A novel dnaC mutation that suppresses priB rep mutant phenotypes in Escherichia coli K-12

The loading of a replisome in prokaryotic and eukaryotic cells at an origin of DNA replication and during replication restart is a highly ordered and regulated process. During replication restart in Escherichia coli, the PriA, PriB, PriC, DnaT and Rep proteins form multiple pathways that bind to repaired replication forks. These complexes are then recognized by DnaC as sites to load DnaB, the replicative helicase. Several dnaC mutations have been isolated that suppress phenotypes of some replication restart mutants. A new dnaC mutation (dnaC824) is reported here that efficiently suppresses priB rep mutant phenotypes. Furthermore, it is shown that dnaC824 will suppress phenotypes of priB priA300, rep priA300 and priB priC strains. Unlike other dnaC suppressors, it can only weakly suppress the absence of priA. Others have reported a different type of dnaC mutation, dnaC1331, is able to mimic priB mutant phenotypes. This is supported herein by showing that like dnaC1331, a priB mutation is synthetically lethal with a dam mutation and this can be rescued by a mutH mutation. Furthermore, priB dam lethality can also be suppressed by dnaC824. Like a priB mutation, a dnaC1331 mutation causes a priA2::kan-like phenotype when combined with priA300. Lastly, we show that dnaC824 is dominant to wild type and that dnaC1331 is recessive to wild type. Several models are discussed for the action of these mutant dnaC proteins in replication restart.     

PriA mutations that affect PriA-PriC function during replication restart

In Escherichia coli, repair and restart of collapsed replication forks is thought to be essential for cell growth. The replication restart proteins, PriA, PriB, PriC, DnaB, DnaC, DnaG, DnaT and Rep, form redundant pathways that recognize repaired replication forks and restart them. Recognition, modulation of specific DNA structures and loading of the replicative helicase by the replication restart proteins, is likely to be important for replication restart. It has been hypothesized that PriB and PriC function with PriA in genetically separate and redundant PriA-PriB and PriA-PriC pathways. In this study, the del(priB)302 or priC303:kan mutations were used to isolate the PriA-PriB and PriA-PriC pathways genetically so that the effects of three priA missense mutations, priA300 (K230R), priA301 (C479Y) and priA306 (L557P), on these pathways could be assessed. In a wild-type background, the three priA mutations had little, if any, effect on the phenotypes of UV resistance, basal levels of SOS expression and cell viability. In the priB mutant, priA300 and priA301 caused dramatic negative changes in the three phenotypes listed above (and others), whereas the third priA mutant allele, priA306, showed very little negative effect. In the priC mutant, all three priA mutations behaved similarly, producing little, if any, changes in phenotypes. We conclude that priA300 and priA301 mostly affect the PriA-PriC pathway and do so more than priA306. We suggest that PriA's helicase activity is important for the PriA-PriC pathway of replication restart.     

Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12

In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Deltarep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3'-5' helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.     

The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair.

The PriA protein, a component of the phiX174-type primosome, was previously shown to be essential for damage-inducible DNA replication in Escherichia coli, termed inducible stable DNA replication. Here, we show that priA::kan null mutants are defective in transductional and conjugational homologous recombination and are hypersensitive to mitomycin C and gamma rays, which cause double-strand breaks. The introduction of a plasmid carrying the priA300 allele, which encodes a mutant PriA protein capable of catalyzing the assembly of an active primosome but which is missing the n'-pas-dependent ATPase, helicase, and translocase activities associated with PriA, alleviates the defects of priA::kan mutants in homologous recombination, double-strand break repair, and inducible stable DNA replication. Furthermore, spa-47, which was isolated as a suppressor of the broth sensitivity of priA::kan mutants, suppresses the Rec- and mitomycin C sensitivity phenotypes of priA::kan mutants. The spa-47 suppressor mutation maps within or very near dnaC. These results suggest that PriA-dependent primosome assembly is crucial for both homologous recombination and double-strand break repair and support the proposal that these processes in E. coli involve extensive DNA replication.     

Differential Suppression of Pria2::Kan Phenotypes in Escherichia Coli K-12 by Mutations in Pria, Lexa, and Dnac

First identified as an essential component of the X174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UV(S) and Rec(-), (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA(+), restores both UV(R) and Rec(+) phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UV(R) Rec(+) revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.     

Requirement for RecFOR-mediated recombination in priA mutant

Restart of arrested replication forks is an important process and PriA, the main Escherichia coli replication restart protein, is essential for viability under any condition that increases the frequency of fork arrest. In priA mutant, replication forks are arrested by spontaneously occurring roadblocks and blocked replication forks persist as a result of the defect in replication restart. In the present work, we analysed how recombination proteins contribute to the viability of the priA mutant. RecFOR-mediated homologous recombination occurs in a large fraction of priA mutant cells, indicating a frequent formation of DNA single strand gaps and their recombinational repair. This high level of homologous recombination renders the proteins that resolve Holliday junctions recombination intermediates essential for viability. When homologous recombination is blocked at early steps by recFOR or recA inactivation, exonuclease V-mediated DNA degradation is required for full viability of priA mutants, indicating that unrepaired gaps are broken and that DNA degradation of the broken DNA allows the formation of viable cells. Models for the formation of single strand DNA gaps consequently to a replication restart defect and for gap processing are proposed.     

Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replication.

Under certain conditions, Escherichia coli cells exhibit either of two altered modes of chromosomal DNA replication. These are inducible stable DNA replication (iSDR), seen in SOS-induced cells, and constitutive stable DNA replication (cSDR), seen in rnhA mutants. Both iSDR and cSDR can continue to occur in the absence of protein synthesis. They are dependent on RecA protein, but do not require DnaA protein or the oriC site. Here we report the requirement for PriA, a protein essential for assembly of the phi X174-type primosome, for both iSDR and cSDR. In priA1(Null)::kan mutant cells, iSDR is not observed after induction by thymine starvation. Replication from one of the origins (oriM1) specific to iSDR is greatly reduced by the priA1::kan mutation. cSDR in rnhA224 mutant cells deficient in RNase HI is also completely abolished by the same priA mutation. In both cases, SDR is restored by introduction of a plasmid carrying a wild-type priA gene. Furthermore, the viability of an rnhA::cat dnaA46 strain is lost at 42 degrees C upon inactivation of the priA gene, indicating the lethal effect of priA inactivation on those cells whose viability depends on cSDR. These results demonstrate that a function of PriA protein is essential for iSDR and cSDR and suggest the involvement of the PriA-dependent phi X174-type primosome in these DnaA/oriC-independent pathways of chromosome replication. Whereas ColE1-type plasmids, known to be independent of DnaA, absolutely require PriA function for replication, DnaA-dependent plasmid replicons such as pSC101, F, R6K, Rts1 and RK2 are able to transform and to be maintained in the priA1::kan strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome.

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single-stranded DNA in a PriA-independent, DnaD- and DnaI-dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.     

Overlapping functions for recF and priA in cell viability and UV-inducible SOS expression are distinguished by dnaC809 in Escherichia coli K-12

The recF and priA genes have roles in DNA repair and homologous recombination. Mutations in these genes also cause decreases in cell viability and alterations in UV-inducible sulAp–lacZ (SOS) expression. To find out if the two genes are in the same or different pathways for viability and SOS expression, the phenotypes of the double mutant strains were studied. The recF priA double mutant showed a lower viability and SOS expression level than either of the single mutants. In the case of cell viability, recF missense mutations decreased viability of a priA2 :: kan strain two to fivefold whereas recF null priA2 :: kan double mutants were not viable at all. dnaC809 , a mutation that suppresses the UV-sensitive (UV^S) and Rec⁻ phenotypes of priA2 :: kan , restored cell viability, but not UV-inducible SOS expression, to a priA recF strain. Since recF is epistatic with recO and recR ( recOR ) for UV resistance, recOR mutations were also tested with priA2 :: kan . No overlap was found between recOR and priA for viability and SOS expression. It is concluded that priA and recF have two different overlapping functions in viability and SOS expression that are distinguishable by the effects of dnaC809 . The role of recF in a priA2 :: kan strain in cell viability is a new function for recF and unlike recF  's other roles in DNA repair and recombination, is independent of recOR . A new role for priA in UV-inducible SOS expression in a recF mutant is also defined.     

Effect of a null mutation in the priA gene on radioresistance of Escherichia coli

According to Kogoma's model of DNA recombination by replication, the PriA protein is involved in the RecBCD pathway of double-strand break (DSB) repair, which is associated with extensive DNA degradation, at the stage of primosome assembly in D-loops (intermediates of strand exchange at the ends of DSB) for the subsequent switch to DSB-induced DNA resynthesis. Comparable data on possible involvement of the PriA protein in the repair of gamma-ray-induced lethal lesions in cells of the wild-type strain of Escherichia coli (strain AB1157) and in two radiation-resistant mutants Gamr445 and Gamr444 were obtained. In all the three strains examined, the null priA2::kan mutation in the structural priA gene was shown to markedly enhance the radiation sensitivity, causing a two- to threefold increase in the slopes of linear dose-survival curves. In the AB1157 strain, the inactivation of PriA is manifested most clearly in the range of low doses (up to 0.15 kGy) when the priA2::kan mutation had only a slight effect on the radiation resistance of Gamr mutants. It can be assumed that, in these mutants with a decreased level of postradiation DNA degradation, the PriA-dependent RecBCD pathway of DSB repair associated with extensive DNA resynthesis is not essential for the repair of lethal lesions at low doses. However, this pathway becomes crucial at higher doses (> 0.5 kGy) even for radiation-resistant strains, especially for the most resistant Gamr444 mutant.     

