Initiation of Chromosome Replication
Chapter
99
WALTER MESSER and CHRISTOPH WEIGEL
The genetic maps of Escherichia coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) are circular. This reflects the physical structure of their single chromosomes, which are DNA rings with a circumference of 1.6 mm (44) that contain 4,720 kbp (149). A large part of the sequence of the E. coli chromosome is known by now.
During balanced growth, by definition, all components of the cell double in one generation. Obviously, the replication of the genome must be regulated in such a way that upon cell division, two entities can be distributed between two identical daughter cells. The replication of the chromosome is regulated at the level of initiation, as is all macromolecular synthesis of cells. This is one of the essential statements in the book by Maaloe and Kjeldgaard (185) that appeared nearly 30 years ago and has had a profound impact on research on the bacterial cell cycle: "We are therefore led to believe that the overall production of DNA, RNA, and protein is regulated by mechanisms that control the frequencies with which the synthesis of individual nucleotide and amino acid chains are initiated."
We set the stage with a brief historical introduction. A more detailed discussion of the two principal components, the replication origin oriC and the initiator protein DnaA, and an analysis of the biochemistry and the control of replication follow. The reader is also referred to a series of excellent recent reviews and monographs and especially the citations in the works (23, 40, 53, 61, 155, 190, 197, 281, 351). Some topics discussed by von Meyenburg and Hansen (313) in the first edition of this monograph are not discussed in the same depth here, and the reader is referred to the first edition.
Besides their basic observation that DNA replicates semiconservatively, Meselson and Stahl (200) were the first to observe that DNA replication is a very regular process. Exactly one generation after the shift of a growing culture of E. coli from a medium containing the heavy isotope 15N to a medium containing 14N, all DNA is found at hybrid density. This shows that any segment of the chromosome in any cell in the population that replicates at any moment does so again exactly one generation later. It implies that successive rounds of replication are initiated at the same point on the chromosome. This basic observation was later corroborated by using pulse-labeling in combination with density shifts(164, 165) (see also reference 313).
In the same year Meselson and Stahl’s article was published (1958), Schaechter et al. (264) showed that the amount of DNA per cell in S. typhimurium in balanced growth is dependent on the growth rate. Similar results were obtained later for different strains of E. coli (reviewed in reference 313). When the growth rate is increased by changing the growth medium from poor to rich (a shift-up experiment), RNA and protein synthesis adjust rapidly. The rate of DNA synthesis, however, is initially unaffected and only gradually approaches the new steady-state rate (41, 139). In the reverse situation, a shift-down, mass increase ceases, whereas DNA synthesis continues after the shift and adjusts only slowly (139). A change in growth rate thus results in a new steady-state rate of DNA synthesis only after some delay.
Kohiyama et al. (151, 152) were the first to isolate temperature-sensitive mutants defective in DNA replication at 42°C. Depending on the kinetics of DNA synthesis following the shift to nonpermissive temperature, they were grouped into immediate-stop, i.e., elongation-type, mutants and delayed-stop mutants. Delayed-stop mutants show the phenotype expected for defects in replication initiation. Upon a temperature shift to 42°C, DNA synthesis gradually ceases and reaches a plateau; in the typical case, this plateau is 1.4 times the amount observed at the time of the shift. The first mutations of this kind, in the dnaA gene, are discussed extensively later. All genuine initiation mutants subsequently isolated were eventually found to map in only two genes, dnaA and dnaC (reviewed in reference 313), with one exception, the dnaB252 mutation (350). The product of the dnaA gene is the initiator protein DnaA, the DnaB protein is the replicative DNA helicase, and DnaC is required to deliver DnaB to the replication complex. Mutants in genes that affect replication initiation under special conditions, dnaJ, dnaK, and dnaR, are discussed later.
The replicon model of Jacob et al. (121) defines the minimally required elements for the regulated duplication of a DNA molecule. The replicator is the site at which replication begins and is synonymous with what we call now a replication origin. An initiator element interacts with the replicator for replication initiation and is coded for by the replicon. The DnaA protein is the initiator. The evidence for this fact and whether it acts alone or in concert with other factors are discussed below.
In principle, the bacterial chromosome could consist of several replicons, i.e., could contain several origins and replicators. This is not excluded by the Meselson-Stahl experiment, which only requires that the same site(s) be used in successive initiations in the same order. However, extensive analysis has shown that the E. coli chromosome has one fixed replication origin, oriC, located at 84.3 min on the E. coli genetic map (see chapter 109), and that replication proceeds bidirectionally from there to the terminus, terC (reviewed in reference 313). Likewise, replication of the S. typhimurium chromosome (75) and the chromosome of Bacillus subtilis, first thought to be unidirectional, proceeds bidirectionally from a fixed origin (reviewed in reference 336).
The DNA segment which was first replicated after release of the replication block in a dnaC mutant (191) had a characteristic restriction fragment pattern which could be localized on F' plasmids carrying the 84-min region of the chromosome (315). The diploidy of the origin region gave such F' strains a particular phenotype (reviewed in reference 313), and it was Hiraga (105) who gave the now precisely mapped chromosomal replication origin the name oriC.
Dissection of the molecular events at the initiation of replication proved to be difficult or impossible, in part because of the large size of the bacterial chromosome. This problem was overcome by the cloning of oriC on plasmids, called minichromosomes. They were selected by their abilities to promote the autonomous replication of small DNA segments by using oriC as their only replication origin (204, 333). Specialized transducing phages λ asn were also shown to replicate autonomously as phage minichromosomes by virtue of oriC (212, 316, 317) Using a similar approach, the origins of members of the family Enterobacteriaceae other than E. coli were cloned and shown to function in E. coli (347, 349).
Minichromosomes replicate bidirectionally (199), show a similar dependence of their replication on protein and RNA synthesis, and respond to all dna mutants in a way similar to the way the chromosome responds (208, 317).
The nucleotide sequence of the oriC region was determined (42, 198, 209, 294). The construction of minichromosomes was the prerequisite for a multitude of molecular and biochemical analyses of the initiation of replication which will be discussed in the following chapters; it opened the gate to "modern" experiments.
The interaction of DnaA protein and oriC results in a local unwinding of the DNA in an AT-rich region of oriC, the basic biochemical reaction in the initiation of replication. However, other proteins assist in this interaction, and oriC contains structural information for subsequent reactions, e.g., positioning of the DnaA primosome or oriC sequestration at the membrane. Likewise, DnaA protein has several additional functions, and understanding its regulation is central to understanding the control of initiation. Therefore, we first discuss oriC and DnaA protein separately and then discuss their interactions in the initiation complex.
Chromosomal Region Containing oriC.
The genes directly surrounding the replication origin oriC and their orientations are shown in Fig. 1 (reviewed in reference 313). asnA codes for one of the two asparagine synthases of E. coli. The product of asnC is a transcriptional activator of asnA and has a positive effect on posttranscriptional expression of gidA (153). The mioC gene codes for a 16-kDa protein of unknown function. The name mioC, for modulation of initiation at oriC (314), derives from the observation that mioC transcription influences the copy numbers of minichromosomes. Mutations in the two genes left of oriC, gidA and gidB, result in glucose inhibition of division (312), and disruption of the gidA gene reduces the growth rate. The unc or atp operon codes for the subunits of the membrane-bound ATP synthase. Slow growth of cells carrying F' plasmids or minichromosomes with this region was originally interpreted to indicate a replication control region but has since been attributed to the overproduction of ATP synthase subunits.
Sequence information for the region around oriC has been provided by several groups (for citations, see GenBank). A 227-kbp segment centered around oriC is currently the largest contiguous DNA sequence available for E. coli (43). This region shows a remarkable symmetry. Genes tend to be transcribed divergently from oriC (see references in references 43 and 336), and Chi octanucleotide (5'-GCTGGTGG-3') recombinational hot spots are oriented symmetrically with respect to oriC (43).
The gene for the initiator protein, dnaA, is close to oriC in most bacteria (336). However, in the case of E. coli, the dnaA gene is located 42 kbp counterclockwise of oriC on the genetic map (see Global Aspects of Origin Structure below).
Structure of oriC.
The minimal DNA segment which promotes autonomous replication was found by deletion analysis of minichromosomes (204, 209) to be contained in a segment 245 bp long (232) (Fig. 1). Later, it was observed that a 12-bp AT-rich segment at the left end of oriC is also required for initiation (AT cluster in Fig. 1) but that it can be replaced by other AT-rich sequences (10). The minimal origin thus consists of 258 bp (coordinates 11 to 268 in Fig. 1).
Sequence analysis of the origins of replication in six other Enterobacteriaceae (347) and comparison to oriC of E. coli revealed that the origins contain segments that are highly conserved and are separated by segments with variable sequences but constant lengths. This finding led to the concept of binding sites separated by spacer regions in oriC (7). Prominent among the conserved regions are four nonpalindromic 9-bp repeats (R1 to R4 in Fig. 1), the consensus binding sites for the initiator protein DnaA, called DnaA boxes (80). DNase I footprint analysis revealed a fifth DnaA box (195), labeled M in Fig. 1. Each of the enterobacterial origins contains 9 to 14 GATC sites, 8 of them conserved in sequence and position among all these origins.
The left side of oriC is an AT-rich region consisting of three similar sequences, each 13 nucleotides long and each starting with GATC (35). As mentioned already, there is an additional essential AT-rich region, the AT cluster (10) (Fig. 1). A region of helical instability, called a DNA unwinding element, was observed in the left part of oriC (156). Helical instability can be detected by partial unwinding in the region of the AT cluster and the left 13-mer in the absence (89) or at low concentrations (326) of Mg2+. Apparently, it is the AT richness and the inherent helical instability that are important in this region. Consequently, this part can be replaced by DNA segments with similar properties but different sequences (10, 156). For the right 13-mer, however, the integrity of the 13-mer sequence is required (10, 35, 118); for the middle 13-mer, it is controversial whether the precise sequence or mere AT richness is important (10, 118).
