Differential binding of Escherichia coli DNA polymerases to the beta-sliding clamp

Escherichia coli strains expressing the mutant beta159-sliding clamp protein (containing both a G66E and a G174A substitution) are temperature sensitive for growth and display altered DNA polymerase (pol) usage. We selected for suppressors of the dnaN159 allele able to grow at 42 degrees C, and identified four intragenic suppressor alleles. One of these alleles (dnaN780) contained only the G66E substitution, while a second (dnaN781) contained only the G174A substitution. Genetic characterization of isogenic E. coli strains expressing these alleles indicated that certain phenotypes were dependent upon only the G174A substitution, while others required both the G66E and G174A substitutions. In order to understand the individual contributions of the G66E and the G174A substitution to the dnaN159 phenotypes, we utilized biochemical approaches to characterize the purified mutant beta159 (G66E and G174A), beta780 (G66E) and beta781 (G174A) clamp proteins. The G66E substitution conferred a more pronounced effect on pol IV replication than it did pol II or pol III, while the G174A substitution conferred a greater effect on pol III and pol IV than it did pol II. Taken together, these findings indicate that pol II, pol III and pol IV interact with distinct, albeit overlapping surfaces of the beta clamp.     

Roles of the Escherichia coli RecA protein and the global SOS response in effecting DNA polymerase selection in vivo

The Escherichia coli beta sliding clamp protein is proposed to play an important role in effecting switches between different DNA polymerases during replication, repair, and translesion DNA synthesis. We recently described how strains bearing the dnaN159 allele, which encodes a mutant form of the beta clamp (beta159), display a UV-sensitive phenotype that is suppressed by inactivation of DNA polymerase IV (M. D. Sutton, J. Bacteriol. 186:6738-6748, 2004). As part of an ongoing effort to understand mechanisms of DNA polymerase management in E. coli, we have further characterized effects of the dnaN159 allele on polymerase usage. Three of the five E.coli DNA polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicate that elevated expression of the dinB-encoded polymerase IV is sufficient to result in conditional lethality of the dnaN159 strain. In contrast, chronically activated RecA protein, expressed from the recA730 allele, is lethal to the dnaN159 strain, and this lethality is suppressed by mutations that either mitigate RecA730 activity (i.e., DeltarecR), or impair the activities of DNA polymerase II or DNA polymerase V (i.e., DeltapolB or DeltaumuDC). Thus, we have identified distinct genetic requirements whereby each of the three different SOS-regulated DNA polymerases are able to confer lethality upon the dnaN159 strain, suggesting the presence of multiple mechanisms by which the actions of the cell's different DNA polymerases are managed in vivo.     

The Escherichia coli dnaN159 mutant displays altered DNA polymerase usage and chronic SOS induction

The Escherichia coli beta sliding clamp, which is encoded by the dnaN gene, is reported to interact with a variety of proteins involved in different aspects of DNA metabolism. Recent findings indicate that many of these partner proteins interact with a common surface on the beta clamp, suggesting that competition between these partners for binding to the clamp might help to coordinate both the nature and order of the events that take place at a replication fork. The purpose of the experiments discussed in this report was to test a prediction of this model, namely, that a mutant beta clamp protein impaired for interactions with the replicative DNA polymerase (polymerase III [Pol III]) would likewise have impaired interactions with other partner proteins and hence would display pleiotropic phenotypes. Results discussed herein indicate that the dnaN159-encoded mutant beta clamp protein (beta159) is impaired for interactions with the alpha catalytic subunit of Pol III. Moreover, the dnaN159 mutant strain displayed multiple replication and repair phenotypes, including sensitivity to UV light, an absolute dependence on the polymerase activity of Pol I for viability, enhanced Pol V-dependent mutagenesis, and altered induction of the global SOS response. Furthermore, epistasis analyses indicated that the UV sensitivity of the dnaN159 mutant was suppressed by (not epistatic with) inactivation of Pol IV (dinB gene product). Taken together, these findings suggest that in the dnaN159 mutant, DNA polymerase usage, and hence DNA replication, repair, and translesion synthesis, are altered. These findings are discussed in terms of a model to describe how the beta clamp might help to coordinate protein traffic at the replication fork.     

Role of Escherichia coli DNA polymerase I in conferring viability upon the dnaN159 mutant strain

The Escherichia coli dnaN159 allele encodes a mutant form of the beta-sliding clamp (beta159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain. In addition, we demonstrate that the beta clamp stimulates the processivity of Pol I in vitro and that beta159 is impaired for this activity. The reduced ability of beta159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the beta clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 DeltapolA strain.     

