RecA protein-dependent R-loop formation in vitro.

The RecA protein of Escherichia coli, which has crucial roles in homologous recombination, DNA damage repair, induction of the SOS response, and SOS mutagenesis, was found to catalyze assimilation of complementary RNA into a homologous region of a DNA duplex (R-loop). The reaction strictly requires a region of mismatch in the duplex, which may serve as a nucleation site for RecA protein polymerization. The optimum conditions for the assimilation reaction resemble those for the previously studied RecA protein-catalyzed homologous pairing and strand exchange reaction between two DNA molecules. Our finding lends strong support to the proposal that RecA protein-catalyzed assimilation of a transcript into duplex DNA results in formation of an R-loop at certain regions of the chromosome and that, when stabilized, the R-loop can serve as an origin of chromosome replication.

Are minichromosomes valid model systems for DNA replication control? Lessons learned from Escherichia coli.

Initiation of chromosome replication is a key event in the life cycle of any organism. Little is known, however, about the regulatory mechanisms of this vital process. Conventionally, the initiation mechanism of chromosome replication in microorganisms has been studied using plasmids in which an origin of chromosome replication has been cloned, rather than using the chromosome itself. The reason for this is that even bacterial chromosomes are so large that biochemical and genetic manipulations become difficult and cumbersome. Recently, the combination of flow cytometry and genetic methods, in which modifications of the replication origin are systematically introduced onto the chromosome, has made possible detailed studies of the molecular events involved in the control of replication initiation in Escherichia coli. The results indicate that requirements for initiation at the chromosomal origin, oriC, are drastically different from those for initiation at cloned oriC.

The absence of effect of gid or mioC transcription on the initiation of chromosomal replication in Escherichia coli.

Despite the widely accepted view that transcription of gid and mioC is required for efficient initiation of cloned oriC, we show that these transcriptions have very little effect on initiation of chromosome replication at wild-type chromosomal oriC. Furthermore, neither gid nor mioC transcription is required in cells deficient in the histone-like proteins Fis or IHF. However, oriC that is sufficiently impaired for initiation by deletion of DnaA box R4 requires transcription of at least one of these genes. We conclude that transcription of mioC and especially gid is needed to activate oriC only under suboptimal conditions. We suggest that either the rifampicin-sensitive step of initiation is some other transcription occurring from promoter(s) within oriC, or the original inference of transcriptional activation derived from the rifampicin experiments is incorrect.

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.

Activation of stable DNA replication in rapidly growing Escherichia coli at the time of entry to stationary phase.

The conditions are described in which DNA replication can occur, in the absence of protein synthesis, in wild-type Escherichia coli cells. Chromosome replication, which is normally inhibited by addition of chloramphenicol, becomes resistant to this drug after nutritional shiftup, e.g. from minimal medium to Luria broth. This replication activity appears transiently when nutritionally upshifted cells enter stationary phase. The activity strictly requires recA+, but it is independent of recB+ and dnaA+. It can occur in the absence of concomitant transcription. Activation of the replication does not result from induction of the SOS response. As the characteristics of this DNA replication resemble those of the previously characterized stable DNA replication, it is termed nutritional shiftup-activatable stable DNA replication, nSDR. Possible mechanisms of the activation of nSDR in rapidly growing cells at the time of entry to stationary phase are discussed.

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.

The DnaA box R4 in the minimal oriC is dispensable for initiation of Escherichia coli chromosome replication.

We have developed a genetic system with which to replace oriC+ on the Escherichia coli chromosome with modified oriC sequences constructed on plasmids. Using this system we have demonstrated that chromosomal oriC can tolerate the insertion of a 2 kb fragment at the HindIII site between DnaA boxes R3 and R4, whereas the same insertion completely inactivates cloned oriC. We have further found that although R4 is essential for the origin activity of cloned oriC, cells carrying a deletion of R4 in chromosomal oriC are viable. These results indicate that the oriC sequence necessary for initiation of chromosome replication is different from the so-called minimal oriC that was determined with cloned oriC. Flow cytometric analyses have revealed that these oriC mutations confer the initiation asynchrony phenotype. Introduction of the R4 deletion into a fis::kan mutant, which lacks the DNA bending protein FIS, renders the mutant cells inviable.

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.

The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli.

The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.

The RecF pathway of homologous recombination can mediate the initiation of DNA damage-inducible replication of the Escherichia coli chromosome.

