Constitutive Stable DNA Replication Research Articles

Topoisomerase I Essentiality, DnaA-Independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli.

Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here, we show that several topA alleles, including ΔtopA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in the absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain (MDS42) or (ii) by an RNA polymerase (RNAP) mutation, rpoB*35, that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) one of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn leads to transcription-replication conflicts and to cSDR. IMPORTANCE In all life forms, double-helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability and suggests that in its absence, aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation.

The bacterium Escherichia coli can initiate replication in the absence of the replication initiator protein DnaA and/or the canonical origin of replication oriC in a ΔrnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of ΔrnhA-ΔdnaA strains followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4 to 0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop-forming regions, and includes a site containing sequence motifs that favor R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion, however, increases the possibility of conflicts at essential mRNA genes, which would utilize only a minuscule fraction of RNA polymerase molecules, most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question.IMPORTANCE The bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics and show that selection favors cSDR initiation predominantly at a region ∼0.6 Mb clockwise of oriC. Initiation from this site will result in more head-on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimized. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance to antibiotics that target initiation of DNA replication.

A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication.

The Dam DNA methylase of Escherichia coli is required for methyl-directed mismatch repair, regulation of chromosomal DNA replication initiation from oriC (which is DnaA-dependent), and regulation of gene expression. Here, we show that Dam suppresses aberrant oriC-independent chromosomal replication (also called constitutive stable DNA replication, or cSDR). Dam deficiency conferred cSDR and, in presence of additional mutations (Δtus, rpoB*35) that facilitate retrograde replication fork progression, rescued the lethality of ΔdnaA mutants. The DinG helicase was required for rescue of ΔdnaA inviability during cSDR. Viability of ΔdnaA dam derivatives was dependent on the mismatch repair proteins, since such viability was lost upon introduction of deletions in mutS, mutH or mutL; thus generation of double strand ends (DSEs) by MutHLS action appears to be required for cSDR in the dam mutant. On the other hand, another DSE-generating agent phleomycin was unable to rescue ΔdnaA lethality in dam+ derivatives (mutS+ or ΔmutS), but it could do so in the dam ΔmutS strain. These results point to a second role for Dam deficiency in cSDR. We propose that in Dam-deficient strains, there is an increased likelihood of reverse replication restart (towards oriC) following recombinational repair of DSEs on the chromosome.

A New Pathway of Transcription‐Coupled Repair

Transcription‐coupled repair (TCR) of damaged DNA in E. coli can involve several different pathways. We propose that one pathway entails release of RNAP at the site of DNA damage without dissociation of the RNA:DNA hybrid. The RNA can serve as a primer for PolI, which initiates the repair process. We are investigating how TCR repairs nalidixic acid‐induced DNA damage. Nalidixic acid, like Etoposide generates a DNA double‐strand break bearing a TopA adduct at each 5′ DNA end. We show that efficient repair requires two transcription factors. The first DksA, is a coiled‐coil protein that inserts into the RNAP secondary channel and, in concert with ppGpp, regulates the activity of certain promoters. The role of DksA in transcription elongation is undefined, although it is known to suppresses replisome – RNAP clashes. DksA plays a direct role in repair, rather than blocking access of GreA to RNAP. Thus, a ΔdksA mutation is not suppressed by a greA deletion. Recent work from the Murakami laboratory reveals that DksA binding distorts the structure of RNAP, opening the pincers, thus potentially reducing the processivity of transcription elongation. DksA in association with ppGpp, however, does not affect RNAP structure. We find that repair is more efficient in strains carrying an RNAP mutation that reduces ppGpp binding to site 2, the location of DksA insertion. We have isolated a suppressor of ΔdksA based on resistance to nalidixic acid. The mutation, rpoBQ148H, phenocopies RNAP bound to DksA in the absence of ppGpp, i.e. it opens the pincers and may favor release of elongating RNAP. These findings suggest that DksA may act as a transcription termination factor that can release RNAP without removing the RNA:DNA hybrid.The second, transcription termination protein Rho, is an RNA‐dependent DNA:RNA helicase. The defects in TCR of rho and dksA mutations are additive, suggesting that they act on different substrates. Whereas Rho is thought to release RNAP by unwinding the RNA:DNA helix, rho mutants that terminate transcription without releasing RNA have been isolated.Another reaction that entails elongation of an RNA:DNA hybrid by PolI is constitutive stable DNA replication (cSDR). In cSDR, elongation of an R‐loop by PolI creates a new origin of replication. We find that cSDR is dependent on DksA. Presumably the mechanisms of nalidixic acid DNA damage and cSDR both involve allowing access of PolI to the 3′ end of an RNA:DNA hybrid.Support or Funding InformationGM037219 GM037219 NIH: GM037219 NIH: GM037219 GM037219This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Constitutive stable DNA replication in Escherichia coli cells lacking type 1A topoisomerase activity