Mutant forms of the Escherichia colibeta sliding clamp that distinguish between its roles in replication and DNA polymerase V-dependent translesion DNA synthesis

The Escherichia colibeta sliding clamp is proposed to play an important role in regulating DNA polymerase traffic at the replication fork. As part of an ongoing effort to understand how organisms manage the actions of their multiple DNA polymerases, we examined the ability of several mutant forms of the beta clamp to function in DNA polymerase V- (pol V-) dependent translesion DNA synthesis (TLS) in vivo. Our results indicate that a dnaN159 strain, which expresses a temperature sensitive form of the beta clamp, was impaired for pol V-dependent TLS at the permissive temperature of 37 degrees C. This defect was complemented by a plasmid that expressed near-physiological levels of the wild-type clamp. Using a dnaN159 mutant strain, together with various plasmids expressing mutant forms of the clamp, we determined that residues H148 through R152, which comprise a portion of a solvent exposed loop, as well as position P363, which is located in the C-terminal tail of the beta clamp, are critically important for pol V-dependent TLS in vivo. In contrast, these same residues appear to be less critical for pol III-dependent replication. Taken together, these findings indicate that: (i) the beta clamp plays an essential role in pol V-dependent TLS in vivo and (ii) pol III and pol V interact with non-identical surfaces of the beta clamp.     

Differential effects of cisplatin and MNNG on dna mutants of Escherichia coli

DNA mismatch repair (MMR) in mammalian cells or Escherichia coli dam mutants increases the cytotoxic effects of cisplatin and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We found that, unlike wildtype, the dnaE486 (alpha catalytic subunit of DNA polymerase III holoenzyme) mutant, and a DnaX (clamp loader subunits) over-producer, are sensitive to cisplatin but resistant to MNNG at the permissive temperature for growth. Survival of dam-13 dnaN159 (beta sliding clamp) bacteria to cisplatin was significantly less than dam cells, suggesting decreased MMR, which may be due to reduced MutS-beta clamp interaction. We also found an elevated spontaneous mutant frequency to rifampicin resistance in dnaE486 (10-fold), dnaN159 (35-fold) and dnaX36 (10-fold) strains. The mutation spectrum in the dnaN159 strain was consistent with increased SOS induction and not indicative of MMR deficiency.     

Analysis of a multicomponent thermostable DNA polymerase III replicase from an extreme thermophile

This report takes a proteomic/genomic approach to characterize the DNA polymerase III replication apparatus of the extreme thermophile, Aquifex aeolicus. Genes (dnaX, holA, and holB) encoding the subunits required for clamp loading activity (tau, delta, and delta') were identified. The dnaX gene produces only the full-length product, tau, and therefore differs from Escherichia coli dnaX that produces two proteins (gamma and tau). Nonetheless, the A. aeolicus proteins form a taudeltadelta' complex. The dnaN gene encoding the beta clamp was identified, and the taudeltadelta' complex is active in loading beta onto DNA. A. aeolicus contains one dnaE homologue, encoding the alpha subunit of DNA polymerase III. Like E. coli, A. aeolicus alpha and tau interact, although the interaction is not as tight as the alpha-tau contact in E. coli. In addition, the A. aeolicus homologue to dnaQ, encoding the epsilon proofreading 3'-5'-exonuclease, interacts with alpha but does not form a stable alpha.epsilon complex, suggesting a need for a brace or bridging protein to tightly couple the polymerase and exonuclease in this system. Despite these differences to the E. coli system, the A. aeolicus proteins function to yield a robust replicase that retains significant activity at 90 degrees C. Similarities and differences between the A. aeolicus and E. coli pol III systems are discussed, as is application of thermostable pol III to biotechnology.     

Genetic interactions between the Escherichia coli umuDC gene products and the beta processivity clamp of the replicative DNA polymerase

The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA-single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD' (which enables TLS). In addition, even modest overexpression of the umuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD(2)C. We have previously reported that overexpression of the epsilon proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of epsilon with UmuD(2)C is important for the DNA damage checkpoint control function of the umuDC gene products. Here, we report that overexpression of the beta processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of the umuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD'C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant beta proteins possibly deficient for physical interactions with the umuDC gene products, we selected for novel dnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD'C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of beta or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaN missense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eight dnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of the umuDC gene products, suggesting that the interactions between UmuD' and beta are a subset of those between UmuD and beta. Taken together, these findings suggest that interaction of beta with UmuD(2)C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD(2)C with the epsilon and the beta subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.     

