Tohru Ogawa

Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli.

Insertion and deletion mutations of the hupB and hupA genes, which encode the HU-1 and HU-2 proteins, respectively, of Escherichia coli, have been constructed in vitro and transferred to the hup loci on the bacterial chromosome. The mutations were constructed by inserting a gene encoding chloramphenicol resistance or kanamycin resistance into the coding region of the hupB or hupA gene, respectively. A complete deletion of the hupA gene was constructed by replacing the entire hupA coding region with the kanamycin resistance gene. Cells in which either the hupB or the hupA gene is defective grow normally, but cells in which both of the hup genes are defective exhibit phenotypes different from the wildtype strain. The hupA-hupB double mutants are cold-sensitive, although their growth rate is normal at 37 degrees C. Furthermore, the viability of the hupA-hupB double mutants is severely reduced when the cells are subjected to either cold shock or heat shock, indicating that the hup genes are essential for cell survival under some conditions of stress. The double mutants also exhibit filamentation when grown in the lower range of permissive growth temperature.

A novel DnaA protein-binding site at 94.7 min on the Escherichia coli chromosome.

There is a DNA sequence which has unusually high affinity for the DnaA protein of Escherichia coli between the glyV and amiB–mutL operons at 94.7 min on the genetic map. Affinity of DnaA protein for DNA was measured in vivo as the activity of β‐galactosidase produced from the lacZ gene on a plasmid fused to the 5′‐terminal portion of the mioC gene, which is under the control of the DnaA protein. The chromosomal DNA segment between the two operons, carried on a compatible plasmid, derepressed the β‐galactosidase activity by titrating DnaA protein. Derepression occurred on the chromosomal dnaA gene as well, since it is autoregulated. Hence, as measured by immunoassays, one plasmid molecule carrying the DnaA‐binding region titrated 370 DnaA molecules, which is a value eightfold higher than that for a plasmid containing the oriC region. We estimate that about 60% of the total cellular DnaA molecules are bound to this site. Four DnaA‐binding sequences (DnaA boxes) and a DnaA‐regulated promoter directing transcription of two small genes were present within a 250 bp stretch in this region but additional long DNA regions, including the fifth DnaA box located about 650 bp downstream, were required for maximum binding. A role for the DnaA‐binding site in controlling DnaA‐protein concentration in the cell cycle is discussed.

Mechanism of DNA chain growth: XVI. Analyses of RNA-linked DNA pieces in Escherichia coli with polynucleotide kinase

An improved method for the isolation of RNA-linked DNA pieces from Escherichia coli has been developed. The following results, obtained by end group labelling with T4 polynucleotide kinase, strongly suggest that the short RNA segment is covalently linked to the 5′ end of DNA. (1) After denaturation by dimethyl sulphoxide, all the labelled RNA bands at the position of DNA in a Cs 2 SO 4 equilibrium density gradient. (2) The number of 5′ termini of RNA equals the number of 5′-hydroxyl termini of DNA produced from the same preparation by alkaline hydrolysis. (3) After digestion with pancreatic DNAase, the 5′-terminally labelled RNA is resistant to periodate oxidation. This suggests that these molecules contain deoxyribonucleotides at their 3′ termini. The size of the majority of the RNA segments thus obtained ranges from mono- to trinucleotides. The cellular abundance of the RNA-linked DNA pieces has been estimated by selective polynucleotide kinase-catalysed labelling of the 5′-hydroxyl ends of DNA generated by alkaline hydrolysis. In the restrictive conditions RNA-linked DNA pieces accumulate in mutants defective in the 5′ → 3′ exonuclease and/or the polymerase activity of DNA polymerase I, but not in a DNA ligase mutant or in the wild-type control. This suggests that the removal of the RNA attached to the nascent DNA pieces requires the concerted action of both the 5′ → 3′ exonuclease and the polymerase activities of DNA polymerase I. The RNA-linked DNA pieces were hydrolysed with alkali and incubated with polynucleotide kinase and [γ- 32 P]ATP. When the DNA thus labelled is degraded to 5′-mononucleotides, the 32 P is found in all four deoxyribonucleotides.

Function of RNase H in DNA replication revealed by RNase H defective mutants of Escherichia coli

SummaryEscherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with [3H]poly(rC)·poly(dG) as substrate. RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells). This mutant formed small colonies at 43 °C. At this temperature, accumulation of nascent fragments was more prominent in the rnh-91·polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43° C. Unlike the 1–3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91·polA4113 cells ranged from one to about ten bases. These results suggest that the 5′→3′ exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5′-portion of longer primers.The rnh mutant supports replication of ColE1-type plasmids. A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed.

The datA locus predominantly contributes to the initiator titration mechanism in the control of replication initiation in Escherichia coli

Replication of the Escherichia coli chromosome is initiated synchronously from all origins (oriC) present in a cell at a fixed time in the cell cycle under given steady state culture conditions. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which titrates exceptionally large amounts of the bacterial initiator protein, DnaA, to prevent overinitiation. Deletion of the datA locus results in extra initiations and altered temporal control of replication. There are many other sites on the E. coli chromosome that can bind DnaA protein, but the contribution of these sites to the control of replication initiation has not been investigated. In the present study, seven major DnaA binding sites other than datA have been examined for their influence on the timing of replication initiation. Disruption of these seven major binding sites, either individually or together, had no effect on the timing of initiation of replication. Thus, datA seems to be a unique site that adjusts the balance between free and bound DnaA to ensure that there is only a single initiation event in each bacterial cell cycle. Mutation either in the second or the third DnaA box (a 9 basepair DnaA‐binding sequence) in datA was enough to induce asynchronous and extra initiations of replication to a similar extent as that observed with the datA‐deleted strain. These DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.