Binding sites for the DNA-bending proteins IHF (integration host factor) (69, 244, 253b).and FIS (factor for inversion stimulation) (70, 88, 253b).are present in oriC (Fig. 1). Both proteins are likely to assist DnaA in the unwinding reaction.
A dimeric protein with subunits of 33 kDa called IciA binds specifically to the AT-rich 13-mer repeats (116, 119, 302). It shares amino acid homology with the LysR family of prokaryotic transcription regulators (302). Binding of IciA inhibits in vitro replication if the protein is added before DnaA protein is added. This effect is due to a block of the DnaA-dependent unwinding of the 13-mer region (118). Once this region is opened by DnaA protein, the IciA protein is without effect. IciA could thus be the long-sought-for negative regulator of initiation. However, cells with an insertion mutation in the iciA gene or that overproduce IciA do not show a striking phenotype, except that cells with excess IciA protein show a longer lag period when diluted into fresh medium (302). Therefore, the function of this protein is not known. This is also true for another protein binding specifically to the oriC region, Rob, for right origin binding. It recognizes a 26-bp site to the right of DnaA box R4 (Fig. 1) (285).
The DNA adjacent to the Rob binding site shows a bend which is considerably stronger in DNA fully methylated by the Dam methyltransferase than in hemimethylated or unmethylated DNA (Fig. 1) (138). Bending to the right of oriC is thus subject to Dam-mediated cyclic changes. Footprint experiments show that the abundant histone-like protein H-NS binds specifically to a site overlapping the Rob binding site in fully Dam methylated DNA, in agreement with its preference for curved DNA (Fig. 1) (M. Falconi, C. Weigel, W. Messer, and C. Gualerzi, unpublished data). Although this region is outside the minimal origin, H-NS binding affects oriC function.
Promoters in and around oriC.
A complicated transcription pattern with an intricate regulation by DnaA protein exists in the origin region. The mioC promoter directs leftward transcription into oriC and is negatively regulated by DnaA owing to an overlapping DnaA box. Most mioC transcripts enter and traverse oriC or are terminated at specific sites within oriC. The mioC promoter requires Dam methylation for maximal activity (reviewed in reference 202). Also, part of the asnC transcripts extend into oriC or beyond, and the passage through the intergenic region between asnC and mioC of these long transcripts is regulated by DnaA (87, 266).
The gidA promoter for leftward transcription (153) has a positive effect on oriC function (10, 225). Two promoters are located in the left half within oriC: Pori-L, transcribing leftward (8, 178, 220, 271), and Pori-R1, with rightward transcription (8, 126). An additional promoter for rightward transcription, Pori-R, is outside of oriC next to DnaA box R4 (8, 178, 271). Pori-L is repressed by DnaA protein (220), but the regulation by DnaA requires the integrity of oriC DnaA boxes R2 and R4 (8). Pori-R1, on the other hand, is activated by DnaA protein. This activation is observed only with Pori-R1 embedded in an intact oriC (8). PmioC, PgidA, and Pori-L are subject to stringent control by ppGpp (225, 253). The importance of a coordinate expression of the different promoters around oriC is stressed by the recent analysis of the timing of transcription in synchronized cultures. mioC transcription is inhibited prior to initiation of chromosome replication, whereas gidA transcription is inhibited after initiation (226, 301).
oriC
Mutants.
Random mutagenesis of oriC gave 18 single-base changes with reduced origin function, 14 of which were in conserved oriC regions (231). However, these mutants showed nearly normal function in constructs with a functional gidA promoter, which again shows the importance of leftward transcription away from oriC (10).
A detailed functional analysis of oriC with mutations in DnaA boxes was done using oligonucleotide-directed mutagenesis. DnaA binding to the different DnaA boxes is either partially or completely abolished. All these mutants have a functional origin (108). Combinations of mutations in two or three DnaA boxes show an impaired origin function in some combinations, whereas mutation of all four DnaA boxes inactivates oriC (U. Langer and W. Messer, unpublished data).
Mutations that modify the distance between DnaA boxes have a much greater effect. Insertions or deletions of 10 bp between DnaA boxes R3 and R4 or boxes R2 and R3 give a functional origin. All changes of distances in these intervals by less or more than one helical turn inactivate oriC, suggesting that the location of the DnaA box with respect to the helix axis is important (207, 326). All 10-bp insertions in the left half of oriC between DnaA boxes R1 and R2 result in nonfunctional origins (207), demonstrating that precise distances are important in this part of oriC. Helical phasing of DnaA boxes is thus an important but not a sufficient condition for oriC function. Inactivation of oriC is also observed for a series of insertions of –6 to +16 bp, in steps of 1 bp, between the right 13-mer and DnaA box R1, because DnaA-mediated unwinding is abolished (110). Apparently, these insertions affect a site which is at the hinge for this process. On the other hand, mutants with insertions or deletions in the R3-R4 region, with functional or nonfunctional origins, show no difference from wild-type oriC organisms with respect to DnaA-mediated helical distortions in the 13-mer region (326).
In summary, base changes in individual DnaA boxes have surprisingly little effect. Any change of distances, however, inactivates oriC, except for modifications by one helical turn in the right half of oriC.
Genetics of dnaA.
The dnaA operon. The gene encoding the initiator protein DnaA has been cloned; its map location has been defined at 83.5 min on the E. coli genetic map (see chapter 109), 42 kbp counterclockwise of oriC; and the nucleotide sequence has been determined (93, 97, 230). DnaA is a basic polypeptide with a molecular weight of 52,500. dnaA is the first gene of an operon (Fig. 2) that also contains dnaN, which encodes the β subunit of DNA polymerase III holoenzyme, and recF, a gene involved in recombination (reviewed in reference 281). The next gene, gyrB, codes for the β subunit of DNA gyrase. gyrB is probably not part of the dnaA transcription unit. The arrangement and sequences of these genes as well as those of surrounding genes are highly conserved among eubacteria (223, 335, 336).
Transcription of this operon occurs from two promoters, dnaA1p and dnaA2p, located 239 and 157 bp, respectively, upstream of the dnaA coding sequence (97). The transcripts extend into recF (238, 251, 261). In addition, both dnaN and recF have their own promoters located within the preceding reading frame (5, 6, 251) (Fig. 2). A DnaA box located between the two promoters dnaA1p and dnaA2p (Fig. 2) is responsible for repression of the operon by DnaA protein (13, 14, 37, 157, 246, 321). Regulation of the dnaA operon is discussed in more detail below.
dnaA mutations. Temperature-sensitive mutations in the dnaA gene were among the first ones isolated that affect DNA replication (reviewed in reference 281). Fine mapping of dnaA mutations showed that mutations with similar phenotypes are clustered (92) (Tables 1, 2, and 3). Sequence analysis (98) of available temperature-sensitive E. coli dnaA mutations revealed that six of them carry the same mutation in the putative ATP binding site (see Fig. 3), i.e., an alanine-to-valine exchange at amino acid position 184. All six carry an additional mutation, which is different in the different alleles (Tables 1, 2, and 3; Fig. 2). Seven other temperature-sensitive alleles contain single mutations; dnaA83 and dnaA508 carry two mutations, at different positions. Four pairs of mutants carry identical mutations: dnaA167 and dnaA603, dnaA601 and dnaA602, dnaA604 and dnaA606, and dnaA203 and dnaA204.
Table 1Characteristics of Ts alleles of dnaA mutants |
Table 2Intragenic suppressors of Ts alleles |
Table 3Other dnaA alleles |
Intragenic suppressors of the heat-sensitive dnaA46 and dnaA508 alleles were isolated. Both are cold sensitive owing to excessive initiation events at 30°C (62, 73, 133, 135). The dnaA46(Cs) allele was shown to contain two additional mutations (38). In dnaA508(Cs), dnaA expression is enhanced due to a mutation of the GUG start codon to an AUG (62) (Table 2). Several nonsense mutations in dnaA were isolated, but only one has been sequenced (136, 137, 269, 324) (Table 2).
Extragenic dnaA suppressors. Several extragenic suppressors of temperature-sensitive dnaA mutations have been isolated (11, 12, 19, 270, 323). Many of these suppressor mutations map in the rpoB gene. The observation of an allele specificity between dnaA and the dnaA-suppressing mutations (Table 1) suggests a physical interaction between the DnaA protein and RNA polymerase (11, 12, 270).
Deletion of the topA gene suppresses the dnaA46 mutation (180). Replication is still DnaA dependent, suggesting either that the dnaA46 product is overexpressed under these conditions or that initiation at oriC requires less DnaA protein in the absence of topoisomerase I.
In attempts to clone dnaA, several plasmids were obtained that contained genes other than dnaA and that were able to suppress the temperature-sensitive defect, apparently because of the high copy numbers of the clones (249, 297). One type of suppressing genes includes groES and groEL, which apparently also exhibit allele specificity in the suppression (66, 125). Suppression results in excessive chromosomal initiations, although the total amount of DnaA is not increased (134). The existence and the properties of diverse suppressors suggest a complicated pattern of interactions of DnaA with other proteins.
Among genes that suppress chromosomal temperature-sensitive dnaA alleles when the genes are present at high copy number are mutant dnaA genes themselves (314). Apparently, most temperature-sensitive dnaA mutants have some residual activity at 42°C, which suffices for initiation at oriC if the amount of functional protein is increased. This may happen by an increased expression, as in the case of dnaA508(Cs), increasing the number of copies of the gene or by the action of chaperones like GroE.
Mutations in a new gene, seqA, were selected on the basis of the interaction of SeqA with hemimethylated oriC DNA. seqA mutations suppress the defects of some temperature-sensitive dnaA mutations and of other mutations with compromised replication initiation like fis and him. Overexpression of SeqA suppresses the cold sensitivity of dnaA46(Cs) and is lethal for fis, him, or dam mutants. Apparently, SeqA protein affects initiation negatively, and it has been suggested that SeqA inhibits the expression or activity of DnaA (181, 311a).