Analysis of the stimulation of DNA polymerase V of Escherichia coli by processivity proteins

Bypass of replication-blocking lesions in Escherichia coli is carried out by DNA polymerase V (UmuC) in a reaction that requires UmuD', RecA, and single-strand DNA-binding protein (SSB). The activity of this four-component basic bypass system is a low-fidelity and low-processivity activity. Addition of the processivity subunits of pol III, the beta subunit sliding DNA clamp, and the five-subunit gamma complex clamp loader increased the rate of translesion replication approximately 3-fold. This stimulation was specific to the lesion bypass step, with no effect on the initiation of synthesis by pol V. The beta subunit and gamma complex increased the processivity of pol V from 3 to approximately 14-18 nucleotides, providing a mechanistic basis for their stimulatory effect. Stimulation of bypass was observed over a range of RecA and SSB concentrations. ATPgammaS, which strongly inhibits translesion replication by pol V, primarily via inhibition of the initiation stage, caused the same inhibition also in the presence of the processivity proteins. The in vivo role of the processivity proteins in translesion replication was examined by assaying UV mutagenesis. This was done in a strain carrying the dnaN59 allele, encoding a temperature-sensitive beta subunit. When assayed in an excision repair-defective background, the dnaN59 mutant exhibited a level of UV mutagenesis reduced up to 3-fold compared to that of the isogenic dnaN(+) strain. This suggests that like in the in vitro system, the beta subunit stimulates lesion bypass in vivo.     

Identification of specific amino acid residues in the E. coli beta processivity clamp involved in interactions with DNA polymerase III, UmuD and UmuD'

Variants of a pentapeptide sequence (QL[S/F]LF), referred to as the eubacterial clamp-binding motif, appear to be required for certain proteins to bind specifically to the Escherichia coli beta sliding clamp, apparently by making contact with a hydrophobic pocket located at the base of the C-terminal tail of each beta protomer. Although both UmuC (DNA pol V) and the alpha catalytic subunit of DNA polymerase III (pol III) each bear a reasonable match to this motif, which appears to be required for their respective interactions with the clamp, neither UmuD not UmuD' do. As part of an ongoing effort to understand how interactions involving the different E. coli umuDC gene products and components of DNA polymerase III help to coordinate DNA replication with a DNA damage checkpoint control and translesion DNA synthesis (TLS) following DNA damage, we characterized the surfaces on beta important for its interactions with the two forms of the umuD gene product. We also characterized the surface of beta important for its interaction with the alpha catalytic subunit of pol III. Our results indicate that although UmuD, UmuD' and alpha share some common contacts with beta, each also makes unique contacts with the clamp. These findings suggest that differential interactions of UmuD and UmuD' with beta impose a DNA damage-responsive conditionality on how beta interacts with the translesion DNA polymerase UmuC. This is formally analogous to how post-translational modification of the eukaryotic PCNA clamp influences mutagenesis. We discuss the implications of our findings in terms of how E. coli might coordinate the actions of the umuDC gene products with those of pol III, as well as for how organisms in general might manage the actions of their multiple DNA polymerases.     

Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA

In Escherichia coli, an interaction between the replication initiator DnaA and the sliding clamp protein, the beta subunit (DnaN) of DNA polymerase III, is required to regulate the chromosomal replication cycle. We report here that colony formation by, and cell division of, the temperature (42 degrees C)-sensitive dnaN59 mutant are inhibited at 34-35 degrees C when DnaA is moderately (4-to 8-fold ) overexpressed, although chromosomal replication and the beta subunit-dependent regulation of DnaA activity are not significantly inhibited. Immunoblotting analysis revealed that the beta subunit is abundant (present at a level of about 5000 dimers per cell) at 34 degrees C, and its concentration per unit cell volume was practically unaffected in the dnaN59 mutant by the overexpression of DnaA. The dnaN mutant cells that overexpress DnaA become filamentous at 34 degrees C via an sfiA-independent pathway, different from that activated by the SOS response. This filamentation is accompanied by inhibition of nucleoid partition and FtsZ ring formation. In the dnaN59 mutant, oversupply of DnaA may disturb the coordinated action of cell cycle-regulating molecules, thus leading to the inhibition of these events.     

The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli

In Escherichia coli, the circular beta sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA. When synthesis is complete, polymerase dissociates from beta and DNA and cycles to a new start site, a primed template loaded with beta. DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments. The clamps left behind remain stable on DNA (t(12) approximately 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand. Here we show that delta, a single subunit of DNA polymerase III holoenzyme, opens beta and slips it off DNA (k(unloading) = 0.011 s(-)(1)) at a rate similar to that of the multisubunit gamma complex clamp loader by itself (0.015 s(-)(1)) or within polymerase (pol) III* (0.0065 s(-)(1)). Moreover, unlike gamma complex and pol III*, delta does not require ATP to catalyze clamp unloading. Quantitation of gamma complex subunits (gamma, delta, delta', chi, psi) in E. coli cells reveals an excess of delta, free from gamma complex and pol III*. Since pol III* and gamma complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the delta subunit probably aids beta clamp recycling during DNA replication.     

