presumably limiting the initiation process. The existence of opened origins led to a moderate SOS response. Lagging strand synthesis, which also requires loading of the β clamp, was apparently unaffected. The result indicates that some aspects of β clamp activity are specific to the origin. It is possible that the origin specific activities of β contribute to regulation of initiation frequency.     

A new ribosomal mutation which affects the two ribosomal subunits in Escherichia coli.

Summary The nif cistrons indentified by complementation analysis in the preceding paper (Dixon et al., 1977) were mapped with respect to hisD and to each other by Pl cotransduction and three-factor reciprocal crosses. The order obtained was hisD nifB nifA (nifL) nifF nifE nifK nifD nifH. Analysis of hisD2-nif cotransduction data by the Wu equation (Wu, 1966) suggested that the nif genes are divided into two clusters: a his-proximal cluster comprising nifBA(L)F and a his-distal group of nifEKDH.     

Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication.

A hepatitis B virus (HBV) genome was cloned from human liver. Numerous mutations in all viral genes define this HBV DNA as a mutant, divergent from all known HBV DNA sequences. Functional analyses of this mutant demonstrated a defect blocking viral DNA synthesis. The genetic basis of this defect was identified as a single missense mutation in the 5' region of the viral polymerase gene, resulting in the inability to package pregenomic RNA into core particles. The replication defect could be trans-complemented by a full-length wild-type, but not by a full-length mutant or 3'-truncated wild-type, polymerase gene construct. Our findings indicate a critical role of the 5' polymerase gene region in the life cycle of the virus and suggest that introducing missense mutations in this region can be a strategy to terminate viral replication in vivo.     

Feedback controls restrain the initiation of Escherichia coli chromosomal replication

In Escherichia coli, initiation of chromosomal replication is activated by a nucleoprotein complex formed primarily between the DnaA protein and oriC (replication origin) DNA. After replicational initiation, this complex has to be inactivated in order to repress the appearance of initiation events until the next scheduled round of initiation. Studies of the mechanisms responsible for this repression have recently revealed direct coupling between these mechanisms and key elements of the replication process, suggesting that feedback-type regulatory loops exist between the factors implicated in initiation and the elements yielded by the replication process. The loading of the ring-shaped beta-subunit of DNA polymerase III onto DNA plays a key role in the inactivation of the DnaA protein. Duplication of oriC DNA results in hemimethylated DNA, which is inert for reinitiation. Titration of large amounts of DnaA protein to a non-oriC locus can repress untimely initiations, and timely duplication of this locus is required for this repression in rapidly growing cells. All these systems functionally complement one another to ensure the maintenance of the interinitiation interval between two normal DNA replication cycles. The mechanisms that link the replication cycle to the progression of the cell cycle are also discussed.     

Regulatory network of the initiation of chromosomal replication in Escherichia coli

The bacterial chromosome is replicated once during the division cycle, a process ensured by the tight regulation of initiation at oriC. In prokaryotes, the initiator protein DnaA plays an essential role at the initiation step, and feedback control is critical in regulating initiation. Three systems have been identified that exert feedback control in Escherichia coli, all of which are necessary for tight strict regulation of the initiation step. In particular, the ATP-dependent control of DnaA activity is essential. A missing link in initiator activity regulation has been identified, facilitating analysis of the reaction mechanism. Furthermore, key components of this regulatory network have also been described. Because the eukaryotic initiator complex, ORC, is also regulated by ATP, the bacterial system provides an important model for understanding initiation in eukaryotes. This review summarizes recent studies on the regulation of initiator activity.     

Nucleotide sequence of the Escherichia coli replication gene dnaZX.

The Escherichia coli 2.2 kilobase dnaZX region contains one 1929 nucleotide reading frame which directs the synthesis of two protein products involved in DNA polymerization. The larger consists of 643 amino acids in a deduced 71,114 dalton chain which could be the tau subunit of DNA polymerase III. The smaller, the DNA polymerase III gamma subunit, is encoded by the same reading frame as the larger. The dnaZX sequence contains a region homologous to ATP binding sites, suggesting that these replication factors are adenine nucleotide binding proteins.     

dnaT, dominant conditional-lethal mutation affecting DNA replication in Escherichia coli.

Normally, bacteria cease DNA replication in the absence of protein synthesis. A variety of treatments, such as thymine starvation or a shift-up to rich medium, lead to continued DNA replication in the absence of protein synthesis. Mutants are described which always terminate replication under these conditions. These conditional lethal mutants, dnaT1 and dnaT2, contransduce with serB and dnaC. The mutation also affects cell division. All aspects of the mutant phenotype (obligatory termination of replication, temperature sensitivity of DNA replication and growth, and aberrant cell division at permissive growth temperatures) were transdominant to the wild-type phenotype. Episomes carrying the dnaT mutation appeared to be unstable. The existence of such a dominant mutation was predicted by a model of chromosome termination proposed by Kogoma and Lark (J. Mol. Biol. 94:243-256, 1975).     

Chromosomal mutation that affects excretion of hemolysin in Escherichia coli

Two types of mutants unable to excrete hemolysin were obtained when E. coli 5K carrying the multicopy hemolytic recombinant plasmid pANN202-312 was mutagenized with Mu d1. One type is altered in the plasmid hly-specific gene, hlyBb, but the other is caused by an insertion of Mu d1 into a chromosomal locus.     

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli.

Summary The conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).     

Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site-directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204-->Gln) decreased to one-third that of the wild-type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild-type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild-type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E. coli cells.