Integrative suppression. The F plasmid (whole F or mini-F) integrated into the chromosome can drive chromosomal replication if replication from oriC is inhibited, e.g., because of a temperature-sensitive dnaA mutation. In such "integrative suppression," chromosome replication starts from the site of plasmid integration. Integrative suppression of dnaA(Ts) mutants can also be obtained by the integration of other plasmids, such as R1, R100, or ColV2, or of phages P1 and P2 (reviewed in reference 313). Integrative suppression is surprising, because plasmids like F themselves require DnaA for replication (94, 140). Apparently, initiation at the F origin requires less active DnaA protein. Consequently, dnaA(Null) mutants cannot be integratively suppressed by F (146), whereas they can be suppressed by the integration of R1 (25). Plasmid and phage replication will be discussed in separate chapters (see chapters 122 and 123).
A different initiation mechanism is DnaA independent, and initiations occur at origins other than oriC in induced and constitutive stable DNA replication (SDR). dnaA mutations can thus be suppressed with respect to their function at oriC by mutations in the gene for RNase H (rnhA) (9, 143, 148, 172, 304). SDR is discussed in a later chapter.
dnaA and other replication mutants. A new temperature-sensitive dna mutation called dnaR (257) is a mutation in prs, the gene for phosphoribosylpyrophosphate synthetase (258, 259). Thermosensitivity of initiation is suppressed by a mutation in the dnaA (dnaA110) or the rpoB gene. The function of DnaR in initiation is different from its synthase activity. The genetic analysis suggests that DnaR and DnaA proteins interact in vivo to initiate DNA replication under special conditions, such as elevated temperatures.
DnaK is one of the major heat shock proteins (86) and is closely related to the Hsp70 family of eukaryotic heat shock proteins (see chapter 88). The dnaK gene is in an operon with another heat shock protein, dnaJ (334), and both proteins play a central role, together with GrpE, in initiation of replication at the origin of phage λ (reviewed in reference 23). Cells with insertions or deletions in dnaK are able to grow at 30°C but are inviable at 42°C (237). Certain dnaK or dnaJ mutations are specifically blocked in initiation at oriC (229, 256), and in vitro replication initiation in cell extracts from dnaK and dnaJ mutants is temperature sensitive (187). Although purified temperature-sensitive DnaA proteins DnaA46 and DnaA5 can be activated by the combined action of GrpE and DnaK (113, 114), in vivo suppression by these gene products has so far not been described. Wild-type DnaA and DnaK proteins associate in vitro (187), and DnaA-phospholipid complexes are activated by DnaK (115). Because DnaK is not required in the oriC-dependent in vitro replication system, we may speculate that the activity of DnaK in initiation is indirect. It could, for instance, be required to maintain DnaA in the active conformation at elevated temperatures.
The dnaX gene encodes both the τ and γ subunits of DNA polymerase III holoenzyme. τ is the full-length product of dnaX, whereas γ is a shorter product produced by a translational frameshift that results in premature termination (29, 71, 305). A temperature-sensitive mutation in dnaX that affects the reading frame common to τ and γ, formerly called dnaZ, can be suppressed by certain dnaA (28, 320). The mechanism of this suppression is not clear at present.
Conservation of the amino acid sequence of DnaA. DnaA genes are ubiquitous among bacteria, and their amino acid sequences are highly conserved (Fig. 3). dnaA genes of Enterobacteriaceae are nearly identical, and even dnaA genes of evolutionarily very distant bacteria like Bacillus, Mycoplasma, or Cyanobacterium spp. are clearly homologous. On the basis of sequence similarities, DnaA proteins have been divided into domains with varying homologies (78, 288, 336). A short N-terminal domain with reasonable homology (domain I) is followed by a stretch that is completely dissimilar in sequence and length among the different DnaA proteins. A highly homologous domain III follows, and this domain is separated from a similarly conserved domain IV by a short stretch with lower homology (Fig. 3). Presumably, this arrangement reflects functional domains of the DnaA protein, but so far there are few data to support this possibility.
A well-conserved region at the beginning of domain III, marked "ATP binding" in Fig. 3, is a motif found in many nucleotide-binding proteins (74; see reference 281 for a review). Starting from the ATP binding motif G-X-X-G-X-G-K-T-X5-V (where X is any amino acid), additional motifs were defined that establish a relationship between DnaA, proteins of the helicase superfamily III, and transcription activators of the NtrC family (154). An especially high similarity between DnaA and NtrC enhancer binding proteins is observed in the end of domain III, with the motif NVRELEGAL (253a).
Fusion proteins consisting of DnaA protein domain IV plus either β-galactosidase or a biotin-target peptide bind oriC DNA with high specificity (Roth and Messer, unpublished data). This demonstrates that the 94 C-terminal amino acids are sufficient for specific binding to DNA. For B. subtilis DnaA protein, DNA binding was also observed in the C-terminal part (336).
Regulation of dnaA expression. Some dnaA(Ts) mutants accumulate a higher level of initiation potential than dnaA + strains under comparable conditions (64, 100, 205, 303), which suggests that DnaA protein controls its own synthesis (100). Evidence for autoregulation of plasmid-borne dnaA genes at the transcriptional level was obtained by using transcriptional and translational fusions to monitor genes (13, 14, 37). Autoregulation of the chromosomal dnaA gene was demonstrated by using comparative S1 mapping (157). Increased levels of DnaA protein made from a DnaA-overproducing plasmid lead to transcriptional repression. A temperature shift to 42°C in dnaA(Ts) mutants results in derepression of transcription. Similarly, the addition of extra DnaA boxes on a plasmid increases dnaA expression, presumably by titrating DnaA protein away from the dnaA promoter (99, 157). Repression of transcription can also be shown in vitro, demonstrating that it is the binding of DnaA protein to the promoter region that is responsible for regulation (321). Evidence for autoregulation of the B. subtilis dnaA gene has also been presented (335), whereas the dnaA genes of Pseudomonas putida and Synechocystis sp. are apparently not autoregulated (120, 252a).
Autoregulation of the E. coli dnaA gene is exerted via a DnaA box located between the two dnaA promoters (93) (Fig. 2), which, however, react differentially to regulatory stimuli. The promoter dnaA1p is responsible for 20 to 30% of the transcripts, and the stronger downstream promoter dnaA2p is responsible for 70 to 80% (52, 157). At the chromosomal location, both promoters are repressed by excess DnaA protein and derepressed upon thermal inactivation of DnaA(Ts) (157). When the dnaA promoter region is located on plasmids, a mutation in the DnaA box between the two promoters results in an increase in dnaA1p and a decrease in dnaA2p transcription, suggesting that under these conditions, dnaA1p is repressed and dnaA2p is activated by interaction of DnaA with the DnaA box (246). In all cases, excess DnaA from a DnaA-overproducing plasmid results in a repression of both promoters (14, 37, 157, 246).
Different behaviors by the two promoters are also observed with respect to their dependence on Dam methylation. Transcription from dnaA2p is nearly completely blocked in the absence of Dam methylation, whereas transcription from dnaA1p is unaffected (39, 157). In addition to this direct effect acting differentially at the level of promoter recognition, the dnaA region becomes sequestered owing to hemimethylation after replication in wild-type cells (46) (see below, Dam Methylation and Replication Initiation). During this time, lasting about one-third of a generation (228), the dnaA promoters are not accessible to RNA polymerase. Consequently, dnaA transcription is inhibited for some time after the initiation of replication (46, 226, 301).
The intracellular signal nucleotide ppGpp, the mediator of the stringent response, seems to be a regulator of dnaA transcription, primarily for the dnaA2p promoter (52). The concentration of ppGpp is inversely related to growth rate (255). Consequently, dnaA transcription is growth rate regulated (52, 246). The dnaA2p promoter is completely inactive during the stationary phase of growth (246). Whether and how this growth rate regulation of transcription is reflected in a growth rate regulation of the intracellular DnaA protein concentration are controversial. Chiaramello and Zyskind (51) reported that the DnaA protein concentration is proportional to growth rate. However, in a subsequent careful analysis of the DnaA protein concentration in five different E. coli strains and in S. typhimurium, Hansen et al. (95) found the DnaA protein concentration to be constant, i.e., independent of growth rate, in all strains tested.
A mutation in the rpoC gene, isolated as a chromosomal copy number mutant with an increased DNA-to-mass ratio (252), causes a more than fivefold increase in dnaA expression (239). Increased dnaA expression may also be the reason for an increased DNAmass ratio in other rpoB and rpoC mutants (299). Such mutants show a reduced rate of RNA synthesis, and consequently, the phenotype of increased DNAmass ratio could be mimicked by a heat shock in the presence of low concentrations of rifampin (91). This treatment also increases expression of dnaA (M. Wende and W. Messer, unpublished data).
The expression of dnaA is induced by DNA lesions that block replication (250). The effect is indirectly related to the SOS response, because the recA1 and lexA1 mutations prevent the induction of dnaA. However, there is no SOS (LexA) box in the promoter region of dnaA. Induction of dnaA by DNA damage may be the explanation for the observed stimulation of reinitiation at oriC after UV irradiation (26, 309).
The observations discussed here demonstrate that dnaA expression is regulated by a complicated control network. Autorepression seems to be the primary control, but in addition, dnaA expression is embedded in several global regulatory systems of the cell.
Biochemical Properties of the DnaA Protein.
DnaA protein was initially expressed from a chromosomal DNA fragment cloned in λ, purified, and shown to bind specifically to oriC DNA (49). With the availability of a test for active DnaA with the oriC-dependent in vitro replication system (81) and the construction of suitable vectors overexpressing DnaA, the purification was successively improved (58, 80, 82, 157, 274).
Purified DnaA protein migrates a little faster in most gel systems than expected from the M r of 52,500 which is inferred from the sequence. It has the N-terminal amino acid sequence SLSLWQQC (W. R. Jueterbock and W. Messer, unpublished data), which defines the GUG codon at nucleotide position 886 (93) as the correct start codon. DnaA protein is active as a monomer but has a strong tendency to aggregate (82).