Type 1A topoisomerases (topos) are ubiquitous enzymes involved in supercoiling regulation and in the maintenance of genome stability. Escherichia coli possesses two type 1A enzymes, topo I (topA) and topo III (topB). Cells lacking both enzymes form very long filaments and have severe chromosome segregation and growth defects. We previously found that RNase HI overproduction or a dnaT::aph mutation could significantly correct these phenotypes. This leads us to hypothesize that they were related to unregulated replication originating from R-loops, i.e. constitutive stable DNA replication (cSDR). cSDR, first observed in rnhA (RNase HI) mutants, is characterized by its persistence for several hours following protein synthesis inhibition and by its requirement for primosome components, including DnaT. Here, to visualize and measure cSDR, the incorporation of the nucleotide analog ethynyl deoxyuridine (EdU) during replication in E. coli cells pre-treated with protein synthesis inhibitors, was revealed by “click” labeling with Alexa Fluor® 488 in fixed cells, and flow cytometry analysis. cSDR was detected in rnhA mutants, but not in wild-type strains, and the number of cells undergoing cSDR was significantly reduced by the introduction of the dnaT::aph mutation. cSDR was also found in topA, double topA topB but not in topB null cells. This result is consistent with the established function of topo I in the inhibition of R-loop formation. Moreover, our finding that topB rnhA mutants are perfectly viable demonstrates that topo III is not uniquely required during cSDR. Thus, either topo I or III can provide the type 1A topo activity that is specifically required during cSDR to allow chromosome segregation.

End of the beginning: elongation and termination features of alternative modes of chromosomal replication initiation in bacteria.

In bacterial cells, bidirectional replication of the circular chromosome is initiated from a single origin (oriC) and terminates in an antipodal terminus region such that movement of the pair of replication forks is largely codirectional with transcription. The terminus region is flanked by discrete Ter sequences that act as polar, or direction-dependent, arrest sites for fork progression. Alternative oriC-independent modes of replication initiation are possible, one of which is constitutive stable DNA replication (cSDR) from transcription-associated RNA-DNA hybrids or R-loops. Here, I discuss the distinctive attributes of fork progression and termination associated with different modes of bacterial replication initiation. Two hypothetical models are proposed: that head-on collisions between pairs of replication forks, which are a feature of replication termination in all kingdoms of life, provoke bilateral fork reversal reactions; and that cSDR is characterized by existence of distinct subpopulations in bacterial cultures and a widespread distribution of origins in the genome, each with a small firing potential. Since R-loops are known to exist in eukaryotic cells and to inflict genome damage in G1 phase, it is possible that cSDR-like events promote aberrant replication initiation even in eukaryotes.

Roles of type 1A topoisomerases in genome maintenance in Escherichia coli.

In eukaryotes, type 1A topoisomerases (topos) act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the E. coli topo I (topA) and III (topB) enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that topA mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting recA or recQ but not by deleting recJ or recO, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-recQ deletion and topo IIII overproduction are epistatic to recA deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an oriC::aph suppressor mutation which is oriC-competent in topA null but not in isogenic topA+ cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double topA rnhA mutants affecting constitutive stable DNA replication, dnaT::aph and rne::aph, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from oriC (topo I alone) and R-loops and (ii) by acting with RecQ.

