Excess SeqA prolongs sequestration of oriC and delays nucleoid segregation and cell division

Following initiation of chromosomal replication in Escherichia coli, newly initiated origins (oriCs) are prevented from further initiations by a mechanism termed sequestration. During the sequestration period (which lasts about one-third of a cell cycle), the origins remain hemimethylated. The SeqA protein binds hemimethylated oriC in vitro. In vivo, the absence of SeqA causes overinitiation and strongly reduces the duration of hemimethylation. The pattern of immunostained SeqA complexes in vivo suggests that SeqA has a role in organizing hemimethylated DNA at the replication forks. We have examined the effects of overexpressing SeqA under different cellular conditions. Our data demonstrate that excess SeqA significantly increases the time oriC is hemimethylated following initiation of replication. In some cells, sequestration continued for more than one generation and resulted in inhibition of primary initiation. SeqA overproduction also interfered with the segregation of sister nucleoids and caused a delay in cell division. These results suggest that SeqA's function in regulation of replication initiation is linked to chromosome segregation and possibly cell division.     

Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.     

Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.     

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.     

The two chromosomes of Vibrio cholerae are initiated at different time points in the cell cycle

The bacterium Vibrio cholerae, the cause of the diarrhoeal disease cholera, has its genome divided between two chromosomes, a feature uncommon for bacteria. The two chromosomes are of different sizes and different initiator molecules control their replication independently. Using novel methods for analysing flow cytometry data and marker frequency analysis, we show that the small chromosome II is replicated late in the C period of the cell cycle, where most of chromosome I has been replicated. Owing to the delay in initiation of chromosome II, the two chromosomes terminate replication at approximately the same time and the average number of replication origins per cell is higher for chromosome I than for chromosome II. Analysis of cell-cycle parameters shows that chromosome replication and segregation is exceptionally fast in V. cholerae. The divided genome and delayed replication of chromosome II may reduce the metabolic burden and complexity of chromosome replication by postponing DNA synthesis to the last part of the cell cycle and reducing the need for overlapping replication cycles during rapid proliferation.     

A cold sensitive dnaA mutant of E. coli which overinitiates chromosome replication at low temperature

Summary A heat resistant mutant of E. coli dnaAts46 was isolated, which grows normally only at temperatures above 39°. After a temperature shift from 42° to 32° the mutant overproduces DNA relative to protein. This is due to overinitiation of rounds of chromosome replication at low temperature, as indicated by hybridization and other experiments. The mutation is cotransduced by Pl with ilv and could not be separated from dnaAts46 by transduction.     

A new in vivo termination function for DNA polymerase I of Escherichia coli K12

Escherichia coli deleted for the tus gene are viable. Thus there must be at least one other mechanism for terminating chromosome synthesis. The tus deletion strain yielded a small fraction of cells that overproduce DNA, as determined by flow cytometry after run-out chromosome replication in the presence of rifampicin and cephalexin. A plasmid, paraBAD tus+, prevented the excess DNA replication only when arabinose was added to the medium to induce the synthesis of the Tus protein. Transduction studies were done to test whether or not additional chromosomal deletions could enhance the excess chromosome replication in the tus deletion strain. A strain containing a second deletion in metE udp overproduced DNA at a high level during run-out replication. Further studies demonstrated that a spontaneous unknown mutation had occurred during the transduction. This mutation was mapped and sequenced. It is polA(G544D). Transduction of polA(G544D) alone into the tus deletion strain produced the high DNA overproduction phenotype. The polA(G544D) and six other polA alleles were then tested in wild-type and in tus deletion backgrounds. The two alleles with low levels of 5'-->3' exonuclease (exo) overproduced DNA while those with either high or normal exo overproduce much less DNA in run-out assays in the wild-type background. In contrast, all seven mutant polA alleles caused the high DNA overproduction phenotype in a tus deletion background. To explain these results we propose a new in vivo function for wild-type DNA polymerase I in chromosome termination at site(s) not yet identified.     

Cloning and characterization of dnaA(Cs), a mutation which leads to overinitiation of DNA replication in Escherichia coli K-12.

The product of the dnaA gene is essential for the initiation of chromosomal DNA replication in Escherichia coli K-12. A cold-sensitive mutation, dnaA(Cs), was originally isolated as a putative intragenic suppressor of the temperature sensitivity of a dnaA46 mutant (G. Kellenberger-Gujer, A. J. Podhajska, and L. Caro, Mol. Gen. Genet. 162:9-16, 1978). The cold sensitivity of the dnaA(Cs) mutant was attributed to a loss of replication control resulting in overinitiation of DNA replication. We cloned and sequenced the dnaA gene from the dnaA(Cs) mutant and showed that it contains three point mutations in addition to the original dnaA46(Ts) mutation. The dnaA(Cs) mutation was dominant to the wild-type allele. Overproduction of the DnaA(Cs) protein blocked cell growth. In contrast, overproduction of wild-type DnaA protein reduced the growth rate of cells but did not stop cell growth. Thus, the effect of elevated levels of the DnaA(Cs) protein was quite different from that of the wild-type protein under the same conditions.     

rctB mutations that increase copy number of Vibrio cholerae oriCII in Escherichia coli