DnaA boxes. DnaA protein binds tightly to four nonpalindromic 9-bp sequences in oriC, the so-called DnaA boxes, with the common sequence 5'-TTAT(CA)CA(CA)A, as shown by footprinting experiments (80, 108, 117, 326). A fifth box with the related sequence 5'-TCATTCACA binds DnaA in vitro with somewhat less efficiency (195). Fragments carrying the DnaA box sequence or a close match to the consensus sequence are specifically retained on nitrocellulose filters by DnaA protein (80, 82). DNase I footprinting or fragment retention was used to demonstrate DnaA binding to many such boxes in various genes: in the mioC and dnaA promoters, in the promoters of the uvrB and rpoH genes, and in M13 and the left inverted repeat of Tn5 (reviewed in reference 281). They are also found in the origins of plasmids F, R1, pSC101, ColE1, R6K, RK2 , and phage P1 (for a review, see reference 36).
The ability of the DnaA-DnaA box complex to block transcribing RNA polymerase in vivo was used to quantify DnaA binding to DnaA boxes (268). This leads to a more relaxed definition of the DnaA box with the following consensus sequence:
Dissociation constants determined in vitro for DnaA boxes R1, R2, and R4 from oriC by using oligonucleotides and purified DnaA protein vary between Kds of 1.0 and 50 nM. Affinity depends on the DnaA box sequence and the sequence context around the box (268a).
Nucleotide binding and interaction with phospholipids. DnaA protein binds ATP and ADP with high affinity, with Kds of 30 and 100 nM, respectively (272). The two forms bind to DNA in a similar fashion, but only ATP-DnaA is active in oriC replication. However, a limiting level of ATP-DnaA can be augmented by the ADP form (54). ATP function must be allosteric, because the analog ATPγS can replace ATP (272). ATP and ADP forms of DnaA are monomers in solution, but the nucleotide-free form tends to form large aggregates (55). The exchange of the bound ADP by ATP is a slow process; about 50% is exchanged in 40 min (272). DnaA is a low-activity ATPase, hydrolyzing ATP to ADP with a half-time of about 40 min. The tight binding of ATP and its slow hydrolysis, the slow exchange of ADP, and the requirement for ATP-DnaA in the initiation reaction all point to a regulatory role for these nucleotides in the initiation process.
A rapid release of bound ADP and exchange by ATP are catalyzed by phospholipids with acidic head groups (273). The state of fluidity of the phospholipid bilayer is essential for the release of nucleotides (47, 339). In the presence of oriC and phospholipids, DnaA protein can bind ATP and thus provide rejuvenation of an otherwise inert initiation complex. In the absence of oriC, phospholipids cause release of nucleotide and inactivation of DnaA (54). Aggregated inactive DnaA protein complexed with phospholipids can be activated with the help of phospholipase A2 or DnaK protein and ATP (115, 273). One report suggests that a rapid ADP-ATP exchange and thus rejuvenation of DnaA protein can also be mediated by cyclic AMP (112). There is evidence that initiation in vivo (72) and phospholipid-mediated nucleotide exchange in vitro (339) are dependent on oleic acid. Presumably, oleic acid in the membranes provides the correct membrane fluidity for the exchange reaction.
Functions of the DnaA Protein.
The primary role of DnaA protein is that of a replisome organizer. It recognizes the origin oriC and directs all other proteins required to form a replisome (a replication complex) to this site. This function is discussed in detail in the section on the initiation complex.
The second important role of DnaA protein in oriC, and in other replication origins, is its function in primosome assembly, DnaA priming. In addition, DnaA protein affects gene expression in many ways. It can act as a repressor, as discussed above for the dnaA gene itself, or as an activator of transcription. Within a transcription unit, binding of DnaA protein to a DnaA box may result in transcription termination. All these functions are discussed below. The proposed function of DnaA protein as a regulator of replication initiation is discussed in the section dealing with initiation control.
DnaA priming. The traditional φX174-type primosome is not responsible for replication from oriC, because the oriC-dependent in vitro replication system assembled from purified proteins (127) does not contain the primosomal proteins PriA, PriB, PriC, and DnaT. In addition, the closest PriA recognition sites are about 2 kb away from oriC (277, 293, 308). Instead, helicase and primase loading in oriC are mediated by DnaA protein. DnaA priming was analyzed in detail in a model system, the origin of plasmid pBR322.
Plasmid pBR322 and other ColE1-like plasmids have a DnaA box between their origin, defined by the site where the native RNA primer is processed by RNase H, and a (φX174-type) primosome assembly site (PAS). DNA polymerase I extends the processed primer, which activates the PAS on the lagging-strand template by converting it from a double-stranded to a single-stranded form (214, 260). In the absence of RNase H, extension of the RNA primer also activates PAS (234). In both cases, the assembly of the φX174-type primosome at the PAS results from the combined actions of PriA, PriB, PriC, and DnaT, which load the DnaB-DnaC (helicase) complex, followed by primase (4).
If PAS is deleted (183, 278) or φX174-type primosomal proteins are inhibited (278), then pBR322 replication becomes completely dependent on DnaA, demonstrating that DnaA protein can fulfill the combined functions of the primosomal proteins PriA (protein n'), PriB (protein n), PriC (protein n″), and DnaT (protein i). For this action, the orientation of the DnaA box and its distance to the origin are important (276). Depending on the presence or absence of RNase H, the DnaA box is in the form of a D loop or an R loop. DnaA protein binds to the double-stranded part of this loop structure, irrespective of whether it is double-stranded DNA or the DNA-RNA hybrid (235). It has been suggested that DnaA protein then displaces single-stranded binding protein (SSB) from the single-stranded part of the unwound structure and loads the DnaB helicase from a DnaB-DnaC complex in trans to the single strand (235).
The DnaA primosome apparently acts also when the PAS is functioning. Thermoinactivation of DnaA in dnaA(Ts) mutants reduces the rate of synthesis of pBR322 plasmids (1, 245), and excess DnaA protein stimulates pBR322 DNA replication in vitro (183, 278) and in vivo (50).
A stable hairpin loop with a DnaA box in the double-stranded stem acts as a DnaA-dependent complementary strand origin in M13 replication (ABC primosome), and since this structure originates from the γ origin of plasmid R6K, it may be used by this plasmid for priming (193). A DnaA box-containing hairpin loop can also be used as a lagging-strand origin for the replication of the B. subtilis plasmid pUB110, suggesting that DnaA priming is also active in B. subtilis (161).
What, then, is the function of PAS for the φX174-type primosome on the E. coli chromosome? If replication forks are stalled between oriC and a PAS about 2 kbp away from oriC, DNA synthesis can resume again at the PAS (277). This suggests that DnaB-helicase continues to unwind the template from these stalled forks and thereby unwinds and activates the PAS. On the basis of this observation, we may speculate that PAS and the φX174-type primosome may be part of a backup system which becomes operative if replication forks are stalled. The phenotype of priA mutants is compatible with this hypothesis (168, 221).
Repression by DnaA protein. Several other genes besides dnaA are negatively regulated at the level of transcription by DnaA protein. All of them have DnaA boxes in their promoter regions. The mioC gene next to oriC is a particularly interesting example because of the possible effects on initiation at oriC (reviewed in reference 281). The rpoH gene, encoding the heat shock σ 32 factor, is repressed by DnaA protein(322), as is drpA, an essential gene for global RNA and DNA synthesis (344). Indirect evidence suggests that DnaA also mediates repression of the uvrB gene (306). Other genes, e.g., polA, have DnaA boxes in their promoter regions, but repression by DnaA has so far not been demonstrated.
DnaA protein-mediated activation of gene expression. Two DnaA boxes with the same orientation are located immediately upstream of the promoter of nrd, the operon encoding the two subunits of ribonucleoside diphosphate reductase. Binding of DnaA protein to these sites activates the expression of the nrd operon (18). A similar situation is found in the promoter region of glpD, which encodes the aerobic glycerol-3-phosphate dehydrogenase. Overexpression of DnaA protein leads to a concomitant increase in the expression of GlpD, and DnaA directly activates the glpD promoter (Jueterbock and Messer, unpublished data). In this case, two DnaA boxes upstream of the promoter overlap a binding site for the cyclic AMP receptor protein. We may therefore speculate that activation is the result of structural alteration, DNA bending, or loop formation in the promoter region.
Transcription termination by DnaA protein. The mioC gene is transcribed from its own promoter as well as from the upstream asnC promoter (153). Some of the asnC transcripts are terminated in the intergenic region between asnC and mioC owing to the interaction of DnaA protein with the DnaA box in the mioC promoter region (87, 266). On the basis of this observation, several different DnaA boxes were cloned between an inducible promoter and a monitor gene, and the resulting block to transcribing RNA polymerase was used to quantify DnaA binding to DnaA boxes (268). Transcription termination is observed in only one orientation of the DnaA box (267). We may speculate that this is due to an interaction with nearby sequence elements, e.g., a DnaA box in the same orientation in the N-terminal part of the monitor gene. Such a requirement for cooperating sequences is probably the reason that only a subset of DnaA boxes leads to transcription termination upon interaction with DnaA protein. Thus, the DnaA box within the reading frame of the E. coli dnaA gene does not promote transcription termination (324).
DnaA-mediated regulation of the gua operon requires two DnaA boxes (300) and is due to transcription termination (265). Transcription termination is also the likely mechanism by which DnaA protein regulates the essential cell division genes ftsQ and ftsA. The genes contain DnaA boxes within their reading frames and are negatively controlled by DnaA (194). However, since the expression of these genes is also influenced by temperature inactivation of dnaC(Ts) mutants and the dnaB252 allele, their regulation may be more complex.
DnaA protein modulates the attenuation of the trp operon (15). The trp attenuator region does not contain a DnaA box, suggesting that the attenuation results from an indirect effect.
DnaA Protein of B. subtilis.