Requirements for Replication Restart Proteins During Constitutive Stable DNA Replication in Escherichia coli K-12

Constitutive stable DNA replication (cSDR) is a mechanism for replisome loading in Escherichia coli K-12. This occurs in a dnaA-independent fashion in an rnhA mutant. cSDR is dependent on recA, priA, and transcription. In this report, it is shown that dnaA rnhA mutants using cSDR for initiation of their DNA replication additionally require priB, but not priC, for viability. Two subtle priA missense mutations either eliminated the ability to grow using cSDR (priA301 C479Y) or resulted in very small colonies (priA300 K230R). DnaC809, a priA suppressor, failed to allow priA or priB mutants to grow using cSDR to initiate DNA replication. Furthermore, unlike dnaC(+) strains, dnaC809 strains require priC for cSDR. DnaC809,820, a priC-independent suppressor of priA2::kan phenotypes, allowed priA and priC (but not priB) mutants to grow using cSDR to initiate DNA replication. It is also shown that rep and rnhA mutations are synthetically lethal. DnaC809 and dnaC809,820 mutations suppress this lethality. Rep is further shown to be required for cSDR in a dnaC809 strain. A model whereby these different sets of replication restart proteins interact preferentially with substrates associated with either RecA or SSB during replication restart and cSDR, respectively, is proposed.

ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination-dependent DNA replication.

PriA protein, a DEXH-type helicase with C2C2 zinc-finger motifs, plays essential roles in RecA-dependent modes of Escherichia coli chromosomal DNA replication, namely inducible and constitutive stable DNA replication (iSDR and cSDR respectively, which may be initiated from a D-loop or R-loop structure), and in repair of double-stranded DNA breaks generated by various genotoxic agents or spontaneously during the course of DNA replication. However, the roles of ATPase/DNA helicase activities in functions of PriA are not well understood. We have generated and characterized mutants of PriA protein carrying amino acid substitutions in its conserved ATPase/DNA helicase motifs, namely the Walker A, B and QXXGRXGR motifs. All these mutants were deficient in ATP hydrolysis and DNA helicase activities, but showed wild-type levels of D-loop DNA binding, except for the Walker B mutant which showed reduced DNA binding activity, suggesting that the helicase motifs are not directly involved in the DNA binding activity of PriA protein. They also rescued the low viability and UV-sensitivity of priA null cells. However, they did not rescue iSDR or cSDR-alternative modes of chromosomal DNA replication of the E. coli genome dependent on recombination functions-to the full extent. ATPase/DNA helicase activities of PriA protein are required for full-level DNA synthesis in recombination-dependent modes of DNA replication in E. coli.

Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: Involvement of ATPase/helicase activity of PriA for inducible stable DNA replication

The E. coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the φX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. B. subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its biochemical properties. BsPriA binds specifically to both n'- pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'- pas albeit with a specific activity about 30% of that of E. coli PriA. However, it is not capable of supporting n'- pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the φX174-type primosome. We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance. Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant. K230D was previously reported to be fully functional in assembly of the φX174-type primosome at a single-stranded n'- pas. Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo.

Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: Involvement of ATPase/helicase activity of PriA for inducible stable DNA replication

The E. coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the φX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. B. subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its biochemical properties. BsPriA binds specifically to both n'-pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'-pas albeit with a specific activity about 30% of that of E. coli PriA. However, it is not capable of supporting n'-pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the φX174-type primosome. We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance. Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant. K230D was previously reported to be fully functional in assembly of the φX174-type primosome at a single-stranded n'-pas. Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo.

PriA-directed Assembly of a Primosome on D Loop DNA

Escherichia coli strains carrying null mutations in priA are chronically induced for the SOS response and are defective in homologous recombination, repair of UV damaged DNA, double-strand break repair, and both induced and constitutive stable DNA replication. This led to the proposal that PriA directed replication fork assembly at D loops formed by the homologous recombination machinery. The demonstration that PriA specifically recognized and bound D loop DNA supported this hypothesis. Using DNA footprinting as an assay, we show here that PriA also directs the assembly of a varphiX174-type primosome on D loop DNA. The ability to load a complete primosome on D loop DNA is a step necessary for replication fork assembly.