DNA damage-inducible DNA replication in SOS-induced Escherichia coli cells, termed inducible stable DNA replication (iSDR), has previously been shown to require either the RecBCD or the RecE pathway of homologous recombination for initiation. Here, we demonstrate that recB recC sbcC quadruple mutant cells are capable of iSDR induction and that a mutation in the recJ gene abolishes the inducibility. These results indicate that the RecF pathway of homologous recombination can also catalyze iSDR initiation.

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)

RecA, Tus protein and constitutive stable DNA replication in Escherichia coli rnhA mutants.

Constitutive stable DNA replication (cSDR), which uniquely occurs in Escherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. The recA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR in rnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations in recB, recD, recJ, ruvA and ruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR in rnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in the ter region of the chromosome, was ruled out because introduction of the tus::kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.

DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions.

Homologous recombination-dependent DNA replication (RDR) of a lambda cos site-carrying plasmid is demonstrated in E. coli cells when the cells express lambda terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot chi. Chi is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the theta mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (oriMs) on the chromosome.

Roles of ruvA, ruvC and recG gene functions in normal and DNA damage-inducible replication of the Escherichia coli chromosome.

Induction of the SOS response in Escherichia coli activates normally repressed DNA replication which is termed inducible stable DNA replication (iSDR). We previously demonstrated that initiation of iSDR requires the products of genes, such as recA, recB and recC, that are involved in the early stages of homologous recombination. By measuring the copy number increase of the origin (oriM1) region on the chromosome, we show, in this study, that initiation of iSDR is stimulated by mutations in the ruvA, ruvC and recG genes which are involved in the late stages of homologous recombination. Continuation of iSDR, on the other hand, is inhibited by these mutations. The results suggest that Holliday recombination intermediates, left on the chromosome due to abortive recombination, arrest replication fork movement. Low levels of iSDR and sfiA (sulA) gene expression were also observed in exponentially growing ruvA, ruvC and recG mutants, suggesting that the SOS response is chronically induced in these mutants. We propose that replication forks are arrested in these mutants, albeit at a low frequency, even under the normal (uninduced) conditions.

Escherichia coli RNA polymerase mutants that enhance or diminish the SOS response constitutively expressed in the absence of RNase HI activity.

Escherichia coli rnhA mutants lacking RNase HI chronically express the SOS response (T. Kogoma, X. Hong, G. W. Cadwell, K. G. Barnard, and T. Asai, Biochimie 75:89-99, 1993). Seventeen rpoB (Rifr) mutant alleles, which encode altered beta subunits of RNA polymerase, giving rise to resistance to rifampin, were screened for the ability to enhance or diminish constitutive expression of the SOS response in rnhA mutants. Two mutations, rpoB3595 and rpoB2, were found to enhance the SOS response 5- and 2.5-fold, respectively, only when RNase HI is absent. These mutations rendered rnhA mutant cells very sensitive to broth; i.e., the plating efficiency of the double mutants was drastically reduced when tested on broth plates. Two mutations, rpoB8 and rpoB3406, were found to diminish constitutive SOS expression in rnhA mutants by 43 and 30%, respectively. It was suggested that RNA polymerase may have a property that influences the size of DNA-RNA hybrids, the frequency of their formation, or both and that the property resides at least in part in the beta subunit of the polymerase.

DNA damage-inducible replication of the Escherichia coli chromosome is initiated at separable sites within the minimal oriC.

When Escherichia coli cells are subjected to genetic stress by exposure to agents or conditions that transiently block DNA replication, the mode of DNA replication is profoundly altered. One of the alterations is the induction of inducible stable DNA replication (iSDR) that does not require the initiator protein, DnaA, and occurs despite the presence of rifampin and chloramphenicol, which inhibit the initiation of usual chromosome replication at oriC. It has been demonstrated that iSDR starts primarily from both the oriC and terC regions of the chromosome. To precisely map the iSDR origin (oriM1) located in the oriC region, various oriC fragments were inserted into a plasmid vector derived from pSC101, and the copy number of these plasmid constructs was measured in the presence of rifampin and chloramphenicol after cells were induced for the SOS response by thymine starvation. The results indicated that there are at least two origins for iSDR within the minimal oriC; one (oriM1A) is located between the BamHI (coordinate +1) and the AvaII(155) sites, and the other (oriM1B) between the AvaII(155) and the HindIII(244) sites. Furthermore, a 263 bp fragment containing oriM1, which was placed at the att lambda site of the chromosome, was found to initiate chromosome replication in the presence of the drugs when cells were starved of thymine. Introduction of additional copies of oriM1 into a cell stimulated initiation of iSDR at oriM1 on the chromosome. The result supported the model that iSDR starts from D-loops created between oriM1 sequences and that the amount of D-loops determines the level of the iSDR activity.