Primosome assembly requirement for replication restart in the Escherichia coli holDG10 replication mutant

In this report, we study the role of pre-primosome proteins in a strain in which the frequency of replication arrest is increased because of a mutation in a replication protein. The holDG10 mutant was used, in which replication restart involves replication fork reversal. As expected, PriA primosome assembly function is essential for growth of the holDG10 mutant. The priA300 mutation, which inactivates only the helicase function of PriA in vitro, and priB inactivation strongly impair viability. In contrast, priC inactivation has no effect. Therefore, PriB is more important than PriC for PriA-dependent replication fork restart in vivo. The gain of function mutation dnaC809 restores the viability of holDG10 priA and holDG10 priB mutants only to some extent. The dnaC809 820 double mutation restores full viability to the holDG10 mutant lacking either PriA or PriB. Similarly to the holDG10 single mutant, the holDG10 priA dnaC809 820 strain is depend-ent on RecBC for viability, indicating that facilitating primosome assembly using the dnaC809 820 mutation does not allow bypass of replication fork reversal.     

Mutation of the priA gene of Neisseria gonorrhoeae affects DNA transformation and DNA repair

In Escherichia coli, PriA is central to the restart of chromosomal replication when replication fork progression is disrupted and is also involved in homologous recombination and DNA repair. To investigate the role of PriA in recombination and repair in Neisseria gonorrhoeae, we identified, cloned, and insertionally inactivated the gonococcal priA homologue. The priA mutant showed a growth deficiency and decreased DNA repair capability and was completely for deficient in DNA transformation compared to the isogenic parental strain. The priA mutant was also more sensitive to the oxidative damaging agents H2O2 and cumene hydroperoxide compared to the parental strain. These phenotypes were complemented by supplying a functional copy of priA elsewhere in the chromosome. The N. gonorrhoeae priA mutant showed no alteration in the frequency of pilin antigenic variation. We conclude that PriA participates in DNA repair and DNA transformation processes but not in pilin antigenic variation.     

Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function

Escherichia coli strains lacking PriA are severely compromised in their ability to repair UV-damaged DNA and to perform homologous recombination. These phenotypes arise because of a lack of PriA-directed replication fork assembly at recombination intermediates such as D-loops. Naturally arising suppressor mutations in dnaC restore strains carrying the priA2::kan null allele to wild-type function. We have cloned one such gene, dnaC810, and overexpressed, purified, and characterized the DnaC810 protein. DnaC810 can support a PriA-independent synthesis of phiX174 complementary strand DNA. This can be attributed to its ability, unlike wild-type DnaC, to catalyze a SSB-insensitive general priming reaction with DnaB and DnaG on any SSB-coated single-stranded DNA. Gel mobility shift analysis revealed that DnaC810 could load DnaB directly to SSB-coated single-stranded DNA as well as to D loop DNA. This explains the ability of DnaC810 to bypass the requirement for PriA, PriB, PriC, and DnaT during replication fork assembly at recombination intermediates.     

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).     

Cells defective for replication restart undergo replication fork reversal

We have studied the fate of blocked replication forks with the use of the Escherichia coli priA mutant, in which spontaneously arrested replication forks persist owing to the lack of the major replication restart pathway. Such blocked forks undergo a specific reaction named replication fork reversal, in which newly synthesized strands anneal to form a DNA double-strand end adjacent to a four-way junction. Indeed, (i) priA recB mutant chromosomes are linearized by a reaction that requires the presence of the Holliday junction resolvase RuvABC, and (ii) RuvABC-dependent linearization is prevented by the presence of RecBC. Replication fork reversal in a priA mutant occurs independently of the recombination proteins RecA and RecR. recBC inactivation does not affect priA mutant viability but prevents priA chronic SOS induction. We propose that, in the absence of PriA, RecBC action at reversed forks does not allow replication restart, which leads to the accumulation of SOS-inducing RecA filaments. Our results suggest that types of replication blockage that cause replication fork reversal occur spontaneously.     

Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication.

Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.     

Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

Tryptophan- and indole-excreting prototrophic mutant of Escherichia coli

Lim, P. G. (Massachusetts Institute of Technology, Cambridge), and R. I. Mateles. Tryptophan- and indole-excreting prototrophic mutant of Escherichia coli. J. Bacteriol. 87:1051-1055. 1964.-A mutant of Escherichia coli K-12, capable of excreting 350 mg of indole and 50 mg of tryptophan per liter when grown on minimal medium, was found to have a level of 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthetase 60% higher than the parent, and to have a 10- to 15-fold elevation of the levels of enzymes in the tryptophan branch of the pathway for aromatic amino acid biosynthesis. Contrary to what previous investigators found in E. coli W, the presence of a tyrosine-repressible component of DAHP synthetase sensitive to end-product inhibition by tyrosine could not be demonstrated in either strain K-12 or the mutant. The mutant strain is an example of a microorganism which excretes biosynthetic end products solely because of genetic derepression, as opposed to most previously reported amino acid accumulators which require a combination of genetic and physiological manipulation to achieve derepression.     

A new role for translation initiation factor 2 in maintaining genome integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA-damaging agent is present.