Escherichia coli DNA polymerase IV (Pol IV), but not Pol II, dynamically switches with a stalled Pol III* replicase

The dnaN159 allele encodes a temperature-sensitive mutant form of the β sliding clamp (β159). SOS-induced levels of DNA polymerase IV (Pol IV) confer UV sensitivity upon the dnaN159 strain, while levels of Pol IV ～4-fold higher than those induced by the SOS response severely impede its growth. Here, we used mutations in Pol IV that disrupted specific interactions with the β clamp to test our hypothesis that these phenotypes were the result of Pol IV gaining inappropriate access to the replication fork via a Pol III*-Pol IV switch relying on both the rim and cleft of the clamp. Our results clearly demonstrate that Pol IV relied on both the clamp rim and cleft interactions for these phenotypes. In contrast to the case for Pol IV, elevated levels of the other Pols, including Pol II, which was expressed at levels ～8-fold higher than the normal SOS-induced levels, failed to impede growth of the dnaN159 strain. These findings suggest that the mechanism used by Pol IV to switch with Pol III* is distinct from those used by the other Pols. Results of experiments utilizing purified components to reconstitute the Pol III*-Pol II switch in vitro indicated that Pol II switched equally well with both a stalled and an actively replicating Pol III* in a manner that was independent of the rim contact required by Pol IV. These results provide compelling support for the Pol III*-Pol IV two-step switch model and demonstrate important mechanistic differences in how Pol IV and Pol II switch with Pol III*.     

Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment

Escherichia coli DNA polymerase IV (pol IV), a member of the error-prone Y family, predominantly generates -1 frameshifts when copying DNA in vitro. T-->G transversions and T-->C transitions are the most frequent base substitutions observed. The in vitro data agree with mutational spectra obtained when pol IV is overexpressed in vivo. Single base deletion and base substitution rates measured in the lacZalpha gene in vitro are, on average, 2 x 10(-4) and 5 x 10(-5), respectively. The range of misincorporation and mismatch extension efficiencies determined kinetically are 10(-3) to 10(-5). The presence of beta sliding clamp and gamma-complex clamp loading proteins strongly enhance pol IV processivity but have no discernible influence on fidelity. By analyzing changes in fluorescence of a 2-aminopurine template base undergoing replication in real time, we show that a "dNTP-stabilized" misalignment mechanism is responsible for making -1 frameshift mutations on undamaged DNA. In this mechanism, a dNTP substrate is paired "correctly" opposite a downstream template base, on a "looped out" template strand instead of mispairing opposite a next available template base. By using the same mechanism, pol IV "skips" past an abasic template lesion to generate a -1 frameshift. A crystal structure depicting dNTP-stabilized misalignment was reported recently for Sulfolubus solfataricus Dpo4, a Y family homolog of Escherichia coli pol IV.     

Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC

Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.     

The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems.

The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y.S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta subunit in lieu of T4 gp45 in the elongation of singly primed phi X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.     

Fidelity of eucaryotic DNA polymerase delta holoenzyme from Schizosaccharomyces pombe

The fidelity of Schizosaccharomyces pombe DNA polymerase delta was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol delta alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol delta holoenzyme (HE) (pol delta + PCNA + RFC) was 4.6 x 10(-4) for T.G mispairs, 5.3 x 10(-5) for G.G mispairs, and 4.5 x 10(-6) for A.G mispairs. The T.G misincorporation frequency for pol delta without the accessory proteins was unchanged. The fidelity of pol delta HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol delta HE with E. coli pol IIIalpha HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.     

Stationary phase induction of dnaN and recF, two genes of Escherichia coli involved in DNA replication and repair.

The beta subunit of DNA polymerase III holoenzyme, the Escherichia coli chromosomal replicase, is a sliding DNA clamp responsible for tethering the polymerase to DNA and endowing it with high processivity. The gene encoding beta, dnaN, maps between dnaA and recF, which are involved in initiation of DNA replication at oriC and resumption of DNA replication at disrupted replication forks, respectively. In exponentially growing cells, dnaN and recF are expressed predominantly from the dnaA promoters. However, we have found that stationary phase induction of the dnaN promoters drastically changes the expression pattern of the dnaA operon genes. As a striking consequence, synthesis of the beta subunit and RecF protein increases when cell metabolism is slowing down. Such an induction is dependent on the stationary phase sigma factor, RpoS, although the accumulation of this factor alone is not sufficient to activate the dnaN promoters. These promoters are located in DNA regions without static bending, and the -35 hexamer element is essential for their RpoS-dependent induction. Our results suggest that stationary phase-dependent mechanisms have evolved in order to coordinate expression of dnaN and recF independently of the dnaA regulatory region. These mechanisms might be part of a developmental programme aimed at maintaining DNA integrity under stress conditions.     

Escherichia coli cells with increased levels of DnaA and deficient in recombinational repair have decreased viability

The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, beta clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, beta clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the beta clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 micro m) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional approximately 100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.     

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.