The DnaA protein of B. subtilis has been purified (79). Like the E. coli protein, it binds ATP with high affinity (Kd ∼ 20 nM) and binds to DnaA boxes of the same consensus sequence (79). E. coli and B. subtilis DnaA proteins are, however, not interchangeable in vivo. Moreover, the B. subtilis dnaA protein, or a hybrid protein consisting of parts of the B. subtilis and E. coli proteins, is deleterious for the growth of E. coli (3).
Histone-like proteins.
Initiation at oriC is stimulated in vitro by low levels of proteins HU or IHF (59, 117, 280). FIS protein may have a similar role. IHF and FIS change the DNA structure in higher-order DNA-protein complexes such as the λ integration and GIN, HIN, and CIN systems (reviewed in reference 253b). Both the IHF and the FIS proteins have one specific binding site in oriC (Fig. 1) and bend oriC upon binding (69, 70, 88, 206, 244, 253b). One dimer of IHF or FIS is enough to induce a strong bend (142, 332). HU protein binds unspecifically to DNA, but several HU dimers result in bending as well (107). Specific concentrations of HU protein stimulate the binding of IHF to its site in oriC (30).
Chromosomal replication is perturbed in mutants deficient in IHF or FIS. Several origins present in the same cytoplasm normally initiate simultaneously in the cell cycle (282), but mutations in fis or him result in asynchronous initiations (34). Mutants with a defect in any one of the genes for histone-like proteins are viable. Even deficiency in two histone-like proteins is possible, although growth of such cells is impaired (129). The defect in the replication of oriC plasmids is more severe in these mutants. Mutants deficient in both subunits of HU (hupA, hupB) are inefficiently transformed by minichromosomes, and oriC plasmids are unstable in such cells (227). A similar observation was made for mutants in fis (88). An IHF mutant (himD) has a more complicated phenotype. Transformation by minichromosomes is inhibited only if polA (69) or hupAB mutations (130) are also present. The functions of both IHF and FIS are required in cis in the oriC complex. Mutation of the IHF or FIS binding site inactivates oriC (253b).
We suggest that the greater sensitivity to defects in histone-like proteins of oriC on plasmids compared to oriC on chromosomes may be due to a stricter dependence on the status of supercoiling. Probably, the binding of HU, IHF, and FIS, the introduction of negative supercoils, and the transcription away from oriC all work synergistically to help DnaA protein unwind oriC (see below).
DNA gyrase.
In agreement with such a function, gyrase was found to be an important component of the initiation machinery. Negative supercoils are a prerequisite for initiation. Additionally, gyrase is required for releasing the superhelical stress introduced by the helicase action in replication forks. Both functions are discussed in more detail below. A specific binding site for gyrase covering the HindIII site within oriC, which is protected from HindIII digestion by gyrase binding, was discovered (179).
Membrane attachment.
The replication origin region binds specifically to membrane fractions (85, 103, 122, 123, 124, 128, 131, 132, 158, 218, 219, 328, 337, 338). The role of the membrane components is not known, except for that of a membrane fraction that binds specifically hemimethylated oriC DNA present directly after replication (48, 103a, 228). This is discussed in more detail later.
Once the chromosomal replication origin was cloned, it became feasible to develop an in vitro replication system. This was accomplished by A. Kornberg and his group, and most of our knowledge of the biochemistry of the initiation process is derived from this oriC-DnaA-dependent in vitro system. Initially, in vitro replication was obtained in a crude system (81) consisting of a cell extract containing replication proteins (fraction II), a supercoiled oriC plasmid template, and DnaA protein.
Eventually, it was possible to obtain in vitro replication from oriC templates in a system assembled from purified proteins (127, 155). In addition to oriC, the system contains DnaA protein, RNA polymerase (RNA-P), gyrase, DNA polymerase III holoenzyme, SSB, HU, DnaB helicase, DnaC protein, and DnaG primase. The reaction was inefficient when RNA-P was present but primase was omitted, more efficient with primase alone, but of highest efficiency when both RNA-P and primase were added (224, 307). We now know that both leading- and lagging-strand primers are synthesized by primase and that RNA-P assists the initiation process by transcriptional activation (reviewed in reference 155).
Adding the different proteins sequentially and making use of environmental requirements allowed us to divide the initiation process into several successive stages, represented by different complexes (initial complex, open complex, prepriming complexes I and II, priming complex), which are followed by bidirectional replication (155) (Fig. 4).
DnaA protein binds to the five DnaA boxes R1 through R4 (80) and M (195) (Fig. 1). Both ADP-DnaA and ATP-DnaA bind to linear or supercoiled DNA. However, for subsequent stages, ATP-DnaA and supercoiled oriC template are required. The ADP form of DnaA shows some activity but only at elevated protein levels and in the presence of a small amount of ATP-DnaA (54).
The size of the initial complex, as seen in the electron microscope, depends on the ratio of DnaA protein to oriC. One distinct structure is correlated with replication activity and localized to oriC (55). This initial complex has a compact ellipsoid structure and a size that could accommodate about 20 DnaA monomers (55, 80, 84). A 220-bp segment of oriC DNA is hidden within such a complex (84). The DNA seems to be wrapped around an ellipsoid with the two duplex strands crossed (55).
The phenotypes of oriC mutants, discussed above, suggest that the initial complex must have an ordered structure. The helical phasing and the importance of conserved distances between DnaA boxes show the importance of a correct spacing of DnaA monomers in the complex (207, 326). The unspectacular phenotypes resulting from base changes in DnaA boxes show that the actual binding efficiency of DnaA protein to individual DnaA boxes is less important (108). A model that accommodates these features as well as the minimal spacing between some DnaA boxes and the positions of IHF and FIS binding sites has been proposed (326). Its important feature is the ordered structure required for a functional initial complex.
The initial complex is converted to an open complex by the addition of a relatively high concentration of ATP (5 mM). In the open complex, the region of the AT-rich 13-mer repeats in the left part of oriC is partially unwound. This was first detected by Bramhill and Kornberg (35), who measured sensitivity to single-strand specific nuclease in this region. Gille and Messer (89) corroborated this observation in vitro and in vivo by using oxidation by KMnO4 as a measure of helical distortion. They also demonstrated that this unwinding is a normal intermediate of replication initiation in vivo. Surprisingly, the two DNA strands are not equally susceptible to single-strand-specific reagents. One of them, the top strand in Fig. 1, shows a smaller region of reactivity, confined to the right 13-mer, with either nuclease or KMnO4 than the other (bottom) strand shows (35, 89, 118, 326). A physical interaction between DnaA protein and the top strand (35, 340) might explain this phenomenon.
It is not known how DnaA protein binding leads to the unwinding reaction. Structural requirements are the specific sequence of the right 13-mer, the AT richness of the left and middle 13-mers, and the AT cluster left of the 13-mers (10, 35, 118). A precise spacing between the 13-mer region and DnaA box R1 is required, and even 10-bp insertions are not tolerated (110). However, variations in the distance between DnaA boxes in the right part of oriC (between R3 and R4) do not affect the unwinding reaction (326).
DnaA protein alone is able to unwind the 13-mer region, provided the reaction temperature is 37°C or higher. At lower temperature, assisting factors are required (280). HU protein at stimulatory levels or IHF protein allows DnaA-mediated strand opening at temperatures as low as 21°C. HU protein shows a sharp concentration optimum in the stimulation of in vitro replication (59, 280), whereas higher IHF concentrations are not inhibitory (280).
In addition to the extended unwinding in the 13-mer region, smaller helical distortions can be detected in the right part of oriC between DnaA boxes R2 and R4 by KMnO4 oxidation or by cleavage with T4 endonuclease VII (326). These distortions have been interpreted as minibulges and may be related to the DnaA protein-mediated positioning of helicase, as discussed below.
DnaB is the helicase of the replicative fork (167) which is recruited by the open complex, eventually resulting in unwinding starting at oriC (22). It is suggestive that DnaB helicase is loaded onto the separated strands in the open complex, but rigorous experimental proof for a precise entry site is missing. Single-stranded DNA complexed with SSB is no substrate for DnaB. Therefore, we must assume that DnaA protein mediates a release of SSB followed by loading of the DnaB-DnaC complex, as has been suggested for the DnaA primosome in the pBR322 origin region (235).
DnaB loading requires a hexameric ring of DnaB complexed with six DnaC monomers, each of which binds one ATP. In the complex with DnaC, the helicase activity of DnaB is blocked (141, 162, 318, 325). A physical interaction between DnaA protein and DnaB is required at this or the following stage, as shown by interference with monoclonal antibodies and by cross-linking studies (192). Prepriming complexes can be isolated. In the electron microscope, they look somewhat bigger than open complexes (84). Besides oriC, they contain DnaA, DnaB, and HU (83, 84, 341). Apparently, two different prepriming complexes exist, some with DnaC (192) and some without (83, 84, 341). Release of DnaC is associated with ATP hydrolysis (318, 319) and seems to activate the helicase activity of DnaB. This reaction is similar to the DnaK-DnaJ-GrpE-mediated release of P protein from DnaB in the λ replication complex (reviewed in reference 190). Most likely, this activation of DnaB is correlated with its positioning by DnaA protein, i.e., the conversion of prepriming complex I to prepriming complex II.
Helicase has to exert its function at the positions where replication forks start, in the right part of oriC, close to DnaA boxes R2, R3, and R4 (Fig. 1) (279). Therefore, helicase must be translocated from the initial entry site to these replication start sites. Translocation could occur simply by helicase activity itself or alternatively by a direct transfer of DnaB between neighboring DNA strands within the prepriming complex from the unwound 13-mer region to minibulges in the region of the start sites (326). We assume that the translocation is coupled to the ATP-driven release of DnaC that results in activation of the helicase. DnaA-mediated priming is dependent on the orientation of the DnaA-DnaA box complex (235, 276). Therefore, we suggest positioning by a physical interaction between DnaA protein and DnaB helicase.
DnaG primase interacts with DnaB helicase, resulting in the priming complex. Primase seems to play a key role in ensuring that initiation occurs in oriC and only in oriC and that two coupled replication forks that are competent for bidirectional replication are formed. An optimal concentration of primase is required, whereas low primase concentrations result in unidirectional replication from sites outside oriC (104).
