Replication fork reactivation in a dnaC2 mutant at non-permissive temperature in Escherichia coli

Replicative helicases unwind double-stranded DNA in front of the polymerase and ensure the processivity of DNA synthesis. In Escherichia coli, the helicase loader DnaC as well as factors involved in the formation of the open complex during the initiation of replication and primosomal proteins during the reactivation of arrested replication forks are required to recruit and deposit the replicative helicase onto single-stranded DNA prior to the formation of the replisome. dnaC2 is a thermosensitive allele of the gene specifying the helicase loader; at non-permissive temperature replication cannot initiate, but most ongoing rounds of replication continues through to completion (18% of dnaC2 cells fail to complete replication at non-permissive temperature). An assumption, which may be drawn from this observation, is that only a few replication forks are arrested under normal growth conditions. This assumption, however, is at odds with the severe and deleterious phenotypes associated with a null mutant of priA, the gene encoding a helicase implicated in the reactivation of arrested replication forks. We developed an assay that involves an abrupt inactivation of rounds of synchronized replication in a large population of cells, in order to evaluate the ability of dnaC2 cells to reactivate arrested replication forks at non-permissive temperature. We compared the rate at which arrested replication forks accumulated in dnaC2 priA(+) and dnaC2 priA2 cells and observed that this rate was lower in dnaC2 priA(+) cells. We conclude that while replication cannot initiate in a dnaC2 mutant at non-permissive temperature, a class of arrested replication forks (PriA-dependent and DnaC-independent) are reactivated within these cells.     

A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation

SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery.     

Replication cycle dependent association of SeqA to the outer membrane fraction of E. coli

The hemimethylated oriC binding activity of the E. coli heavy density membrane fraction (outer membrane) was investigated by DNase I footprinting experiments using membranes obtained from different replication stages of PC-2 (dnaCts) cells. The maximal binding activity was found at the beginning of replication cycle and then decreased gradually. The same pattern of variation was observed with SeqA protein detected in the membranes by immunoblotting. Both binding activity and the presence of SeqA were conserved in the outer membrane even after floating centrifugation of the heavy density membrane fraction in a sucrose gradient, indicating that SeqA in fact can associate with the membrane and that this association varies according to replication cycle. Site specific binding to hemimethylated oriC, of the heavy density membrane obtained from seqA mutant, could be restored by addition of a low amount of His-tagged SeqA protein.     

Identification of functional domains of the Escherichia coli SeqA protein

The Escherichia coli SeqA protein, a negative regulator of chromosomal DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated G-mA-T-C sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the functional domains responsible for the DNA-binding and self-association activities, we performed a deletion analysis of the SeqA protein and found that the N-terminal (amino acid residues 1-59) and the C-terminal (amino acid residues 71-181) regions form structurally distinct domains. The N-terminal domain, which is not involved in DNA binding, has the self-association activity. In contrast, the C-terminal domain, which lacks the self-association activity, specifically binds to the hemimethylated G-mA-T-C sequence. Therefore, two essential SeqA activities, self-association and DNA-binding, are independently performed by the structurally distinct N-terminal and C-terminal domains, respectively.     

Escherichia coli SeqA Structures Relocalize Abruptly upon Termination of Origin Sequestration during Multifork DNA Replication

The Escherichia coli SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, E. coli cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration. Then, two abrupt relocalization events occurred within 2–6 minutes and resulted in SeqA foci localized at each of the cell’s quarter positions. Imaging of cells containing an additional fluorescent tag in the origin region showed that SeqA colocalizes with the origin region during sequestration. This indicates that the newly replicated DNA of first one chromosome, and then the other, is moved from midcell to the quarter positions. At the same time, origins are released from sequestration. Our results illustrate that newly replicated sister DNA is segregated pairwise to the new locations. This mode of segregation is in principle different from that of slowly growing bacteria where the newly replicated sister DNA is partitioned to separate cell halves and the decatenation of sisters a prerequisite for, and possibly a mechanistic part of, segregation.     