Mechanism of DNA chain growth. XV. RNA-linked nascent DNA pieces in Escherichia coli strains assayed with spleen exonuclease.

Abstract A new method for the detection and assay of RNA-linked nascent DNA pieces has been developed. The method relies on selective degradation by spleen exonuclease of radioactive 5′-OH terminated DNA produced from the pulse-labelled nascent pieces upon alkaline hydrolysis. Analysis with this method in wild type Escherichia coli has shown relatively high proportions of the RNA-linked molecules after shorter pulses and in the smaller pieces, supporting the transient nature of the RNA attachment to the nascent pieces. The RNA-linked nascent DNA pieces are accumulated by both E. coli pol Aex1 (defective in 5′ → 3′ exonuclease of DNA polymerase I) and E. coli pol A12 and pol A1 (defective in polymerase of DNA polymerase I), suggesting the requirement of the concerted action of both 5′ → 3′ exonuclease and polymerase of DNA polymerase I for the removal of the RNA attached to the nascent pieces. Most of the nascent DNA pieces accumulated by E. coli ligts 7 (defective in DNA ligase) are not linked to RNA, as expected from the direct role of DNA ligase in joining of the pieces. The analysis also has shown that a large portion of the nascent DNA pieces present in the cell under the normal steady-state conditions are not linked to RNA and that the level of the RNA-free DNA pieces is also increased in pol A mutants. These findings suggest that the removal of RNA from the nascent pieces is a relatively rapid process and the joining reaction is a rate-limiting step that requires the concurrent action of DNA polymerase and DNA ligase.

Multiple pathways regulating DnaA function in Escherichia coli: Distinct roles for DnaA titration by the datA locus and the regulatory inactivation of DnaA

Escherichia coli DnaA protein forms a multimeric complex at the chromosomal origin of replication (oriC), where a series of initiation reactions occurs and DNA polymerase III holoenzyme is loaded. The ATP-bound form of DnaA, which is active for initiation, is converted to the inactive ADP-bound form through interaction with the sliding clamp, the beta subunit of DNA polymerase III holoenzyme loaded on DNA. This negative regulation, termed RIDA, is required for preventing untimely initiations. Here, we asked if RIDA is functionally related to another negative regulation, DnaA titration by the datA site. The datA site can harbor hundreds of DnaA molecules, and is also required for preventing untimely initiations. We reveal here that, in growing cells of the datA(+) and datA-deleted strains, the ATP-DnaA levels were both maintained in a limited range of about 20-30% of the total ATP- plus ADP-DnaA molecules. This indicates that RIDA functions in the absence of datA. In synchronized datA-deleted cells, the ATP-DnaA level fluctuated in a manner similar to that observed in datA(+) cells. This suggests that RIDA operates independent from DnaA titration to datA. We suggest that these two mechanisms may play complementary roles during the cell cycle to prevent untimely initiations and thus ensure the scheduled initiation.

Assay of RNA-linked nascent DNA pieces with polynucleotide kinase☆☆☆

Abstract The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-32P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, E. coli pol Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type.

Determination of the minimum domain II size of Escherichia coli DnaA protein essential for cell viability

The DnaA protein is the bacterial initiator of replication at a unique chromosomal site, oriC. It is present in all bacterial species and has a conserved structure with four domains. The structures of domains I and III-IV have been solved recently for some bacterial species, and the molecular process leading to the initiation event has been investigated in detail. On the other hand, domain II appears to have no rigid structure and is assumed to be a flexible linker connecting the N-terminal domain I and the C-terminal domains III-IV. It differs significantly in length and amino acid sequence among bacterial species. Whether or not domain II has any function(s) to initiate replication is unknown. The precise borders at both of its ends as well as its essential portions for cell viability are also unknown. In this study, we introduced systematic deletions into the domain II region on the chromosomal dnaA gene of Escherichia coli and examined their effect on cell physiology. Stretches of 30-36 consecutive amino acid residues could be deleted from various portions between the 78th and the 136th residues without affecting cell viability. We propose that domain II of E. coli DnaA is from the 79th to the 135th residues and at least 21-27 residues are required as a spacer to keep domains I and III-IV in the correct positions.

Initiator titration complex formed at datA with the aid of IHF regulates replication timing in Escherichia coli

The initiation of replication in Escherichia coli is negatively controlled by a mechanism referred to as ‘initiator titration’, a process by which the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome to reduce the initiation potential for replication. Initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules to prevent aberrant initiations. We found that this was enabled by integration host factor (IHF). Within datA, there is a consensus IHF recognition sequence between the two DnaA recognition sequences (DnaA boxes) essential for its function. Binding of IHF to this site was demonstrated both in vitro and in vivo. Disruption of the core sequence in the consensus of the IHF‐binding resulted in increased origin concentration as observed in ΔdatA cells. Furthermore, the number of DnaA molecules bound to datA was reduced in cells carrying a disruption in the IHF‐binding core sequence. The IHF‐binding site and the essential DnaA boxes had to be located at a proper distance and orientation to maintain the accurate initiation timing. Therefore, IHF is a unique element in the control of replication initiation that acts negatively at datA, while known to act as a positive regulator at oriC.