At this stage, DnaA protein is no longer required and may be recycled to bind to a challenge oriC template, indicating that it may be reused (341). It has been suggested that the coordinated assembly of two coupled replication forks (104) involves the synthesis by one replication fork of a primer that then becomes the leading-strand primer for the other fork moving in the opposite direction (211). Primase acts distributively. After synthesis of the primers, it leaves the replication fork and is replaced by a new primase molecule during the next cycle of Okazaki fragment synthesis (330, 343).
A dimeric complex of DNA polymerase III holoenzyme assembles at each fork in an ATP-dependent mode by clamping the polymerase to the primer terminus (sliding clamp) (155, 186, 196, 222). Primers are extended, resulting in coordinate leading- and lagging-strand synthesis. DNA gyrase is required to relieve the superhelical stress.
There are conflicting reports with respect to the precise positions of the replication start sites within oriC, the transition sites from RNA primers to DNA. Start sites for bidirectional leading-strand synthesis were found close to DnaA boxes R2, R3, and R4 when the in vitro replication system was used (279). Mapping of chromosomal start sites in vivo, however, gave transition sites only for one leading strand, the one with the 5'→3' orientation pointing leftward. These sites were located at several positions in the left half of oriC, and a few were found outside close to the left border of oriC. Obviously, replication in these experiments was unidirectionally leftward (106, 150). Initiation at oriC was aligned by temperature shifts in a dnaC(Ts) mutant strain. This treatment was subsequently shown to result in unidirectional synthesis leftward both in chromosomes and minichromosomes (337, 338). In contrast, without alignment, minichromosomes replicate in vivo bidirectionally from oriC (199). It was therefore suggested that there are different modes of replication from oriC (202). The dependence of direction and position of replication start sites on primase concentration is a possible explanation for unidirectional replication start sites as well as for other reports of replication starts outside of oriC (296). If primase is missing but gyrase is present, oriC plasmids are extensively unwound by the action of helicase (22). In the absence of both primase and gyrase, complexes that contain a small rather homogeneous bubble of about 400 bp can be isolated (20).
Table 4 lists the proteins involved in the chromosomal replication of E. coli under standard conditions, the respective genes, and the abundance of the proteins in cells.
Table 4Proteins involved in replication of the E. coli chromosome |
Initiation of replication is regulated at three different but interrelated levels: (i) initiation is precisely timed in the cell cycle; (ii) each origin initiates once and only once per cell cycle; and (iii) all origins present in the same cell initiate replication synchronously. A series of physiological experiments, to be discussed first, defined the prerequisites for initiation and culminated in the observation that bacteria initiate replication at a given cell mass per origin, called the initiation mass.
In principle, many of the actors involved in replication initiation might contribute to a regulatory element that may be considered a molecular clock (40). However, evidence is accumulating that DnaA protein is the best and only candidate molecule for the role of a molecular pacemaker. In order to exert its clock function properly, DnaA protein requires the assistance of accessory proteins, e.g., histone-like proteins, and the correct DNA topology, which in turn depends on local transcription in the oriC region. We shall discuss these accessory functions and then the regulatory role of DnaA protein and the different levels of control.
When protein synthesis is blocked in a culture growing in glucose-minimal medium either by starving the culture for a required amino acid or by adding chloramphenicol, the accumulation of DNA gradually stops and reaches a plateau at about 1.4 times the amount present at the start of starvation. This increment corresponds to the amount calculated from the assumption that all rounds of replication continue to the end but no new replication rounds are initiated (184, 263). This interpretation was verified by using density shift experiments in combination with starvation (27, 165). The experiments show that protein synthesis is required for the initiation of replication but not for subsequent DNA polymerization. If protein synthesis is allowed to restart in the starved cultures, DNA synthesis lags behind protein synthesis by about one generation time (263), suggesting that one generation’s worth of protein synthesis is required per initiation event.
In addition to the requirement for protein synthesis, the initiation of replication in vivo requires the action of RNA polymerase. Initiation is sensitive to the action of rifampin at a time when all required proteins are accumulated (163, 201, 348). Initiation thus depends also on untranslated de novo-synthesized RNA. A similar step was observed for replication initiation in B. subtilis (166).
Uncoupling of DNA and protein synthesis reveals that bacteria can accumulate the potential for replication initiation under conditions of blocked DNA synthesis with ongoing RNA and protein synthesis (reviewed in reference 313), and that the accumulated initiation potential is stable until being consumed in an initiation event. Accumulation of initiation potential can be achieved most efficiently by incubating initiation mutants at the nonpermissive temperature. Upon a shift back to the permissive temperature, all chromosomes initiate replication. Such treatment has been widely used for the characterization of initiation mutants and for aligning cells at the initiation event to achieve synchronous initiations.
oriC
Topology.
Topoisomerases, particularly the counteracting activities of topoisomerase I and topoisomerase II (DNA gyrase), maintain the negative superhelicity of the bacterial chromosome. About half of the supercoils are constrained by chromatin-associated (histone-like) proteins, while the remaining unconstrained supercoils are available to facilitate transcription, replication, and site-specific recombination (241, 291, 329). It has been proposed that the E. coli and S. typhimurium chromosomes are composed of 40 to 50 distinct supercoiled domains that do not seem to vary much from each other in the degree of superhelicity, with average values of ∼25 superhelical turns per kbp (213, 236, 329).
E. coli minichromosomes are maintained at a superhelical density comparable to that of the chromosome (170). As discussed above, it is difficult to maintain minichromosomes in host strains which are defective in histone-like protein HU, IHF, or FIS. These strains are viable, although with a slightly disturbed DNA replication. Since these proteins affect the local DNA topology, we may assume that oriC plasmids have more stringent topological requirements because they lack the possibility of balancing superhelical changes along a larger domain (170).
Deletion mutations in topA suppress dnaA(Ts) mutations (180). A possible explanation might be that oriC in such strains attains a topology which requires less DnaA protein for initiation. Some temperature-sensitive mutations in gyrB, the gene for the B subunit of DNA gyrase, exhibit an initiation phenotype (67, 68, 233). These observations stress the importance of DNA topology for the initiation event.
The precise topological requirements for initiation at oriC are not known, but in contrast to oriC, the topological requirements for DNA inversion catalyzed by HIN recombinase in S. typhimurium have been studied in great detail in vitro (see reference 171 and references therein). This system is likely to provide useful guidelines for future oriC studies. HIN binding to hix sites and subsequent protein-protein pairing of HIN bound to hix sites can be accomplished at a low superhelical (σ = 0.02) density of the template DNA. On the other hand, invertasome formation and DNA inversion can be observed only at a higher superhelical density (σ = 0.06) (171). Interestingly, like in vitro open-complex formation at oriC, invertasome formation for HIN-mediated DNA inversion requires HU and FIS.
Transcriptional Activation.
Purified oriC plasmids have a superhelical density that is quite permissive for replication in vitro. In vivo, however, free superhelicity is partially constrained by bound proteins (reviewed in reference 240). Therefore, initiation of replication requires activation by transcription in vivo (163, 201, 348) and in the crude (fraction II) in vitro replication system (81). If free superhelicity is restrained by about half by protein HU, the purified in vitro replication system must also be activated by transcription by RNA-P (21, 280).
Transcriptional activation can occur by two different mechanisms. One of them is the generation of an R loop, which helps DnaA in the unwinding of the strands. The R loop may be several hundred base pairs from oriC, provided the region in between is not particularly difficult to melt (21, 280). These features suggest that the mechanism of activation is a propagation of base unpairing from the R loop to the 13-mer region in oriC (280). The second activation mechanism is due to the generation of positive supercoils in front of transcribing RNA polymerase and negative supercoils behind it (173). Transcription away from the 13-mers would thus be expected to assist in the unwinding. Apparently, it is not simple coincidence that promoters that provide transcription into oriC are repressed by DnaA, whereas promoters that drive transcription away from the 13-mers are activated (8, 10, 177, 220, 253, 271).
The contributions of the different promoters and transcripts are not clear. This is particularly true for the mioC transcription unit, which has all the features of an important regulatory element. It is repressed by DnaA protein (177, 220, 253, 271), and in minichromosomes, this repression has a positive effect on initiation at oriC (177, 292). This is reflected in a decrease in minichromosome copy number upon deletion of the mioC promoter (314) or upon replacement by a promoter that is not DnaA regulated (292, 298). Additionally, the mioC promoter is under stringent control, as are PgidA and Pori-L (225, 253). However, minichromosomes without the mioC region initiate at the same time in the cell cycle as those with mioC (101, 169). Different deletion mutations of the mioC promoter region were transferred from plasmids to the chromosome, and the cell cycle parameters of such cells were measured by flow cytometry. There was no difference between mutants and wild type, indicating that for chromosomal replication under balanced growth conditions, mioC transcription is dispensable (174).
In favor of a regulatory role of mioC and other transcripts around oriC is the observation of an intricate control with respect to the timing of replication. In synchronized cultures, mioC transcription is inhibited shortly before initiation of replication, while gidA transcription is stimulated. Following initiation, PgidA is inhibited and PmioC is stimulated (226, 301). These authors point to a possible correlation of the periodicity in promoter activity with the sequestration of oriC following replication initiation (46). DnaA-regulated transcription thus may amplify the action of DnaA protein at oriC.
C and D Times.
Helmstetter and Cooper used a synchronization technique which does not disturb the physiology of the cells, called the baby machine, for measurements of cell cycle parameters in E. coli Br, particularly cell division and rate of DNA replication (see chapter 102). They found that initiation occurs at a characteristic time in the cell cycle that differs with growth rate. The time between initiation and termination of replication, the C time (40 min), is constant irrespective of the growth rate, as is the time between termination and cell division, the D time (20 min), for cells growing with a generation time shorter than 60 min at 37°C. In fast-growing cells, chromosome initiation must therefore occur before the previous round of replication reaches the terminus, resulting in dichotomously branched chromosomes.
