Multiple roles for DNA polymerase I in establishment and replication of the promiscuous plasmid pLS1

The polymerase activity of DNA polymerase I is important for the establishment of the pLS1 replicon by reconstitutive assembly in Streptococcus pneumoniae after uptake of exogenous pLS1 plasmid DNA. In polA mutants lacking the polymerase domain, such establishment was reduced at least 10-fold in frequency. Chromosomally facilitated establishment of pLS1-based plasmids carrying DNA homologous to the host chromosome was not so affected. However, both types of plasmid transfer gave mostly small colonies on initial selection, which was indicative of a defect in replication and filling of the plasmid pool. Once established, the pLS1-based plasmids replicated in polA mutants, but they showed segregational instability. This defect was not observed in strains with the wild-type enzyme or in an S. pneumoniae strain that encodes the polymerase and exonuclease domains of the enzyme on separate fragments. The role of DNA polymerase I in stably maintaining the plasmids depends on its polymerizing function in three separate steps of rolling-circle replication, as indicated by the accumulation of different replication intermediate forms in polA mutants. Furthermore, examination of the segregational stability of the pLS1 replicon in an Escherichia coli mutant system indicated that both the polymerase and the 5′-to-3′ exonuclease activities of DNA polymerase I function in plasmid replication.     

The involvement of DNA polymerase I in the postreplication repair of ultraviolet radiation-induced damage in Escherichia coli K-12.

Summary A deficiency in DNA polymerase I increased the ultraviolet (UV) radiation sensitivity of a uvrA strain of Escherichia coli K-12 when plated on minimal growth medium. The slope of the survival curve for the uvrA polA strain was 2.0-times greater than that for the uvrA strain. The fluence-dependent yield of unrepaired deoxyribonucleic acid (DNA) parental-strand breaks following UV irradiation and incubation in minimal growth medium was similar in both strains. However, the fluence-dependent yield of unrepaired DNA daughter-strand gaps observed following UV irradiation was 1.8-fold greater in the uvrA polA strain than in the uvrA strain. These results suggest that DNA polymerase I is involved in the filling of at least some daughter-strand gaps during postreplication repair. Also, the uvrA polA strain was sensitized by a post-UV treatment with chloramphenicol (CAP) to a similar extent as was the uvrA strain, indicating that DNA polymerase I is not involved in the CAP-inhibitable pathway of postreplication repair.     

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

Summary Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA ⁺ gene. When the dnaA gene was induced from either the plac or the pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter pL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.     

Isolation of a DNA polymerase I (polA) mutant of Rhizobium leguminosarum that has significantly reduced levels of an IncQ-group plasmid

A population of Tn5 mutagenised Rhizobium leguminosarum cells was screened for mutants affected in protein secretion by introducing a plasmid carrying the Erwinia chrysanthemi prtB gene and screening for mutants defective in secretion of the protease PrtB. One such mutant (A301) also appeared to be defective in secretion of the R. leguminosarum nodulation protein NodO. Genetic analysis showed that the defect in A301 was caused by the Tn5 insertion. However the DNA sequence adjacent to the site of Tn5 insertion had significant homology to the Escherichia coli polA gene, which encodes DNA polymerase I. The mutant A301 showed increased sensitivity to ultraviolet light, a characteristic of polA mutants of E. coli. The apparent defect in secretion by A301 was due to a large decrease in the copy number of the IncQ group replicon on which prtB and nodO were cloned and this decreased the total amounts of PrtB or NodO protein synthesised and secreted by the polA mutant. The polA mutant had a lower growth rate than the parent strain on both rich and minimal media, but there was no obvious effect of the polA mutation on the symbiosis of R. leguminosarum bv. viciae with pea.     

Initiation of chromosome replication after induction of DnaA protein synthesis in a dnaA(null) rhn mutant of Escherichia coli

The kinetics of initiation of chromosome replication after induction of DnaA protein synthesis was studied in a dnaA(null) rnh mutant of Escherichia coli. DnaA protein synthesis was induced to different extents using the wild-type dnaA gene controlled by a lac promoter. Initiation of chromosome replication from oriC, measured as an increase in origin to terminus ratio, took place at different times after addition of an inducer dependent on the DnaA protein synthesis rate. The first initiations always occurred when DnaA protein had accumulated approximately to the average wild-type concentration (24 ng of DnaA protein per ml cells at OD₄₅₀= 1.0) At a low DnaA protein accumulation rate one synchronous round of replication was obtained after 30min of induction. The initiation kinetics obtained when DnaA protein accumulated rapidly was complicated and indicated that other factors might also be involved.     