PriA: At the Crossroads of DNA Replication and Recombination

PriA is a single-stranded DNA-dependent ATPase, DNA translocase, and DNA helicase that was discovered originally because of its requirement in vitro for the conversion of bacteriophage phi X174 viral DNA to the duplex replicative form. Studies demonstrated that PriA catalyzes the assembly of a primosome, a multiprotein complex that primes DNA synthesis, on phi X174 DNA. The primosome was shown to be capable of providing both the DNA unwinding function and the Okazaki fragment priming function required for replication fork progression. However, whereas seven proteins, PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG, were required for primosome assembly on phi X174 DNA, only DnaB, DnaC, and DnaG were required for replication from oriC, suggesting that the other proteins were not involved in chromosomal replication. Strains carrying priA null mutations, however, were constitutively induced for the SOS response, and were defective in homologous recombination, repair of UV-damaged DNA, and double-strand breaks, and both induced and constitutive stable DNA replication. The basis for this phenotype can now be explained by the ability of PriA to load replication forks at a D loop, an intermediate that forms during homologous recombination, double-strand break-repair, and stable DNA replication. Thus, a long-theorized connection between recombination and replication is demonstrated.

Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.

DNA polymerase I in constitutive stable DNA replication in Escherichia coli.

We examined the effects of mutations in the polA (encoding DNA polymerase I) and polB (DNA polymerase II) genes on inducible and constitutive stable DNA replication (iSDR and cSDR, respectively), the two alternative DNA replication systems of Escherichia coli. The polA25::miniTn10spc mutation severely inactivated cSDR, whereas polA1 mutants exhibited a significant extent of cSDR. cSDR required both the polymerase and 5'-->3' exonuclease activities of DNA polymerase I. A similar requirement for both activities was found in replication of the pBR322 plasmid in vivo. DNA polymerase II was required neither for cSDR nor for iSDR. In addition, we found that the lethal combination of an rnhA (RNase HI) and a polA mutation could be suppressed by the lexA(Def) mutation.

Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.

ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase.

The RecG protein of Escherichia coli is a DNA helicase that promotes branch migration of the Holliday junctions. We found that overproduction of RecG protein drastically decreased copy numbers of ColE1-type plasmids, which require R-loop formation between the template DNA and a primer RNA transcript (RNA II) for the initiation of replication. RecG efficiently inhibited in vitro ColE1 DNA synthesis in a reconstituted system containing RNA polymerase, RNase HI and DNA polymerase I. RecG promoted dissociation of RNA II from the R-loop in a manner that required ATP hydrolysis. These results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R-loops formed at the replication origin region of these plasmids with its unique helicase activity. The possibility that RecG regulates the initiation of a unique mode of DNA replication, oriC-independent constitutive stable DNA replication, by its activity in resolving R-loops is discussed.

Escherichia coli RecG and RecA proteins in R-loop formation.

Escherichia coli rnhA mutants devoid of RNase HI exhibit constitutive stable DNA replication, cSDR, which is thought to be initiated from R-loops stabilized in the absence of RNase HI. We found that a combination of an rnhA and a recG mutation is lethal to the cell. recG mutations that inactivate the helicase activity of RecG protein and inhibit reverse branch migration of Holliday junctions impart phenotypes resembling those of rnhA mutants. Thus, recG mutants display cSDR activity, and recG polA double mutants are inviable as are rnhA polA double mutants. These results suggest that the RecG helicase has a role in preventing R-loop formation. A model that R-loops are formed by assimilation of RNA transcripts into the duplex DNA is discussed. The model further postulates that RecA protein catalyzes this assimilation reaction and that RecG protein counteracts RecA in this reaction, resolves R-loops by its helicase activity, or does both.

In vivo evidence for the involvement of anionic phospholipids in initiation of DNA replication in Escherichia coli.

In vitro, anionic phospholipids can reactivate inactivated DnaA protein, which is essential for initiation of DNA replication at the oriC site of Escherichia coli [Sekimizu, K. & Kornberg, A. (1988) J. Biol. Chem. 263, 7131-7135]. Mutations in the pgsA gene (encoding phosphatidylglycerophosphate synthase) limit the synthesis of the major anionic phospholipids and lead to arrest of cell growth. We report herein that a mutation in the rnhA gene (encoding RNase H) that bypasses the need for the DnaA protein through induction of constitutive stable DNA replication [Kogoma, T. & von Meyenburg, K. (1983) EMBO J. 2, 463-468] also suppressed the growth arrest phenotype of a pgsA mutant. The maintenance of plasmids dependent on an oriC site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein.

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)