Refined analysis has since shown that the C time gradually increases with decreasing growth rate in both E. coli and S. typhimurium. More recently, flow cytometry was used to measure cell cycle parameters in exponentially growing populations (2, 284), corroborating and extending the results obtained with synchronized cultures. A more extensive description of cell cycle events is given in chapter 102.
Initiation Mass.
Donachie (60) combined the classical data on DNA concentration as a function of growth rate (264) with the time of initiation observed in the Cooper and Helmstetter experiments. He concluded that cell mass per replication origin at the time of initiation is constant, irrespective of the growth rate of the cells. This initiation mass, or initiation volume, has since become the basis of biochemical models trying to explain the regulation of the time of initiation in the cell cycle. A recent report, however, suggests that the initiation volume may not be quite as constant as anticipated by the Donachie rule (327) (see chapter 97 for additional discussion).
Control Circuits Based on Initiation Mass.
The two basic models proposed for the control of replication in E. coli are reflected by two aspects of the constancy of initiation mass. Initiation may be envisioned to occur when the origin concentration (origin per mass) has decreased sufficiently or, conversely, when the mass per origin has increased sufficiently. The model for the first view, the inhibitor dilution model, was proposed by Pritchard et al. (248). It suggests the existence of an inhibitor of initiation that is synthesized at the time of initiation or shortly thereafter. Increase in cell volume results in the dilution of the inhibitor, and after its concentration is below the threshold level of 0.5, initiation can occur. Variants of this model suggest an unstable inhibitor which is synthesized constitutively (205, 247).
Although factors that affect initiation negatively have been described, e.g., IciA (119) and SeqA (181), a molecule that could be a genuine negative regulator has never been found. This makes the second type of model more attractive. Sompayrac and Maaloe (290) suggested that accumulation of an initiator is coupled to mass increase via an autoregulatory control loop. This model was later analyzed by computer simulation (189). The most likely candidate for such an initiator is DnaA protein.
It has recently been found that a mutant of B. subtilis that confers resistance to a protein kinase C inhibitor has an altered initiation mass (275). No connection between initiation and protein phosphorylation has been detected in E. coli.
DnaA Protein Regulates the Initiation Mass.
Overproduction of DnaA protein from strong promoters results in a burst of initiation events, suggesting that DnaA regulates initiation positively (16, 17, 242, 243, 283, 331). The significance of these observations for the regulatory function of DnaA protein has been challenged with two arguments (see also reference 40 for a discussion): (i) in many experiments, the increased initiations did not result in an increase in the overall rate of DNA synthesis per cell; and (ii) an initiator is supposed to oscillate in order to exert its function, but such oscillation is unlikely for an autoregulated protein. However, both of these arguments have been ruled out by recent experiments. It is at least possible that the concentration of DnaA protein oscillates because the dnaA gene is exempt from the autoregulatory loop for about one-third of the cell cycle owing to sequestration of hemimethylated DNA (46, 301). In addition, an excessive concentration of DnaA protein results in stalling of the supernumerary newly initiated replication forks within about 50 kbp of oriC (17) or reduces the rate of replication fork progression. The overall rate of DNA synthesis is thereby reduced and does not reflect the number of initiated replication origins (16). In B. subtilis, an additional postinitiation regulatory step is operative, with replication forks stalled at discrete distances on either side of oriC (102). For E. coli, no such mechanism has been found.
At concentrations of DnaA below wild-type levels, the initiation mass is increased; i.e., cells initiate replication later in the cell cycle. Some dnaA(Ts) mutants show an increased initiation mass even at permissive temperature (100, 182, 303). The most convincing experiment was the change of initiation mass by the controlled expression of dnaA from an inducible promoter (176). We can thus safely conclude that the time of initiation in the cell cycle depends on the activity of DnaA protein in the cell.
The Initiator Titration Model.
How DnaA protein might regulate initiation mass was proposed in the initiator titration model (96). It is the legal offspring of the replicon model, the autorepressor model, and later improvements of these prototype models (121, 290). At the same time, however, it represents a true second-generation model in that it uses stochastic methods for computer-based simulations of the bacterial cell cycle. In this model, all newly synthesized DnaA protein is titrated by DnaA boxes on the chromosome (initiator titration). As soon as the number of DnaA molecules exceeds the number of DnaA boxes, which also increases because of replication, initiation is supposed to occur.
Two assumptions, supported by experimental evidence, are important. (i) The final switch that triggers initiation due to suddenly available free DnaA has to be of a different kind or quality than the binding to an average DnaA box before that time point. Binding to a DnaA box with much lower affinity is an obvious possibility. The observation that the oriC DnaA boxes R1, R2, and R4 but not box R3 are protected by DnaA protein against methylation by dimethyl sulfate in vivo suggests that R3 might have such a pivotal role in initiation (262). (ii) Once initiation is triggered at a single oriC, DnaA is ejected. The sudden increase in concentration of free DnaA then leads to successive initiations at all origins in the cell in the form of a cascade (175). DnaA has to be proficient for more than one initiation, as suggested from results in in vitro initiation (341). Membrane sequestration of newly replicated oriC excludes them from an immediate second initiation (see below). The cascade model can, in addition, explain the synchronous initiation of minichromosomes and the chromosome.
Initiation Synchrony.
Rapidly growing E. coli cells with multiple origins initiate the origins in each individual cell almost simultaneously (282). There are several conditions in which initiation synchrony is disturbed (reviewed in reference 281). In dam, hobH, and seqA mutants, sequestration is impaired; hence, initiation synchrony is disturbed (32, 33, 103a, 181). Temperature-sensitive dnaA mutants which are defective in the ATP binding site (Table 1) seem to initiate their origins at random in the cell cycle even at fully permissive temperature (282, 286). Other dnaA mutants are less affected in initiation synchrony (Table 1). As discussed above, mutations which affect functions that assist DnaA protein in its initiator function result in an asynchrony phenotype. This includes fis and him mutations (34) and mutations affecting the status of superhelicity in the cell (310, 311). We can conclude that optimal function of the initiation machinery is required for an uninhibited initiation cascade, which is essential for initiation synchrony.
The E. coli Dam methyltransferase modifies A in its recognition sequence 5'-GATC. When fully methylated DNA becomes temporarily hemimethylated upon replication, the lack of a cognate restriction enzyme in the Dam system provides the cell with a molecular monitor for newly replicated DNA. The Dam system is therefore ideally suited to ensure that all origins in an individual cell initiate once before any origin initiates a second time. In addition, Dam methylation plays a major role in cellular repair processes (mismatch repair) and is involved in the regulation of various promoters (see chapter 53 for details on Dam methylation).
The clustering of Dam sites and their positional conservation among enterobacterial replication origins suggest that Dam methylation has a role in replication control. Initially, Messer and his colleagues and Smith and colleagues found that unmethylated or fully methylated minichromosomes transform dam mutant strains poorly if at all (203, 289). Subsequently, it was discovered that it is hemimethylated DNA which cannot replicate in vivo and that hemimethylated DNA accumulates in dam mutants upon transformation with methylated plasmids (254). Whether unmethylated oriC plasmids are able to transform dam mutants is still controversial (46, 160, 203, 254, 289). Unmethylated or hemimethylated oriC plasmids nevertheless act as substrates for in vitro replication, albeit less efficiently than fully methylated DNA (31, 112, 160, 203, 289). dam mutants initiate replication at the chromosomal oriC, but initiation is poorly controlled. Interinitiation times are variable, as shown by density shift analysis (24) and flow cytometry (32, 175). Under conditions of controlled Dam expression in a dam host, the asynchrony phenotype was also revealed by flow cytometry at Dam methyltransferase concentrations that were either lower or higher than that of wild type (32). All these results emphasize that Dam methylation participates in initiation control (210).
A solution to this puzzling scenario came when Schaechter and his colleagues found that hemimethylated but not fully methylated or unmethylated oriC DNA specifically binds to an E. coli membrane fraction in vitro (228). They observed that in rapidly growing cells, oriC DNA remains hemimethylated after replication fork passage for about one-third of a generation before it is rapidly remethylated. Like oriC DNA, the promoter region of the dnaA gene shows a similar delay of remethylation following replication, concomitant with drastically reduced dnaA transcription (46). In contrast, other parts of the genome analyzed so far become remethylated within 1 to 2 min (46, 228, 295). The interpretation of these observations is that hemimethylated oriC becomes sequestered in the membrane and is thus inaccessible to Dam methyltransferase. The period during which a newly replicated oriC is refractory to a second initiation is termed the eclipse period. The autoregulatory control of dnaA expression is also suspended during this eclipse period. Rapid cessation of DnaA synthesis may contribute considerably to preventing secondary initiations (46, 281). Note that membrane sequestration of oriC not only prevents secondary initiation but also may shield the newly replicated DNA from the action of the mutH-mutS repair system.
A shortening of the eclipse period is the prominent phenotype of a newly discovered mutation in E. coli, seqA (181, 311a). Accordingly, seqA mutants exhibit an asynchrony phenotype that is most pronounced when they are grown in rich medium. Although direct evidence has not yet been obtained, Kleckner and colleagues propose for SeqA the role of a negative modulator of replication initiation in a sequestration step that precedes sequestration of newly replicated oriC into the membrane (181). In contrast, von Freiesleben et al. (311a) argue that a possible negative modulation of DnaA activity by SeqA protein may not depend on the methylation status of oriC and consequently may not be related to membrane sequestration. They found that the lethal effect of SeqA overproduction is independent of the state of methylation and that minichromosomes are more unstable in dam single mutants than in dam seqA double mutants (311a).
Sequestration of hemimethylated oriC DNA into the membrane was recently shown by Kohiyama and coworkers to involve HobH protein (103a). HobH was identified in membrane fractions and binds specifically to the methylated parental strand at specific sites in hemimethylated oriC DNA (Fig. 1). Comparable to the situation in dam mutants, the reduction of oriC membrane attachment in the hobH deletion mutant leads to asynchronous initiation of replication, in this case because of a missing receptor. However, the residual initiation synchrony in the hobH mutant precludes any key regulatory role of HobH for initiaton.