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 polymerase III-dependent repair synthesis in response to bleomycin in toluene-treated Escherichia coli

Summary Bleomycin (BLM) is an antitumor drug which interacts with and damages DNA. We have reported a repair response dependent on DNA polymerase I in toluene-treated Escherichia coli. We report here that DNA polymerase III can also catalyze a repair response in toluene-treated E. coli following exposure to BLM. Polymerase III-mediated synthesis differs because it is ATP-dependent, whereas polymerase I-mediated repair synthesis is not. Polymerase III repair synthesis is independent of replicative synthesis, as demonstrated in a polA ^-, dnaB _ts strain, or use of Novobiocin to inhibit replication, and replication persists in the presence of repair synthesis. It appears that ATP-dependent repair synthesis in response to BLM is also present in polA ⁺ strains. Repair synthesis does not require the uvrA gene product.This research was supported by Public Health Service grants GM-19122 and GM-24711 from the National Institute of General Medical Sciences, Robert A. Welch Foundation grant Q-543 and American Cancer Society BC-290     

Establishment of Escherichia coli cells with an integrated high copy number plasmid

Summary The dnaA46 cells can grow at high temperature when a high copy number plasmid pKY31, a derivative of pBR322 carrying a segment of the E. coli chromosome, integrates into the bacterial chromosome. In contrast, the dnaA46 polA ^- cells with the integrated plasmid can not grow at high temperature. Therefore, integration of the plasmid can suppress the dnaA mutation and this suppression requires DNA polymerase I which has been known to be required for plasmid replication. Full reversion of polA or lysogenization of polA ⁺ is lethal for the dnaA46polA ^- bacteria that carry the plasmid only in integrated state. Partial reversion of polA allows these cells to grow at both low and high temperatures. Introduction of the plasmid pBR322 into cytoplasm of these bacteria suppresses the lethal effect caused by full reversion of polA or lysogenization of polA ⁺. This lethal effect expresses independent of the presence or absence of the dnaA mutation. In partial revertants of polA which have only integrated plasmid, the number of copies of a region near the replication origin of integrated plasmid increases. The number is reduced by the presence of extrachromosomal pBR322. It is suggested that the lethal effect of normal levels of DNA polymerase I in strains that carry only the integrated plasmid is due to excessive initiation of replication of the bacterial chromosome from the plasmid origin and high potential of initiation can be absorbed in many copies of cytoplasmic plasmid, probably, in their replication origins.Abbreviations Amp^r ampicillin resistant (resistance) - Tet^s tetracycline sensitive - Tet^r tetracycline resistant - MMS^r methyl methane sulfonate resistant (resistance) - ts temperature sensitive - Kb kilobase pairs     

The DnaA inhibitor SirA acts in the same pathway as Soj (ParA) to facilitate oriC segregation during Bacillus subtilis sporulation

DNA replication and chromosome segregation must be carefully regulated to ensure reproductive success. During Bacillus subtilis sporulation, chromosome copy number is reduced to two, and cells divide asymmetrically to produce the future spore (forespore) compartment. For successful sporulation, oriC must be captured in the forespore. New rounds of DNA replication are prevented in part by SirA, a protein that utilizes residues in its N‐terminus to directly target Domain I of the bacterial initiator, DnaA. Using a quantitative forespore chromosome organization assay, we show that SirA also acts in the same pathway as another DnaA regulator, Soj, to promote oriC capture in the forespore. By analyzing loss‐of‐function variants of both SirA and DnaA, we observe that SirA's ability to inhibit DNA replication can be genetically separated from its role in oriC capture. In addition, we identify substitutions near the C‐terminus of SirA and in DnaA Domain III that enhance interaction between the two proteins. One such variant, SirA_P141T, remained functional in regard to inhibiting replication, but was unable to support oriC capture. Collectively, our results support a model in which SirA targets DnaA Domain I to inhibit DNA replication, and DnaA Domain III to facilitate Soj‐dependent oriC capture in the forespore.     

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.     

DNA polymerase I is not required for replication of linear chromosomes in streptomyces

Both polA (encoding DNA polymerase I; Pol I) and a paralog were deleted from Streptomyces strains. Despite the UV sensitivity and slow growth caused by the ΔpolA mutation, the double mutant was viable. Thus, in contrast to a previous postulate, Pol I and its paralog are not essential for replication of Streptomyces chromosomes.