RctB serves as the initiator protein for replication from oriCII, the origin of replication of Vibrio cholerae chromosome II. RctB is conserved between members of Vibrionaceae but shows no homology to known replication initiator proteins and has no recognizable sequence motifs. We used an oriCII based minichromosome to isolate copy-up mutants in Escherichia coli. Three point mutations rctB(R269H), rctB(L439H) and rctB(Y381N) and one IS10 insertion in the 3'-end of the rctB gene were obtained. We determined the maximal C-terminal deletion that still gave rise to a functional RctB protein to be 165 amino acids. All rctB mutations led to decreased RctB-RctB interaction indicating that the monomer is the active form of the initiator protein. All mutations also showed various defects in rctB autoregulation. Loss of the C-terminal part of RctB led to overinitiation by reducing binding of RctB to both rctA and inc regions that normally serve to limit initiation from oriCII. Overproduction of RctB(R269H) and RctB(L439H) led to a rapid increase in oriCII copy number. This suggests that the initiator function of the two mutant proteins is increased relative to the wild-type.     

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.     

Precocious Meiotic Centromere Separation of a Novel Yeast Chromosome

In Saccharomyces cerevisiae, a reciprocal translocation between chromosome II and a linear plasmid carrying a centromere (CEN6) has split chromosome II into two fragments: one, approximately 530 kilobase pairs (kbp) in size, has the left arm and part of the right arm of chromosome II; the other, a telocentric fragment approximately 350 kbp in size, has CEN6 and the rest of the right arm of chromosome II. A cross of this yeast strain with a strain containing a complete chromosome II exhibits a high frequency of precocious centromere separation (separation of sister chromatids during meiosis I) of the telocentric fragment. Precocious centromere separation is not due to the position of the centromere per se, since diploids that are homozygous for both fragments of chromosome II segregate the telocentric fragment with normal meiotic behavior. The precocious centromere separation described here differs from previously described examples in that pairing and synapsis of this telocentric chromosome seem to be normal. One model of how centromeres function in meiosis is that replication of the centromere is delayed until the second meiotic division. Data presented in this paper indicate that replication of the centromere is complete before the first meiotic division. The precocious separation of the centromere described here may be due to improper synapsis of sequences flanking the centromere.     

Overexpression of the dnaA gene in Escherichia coli B/r: chromosome and minichromosome replication in the presence of rifampin.

The replication of chromosomes and minichromosomes in Escherichia coli B/r was examined under conditions in which the dnaA gene product was overproduced. Increased levels of the DnaA protein were achieved by thermoinduction of the dnaA gene, under the control of the lambda pL promoter, or by cellular maintenance of multicopy plasmids carrying the dnaA gene under the control of its own promoters. Previous work has shown that overproduction of DnaA protein stimulates replication of the chromosomal origin, oriC, but that the newly initiated forks do not progress along the length of the chromosome (T. Atlung, K. V. Rasmussen, E. Clausen, and F. G. Hansen, p. 282-297, in M. Schaechter, F. C. Neidhardt, J. L. Ingraham, and N. O. Kjeldgaard, ed., The Molecular Biology of Bacterial Growth, 1985). In the present study, it was found that overproduction of DnaA protein caused both a two- to threefold increase in the amount of residual chromosome replication and an extended synthesis of minichromosome DNA in the presence of rifampin. The amount of residual chromosome replication was consistent with the appearance of functional replication forks on the majority of the chromosomes. Since the rate of DNA accumulation and the cellular DNA/mass ratios were not increased significantly by overexpression of the dnaA gene, we concluded that the addition of rifampin either enabled stalled replication forks to proceed beyond oriC or enabled new forks to initiate on both chromosomes and minichromosomes, or both.     

Discontinuity in DNA replication during expression of accumulated initiation potential in dnaA mutants of Escherichia coli.

Potential for initiation of chromosome replication present in temperature-sensitive, initiation-defective dnaA5 mutants of Escherichia coli B/r incubated at nonpermissive temperature was expressed by shifting to a more permissive temperature (25 degrees C). Upon expression of initiation potential, the rate of [3H]thymidine incorporation varied in a bimodal fashion, i.e., there was an initial burst of incorporation, which lasted 10 to 20 min, then a sudden decrease in incorporation, and finally a second rapid increase in incorporation. Analyses of this incorporation pattern indicated that a round of replication initiated upon expression of initiation potential, but DNA polymerization stopped after replication of 5 to 10% of the chromosome. This round of replication appeared to resume about 30 min later coincident with initiation of a second round of replication. The second initiation was unusually sensitive to low concentrations of novobiocin (ca. 1 microgram/ml) when this inhibitor was added in the presence of chloramphenicol. In the absence of chloramphenicol, novobiocin at this concentration had no detectable effect on DNA replication. It is suggested that cis-acting inhibition, attributable to an attempted second initiation immediately after the first, caused the first round to stall until both it and the second round could resume simultaneously. This DNA replication inhibition, probably caused by overinitiation, could be a consequence of restraints on replication in the vicinity of oriC, possibly topological in nature, which limit the minimum interinitiation interval in E. coli.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.     

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.     

Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif

In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37 °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37°C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.     

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.