The discovery of specific binding of hemimethylated oriC DNA by HobH and the isolation of seqA as a suppressor mutation that allows efficient transformation of a dam host by minichromosomes provide new insight into the role of oriC membrane attachment (103, 181). (i) We may deduce that membrane sequestration of hemimethylated DNA is an active process likely to be regulated. (ii) A receptor mechanism which is not exhausted by the elevated copy number of minichromosomes, in addition to the chromosomal origins, can hardly contribute to any stringent chromosome or origin copy number control. (iii) Since the number of HobH-containing membrane regions does not seem to be strictly limited, there might be an additional use for these regions in the wild-type situation. They could be involved in a sequential membrane attachment of newly replicated chromosome domains, from origin to terminus, before nucleoid segregation and cell division (121). It remains to be seen whether HobH-mediated membrane sequestration is permitted or necessary for chromosome regions other than oriC and dnaA.
Following the eclipse period, oriC might be detached from the membrane solely by the progressive invasion of Dam methyltransferase preventing further membrane contact of then fully methylated GATC sites. The observation that Dam methyltransferase overexpression leads to a shortening of the eclipse period (203) and the observation that in dam mutants hemimethylated DNA is permanently sequestered and blocked in replication (46, 255) clearly argue for an active role of Dam methyltransferase prior to a new round of replication initiation. Dam-mediated initiation cycling in E. coli might thus allow a "sloppy" initiation control, a sort of "open the door for all origins waiting" mechanism. The finding of residual synchrony in the hobH and seqA single mutants supports this view.
Besides in E. coli, Dam methylation is found in S. typhimurium and other Enterobacteriaceae but not in B. subtilis. Intriguingly, minichromosomes can be established only at copy numbers below 1 in B. subtilis because of incompatibility, i.e., competition for initiation proteins (215). Apparently, a more stringent copy number control of oriC operates in B. subtilis than in E. coli. The recent discovery of cell cycle-regulated adenine methylation (GmANTC) in Caulobacter crescentus shows that DNA replication control by monitoring the methylation status of the replication origin is not a unique property of enteric bacteria (345).
When normal chromosome replication is prevented by DNA damage at extreme physiological conditions or in a particular genetic background, E. coli cells can sustain replication by either of two bypass phenomena, termed damage-inducible SDR (iSDR) and constitutive SDR (cSDR), respectively. Neither of the two SDR pathways requires DnaA for initiation of chromosome replication. cSDR is, in addition, independent of oriC and adjacent sequences (for a recent review, see reference 9).
Upon induction of the SOS response in E. coli cells, initiation of DNA replication in the iSDR pathway depends on D-loop formation by the recombinase activity of RecA and the helicase activity of recBC at specific sequences, called oriM. Of the genes activated by the SOS response via the LexA regulon, solely RecA is necessary and sufficient to induce iSDR. Consequently, iSDR cannot be induced in recombinase-deficient recA mutants and in recBC mutants, while in recD mutants (lacking the exonuclease V activity of RecBCD enzyme), iSDR is increased. iSDR activity is, in addition, greatly increased in ruvA, ruvB, ruvC, and recG mutants, which are thought to accumulate unresolved D loops. Despite considerable effort, the initiator nuclease necessary to introduce the nick or double-stranded break at oriM sites for D-loop formation has not yet been found (reviewed in reference 9).
In E. coli, two separable oriM sites reside within oriC, while one other oriM is found in the ter region. Any distinct DNA sequence requirements for oriM function or additional oriM sites on the chromosome are presently unknown. iSDR is active in a dnaA::Tn10 strain, does not require concomitant transcription and translation, and can drive DNA replication for several hours (reviewed in reference 9).
In contrast to iSDR, initiation of DNA replication in the cSDR pathway is crucially dependent on transcription and on RNase H deficiency but likewise requires recombinase activity of RecA (147). RNase H is not essential for replication initiation at oriC (109). Transcripts originating at various sites on the chromosome, termed oriK, are thought to be stabilized in the absence of RNase H, which specifically degrades RNA in RNA-DNA hybrids. While R-loop formation is sufficiently promoted by RecA in a recA + background, cSDR becomes dependent on derepression of the LexA regulon and the nick translation activity of DNA polymerase I, in particular in recombinase-deficient recA mutants (reviewed in reference 9). These findings emphasize the dependence of both known SDR pathways on SOS functions. The cSDR type of replication initiation may in addition resemble the polA-dependent replication mode of ColE1-type plasmids (144).
The oriC region does not contain oriK sequences, but at least four separate oriK sites have been mapped on the E. coli chromosome (56). Any distinct DNA sequence requirements for oriK function are presently unknown. rnhA strains can dispense with oriC and DnaA protein but grow poorly under these conditions (145, 148). Such mutants therefore provide an alternative to integratively suppressed strains for genetic studies.
Because iSDR and cSDR are blocked in a priA::kan mutant, assembly of a φX174-type primosome is thought to follow duplex opening (T. Masai and T. Kogoma, personal communication). The φX174-type primosome then loads DnaB helicase from the DnaBC complex. Priming of bidirectional replication is carried out by DnaG primase, while DNA polymerase III holoenzyme promotes chain growth (reviewed in reference 9). It is thus the where and how of duplex opening and the primosome type that discriminate iSDR and cSDR from each other and from normal replication. In cells forced to grow with cSDR, replication timing and frequency are poorly regulated (9, 148). This finding emphasizes the pivotal roles of DnaA protein and regulation of its expression for correct replication under normal growth conditions.
Although iSDR and cSDR are observed under rather extreme physiological or genetic conditions, they are not mere genetic artifacts. iSDR is part of the complex SOS response that enables cells to meet environmental challenges on viability. A cSDR-like activity was recently found in rnh + cells under certain growth conditions (108a). Kogoma and his colleagues therefore propose that iSDR and cSDR represent stress- or growth-related DNA replication activities (9). iSDR and cSDR may also be seen as the consequence of a functional and regulatory overlap of transcription, replication, and recombination. Free 3'-OH ends as created by transcription in RNA-DNA hybrids (R loops) or in double-stranded DNA (D loops) are substrates for the initial steps in either DNA replication or recombinational repair processes. To avoid or minimize errors, the competing pathways would have to allow for a switch to the other pathway during their initial steps. Concomitantly, they would provide a mutual backup system.
The arrangement of genes and the direction of their transcription have been remarkably well preserved among eubacteria even of very distant evolutionary relationships. In a comparison of sequences and transcription units in more than 20 kbp around oriC and dnaA of B. subtilis and P. putida, the major genes were conserved in sequence, position, and direction of transcription away from dnaA and oriC (223, 336). Most eubacteria seem to possess the gene sequence dnaA-dnaN-recF-gyrB, with oriC within 2 to 3 kbp of dnaA (77, 335). We might consider this to be the primordial origin. There are so far three exceptions to this arrangement, in which one or more of these elements have been translocated to another region.
1. In E. coli, the oriC region is located 42 kbp from dnaA, and gid transcription is directed toward dnaA, thus violating the rule that transcription is away from dnaA (and oriC). It looks as if the E. coli oriC region has been transposed and inverted compared to the primordial origin (335, 336). The dnaA region of E. coli, however, matches perfectly the standard arrangement.
2. In C. crescentus, the origin is close (2 kbp) to the dnaA gene, but both are in a completely different environment. oriC is located between the hemE gene and a homolog of the ribosomal protein gene rpsT. The dnaK and dnaJ genes are downstream of dnaA. Transcription in this limited set of genes is away from oriC (188, 346). C. crescentus homologs of the dnaN, recF, and gyrB genes have also been cloned and identified. As in E. coli and other bacteria, they form a cluster, but this cluster is approximately 150 kbp away from dnaA (346).
3. The dnaA gene of the cyanobacterium Synechocystis sp. has been cloned, sequenced, and identified by virtue of its similaritiy to the E. coli gene (252a). It is located next to genes that are part of photosynthesis system II (psbD, psbC). No homologs of other genes normally close to dnaA are found. Likewise, there is no structure resembling a replication origin within 2.5 kbp. Thus, E. coli (and other Enterobacteriaceae) and Synechocystis sp. are so far the only bacteria in which oriC and dnaA are not next to each other.
The binding sites for DnaA protein, DnaA boxes, are also highly conserved among bacteria. In all cases analyzed, DnaA protein recognizes the same consensus sequence. The only variation is found in organisms with a high G+C content in their DNA, e.g., Micrococcus luteus (77), Streptomyces lividans (342), and Streptomyces coelicolor (45). DnaA boxes of these organisms frequently have G instead of A or T at the third position in the box.
The general structure of the bacterial chromosomal replication origin, oriC, is found in many prokaryotic origins; in plasmids like pSC101, F, R1, R6K, and RK2; and in P1, P4, λ, and lambdoid phages (reviewed in reference 36). In all these cases, iterated binding sites for an initiation protein are next to AT-rich regions. Many of these plasmids and phages contain in their origins, in addition to binding sites for their replicon-specific initiator proteins, DnaA boxes. For some of these origins, DnaA protein has a role in open-complex formation (90, 217).
Also, some eukaryotic viruses, like simian virus 40 or polyoma, show an outline in their origins similar to that of oriC (reviewed in reference 65). Common to all origins of this type is the formation of a higher-order complex between origin and initiator protein, the initial complex, followed by an unwinding reaction at the AT-rich region, the open complex. Because of the similarity of initiation complexes at a variety of origins and the nucleoprotein structures involved in transcription regulation or site-specific recombination, Echols proposed a common term, SNUP, for specialized nucleoprotein structure (63).
The replication origin of the bacterial chromosome, particularly oriC of E. coli, is the best-known cellular origin. However, despite intensive research over more than three decades by many groups, its complex function is not completely understood.
We thank many of our colleagues for helpful suggestions and for communicating unpublished information.
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