Directionality of DNA replication fork movement strongly affects the generation of spontaneous mutations in Escherichia coli

Using a pair of plasmids carrying the rpsL target sequence in different orientations to the replication origin, we analyzed a large number of forward mutations generated in wild-type and mismatch-repair deficient (MMR(-)) Escherichia coli cells to assess the effects of directionality of replication-fork movement on spontaneous mutagenesis and the generation of replication error. All classes of the mutations found in wild-type cells but not MMR(-) cells were strongly affected by the directionality of replication fork movement. It also appeared that the directionality of replication-fork movement governs the directionality of sequence substitution mutagenesis, which occurred in wild-type cells at a frequency comparable to base substitutions and single-base frameshift mutations. A very strong orientation-dependent hot-spot site for single-base frameshift mutations was discovered and demonstrated to be caused by the same process involved in sequence substitution mutagenesis. It is surprising that dnaE173, a potent mutator mutation specific for sequence substitution as well as single-base frameshift, did not enhance the frequency of the hot-spot frameshift mutation. Furthermore, the frequency of the hot-spot frameshift mutation was unchanged in the MMR(-) strain, whereas the mutHLS-dependent mismatch repair system efficiently suppressed the generation of single-base frameshift mutations. These results suggested that the hot-spot frameshift mutagenesis might be initiated at a particular location containing a DNA lesion, and thereby produce a premutagenic replication intermediate resistant to MMR. Significant numbers of spontaneous single-base frameshift mutations are probably caused by similar mechanisms.     

The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein

In wild-type Escherichia coli cells, initiation of DNA replication is tightly coupled to cell growth. In slowly growing dnaA204 (Ts) mutant cells, the cell mass at initiation and its variability is increased two- to threefold relative to wild type. Here, we show that the DnaA protein concentration was two- to threefold lower in the dnaA204 mutant compared with the wild-type strain. The reason for the DnaA protein deficiency was found to be a rapid degradation of the mutant protein. Absence of SeqA protein stabilized the DnaA204 protein, increased the DnaA protein concentration and normalized the initiation mass in the dnaA204 mutant cells. During rapid growth, the dnaA204 mutant displayed cell cycle parameters similar to wild-type cells as well as a normal DnaA protein concentration, even though the DnaA204 protein was highly unstable. Apparently, the increased DnaA protein synthesis compensated for the protein degradation under these growth conditions, in which the doubling time was of the same order of magnitude as the half-life of the protein. Our results suggest that the DnaA204 protein has essentially wild-type activity at permissive temperature but, as a result of instability, the protein is present at lower concentration under certain growth conditions. The basis for the stabilization in the absence of SeqA is not known. We suggest that the formation of stable DnaA-DNA complexes is enhanced in the absence of SeqA, thereby protecting the DnaA protein from degradation.     

Impediment to replication fork movement in the terminus region of the Bacillus subtilis chromosome

The terminus regions of the chromosomes of three strains of Bacillus subtilis 168 were radioactively labelled by supplying [3H]thymine towards the end of a round of replication. These strains lacked or contained the prophage SP beta c2. Following restriction endonuclease digestion of the purified DNA and fluorography, an SP beta c2-related perturbation of the terminus-labelling profile was observed, which was completely consistent with the previously suggested existence of an impediment to replication fork movement (terC) within a BamHI 24.8 X 10(3) base fragment (Weiss & Wake, 1983). The present data suggest that terC is located within the 11.4 X 10(3) base BamHI + SalI double-digest portion of this BamHI fragment.     

Stimulation of deoxyribonucleic acid replication fork movement by spermidine analogs in polyamine-deficient Escherichia coli.

We examined the rate of deoxyribonucleic acid (DNA) replication fork movement in polyamine-deficient cells of Escherichia coli by two independent techniques. DNA autoradiography was used to directly visualize the length of DNA produced during a given time interval, and replication rates were calculated. The amount of DNA synthesized after blocking protein synthesis also allowed calculation of replication rates. We found that the DNA chain elongation rate in polyamine-deficient cells was about half that of putrescine- or spermidine-supplemented cells. We also found that spermidine homologs of increasing chain length, when present at equal intracellular concentrations, exhibited a decreasing ability to support growth and the rate of DNA replication fork movement. The kinetics of recovery of DNA synthesis from the polyamine-deficient state were also investigated. A new rate of DNA synthesis was reached about 20 min after addition of spermidine to polyamine-limited cells. The rise in the rate of DNA synthesis was preceded by a rise in the intracellular concentration of spermidine.     

PriA-directed replication fork restart in Escherichia coli

The encounter of a replication fork with either a damaged DNA template, a nick in the template strand or a 'frozen' protein-DNA complex can stall the replisome and cause it to fall apart. Such an event generates a requirement for replication fork restart if the cell is going to survive. Recent evidence shows that replication fork restart is effected by the action of the recombination proteins generating a substrate for PriA-directed replication fork assembly.     

The Escherichia coli SeqA protein binds specifically to two sites in fully and hemimethylated oriC and has the capacity to inhibit DNA replication and affect chromosome topology

The SeqA protein was identified as a factor that prevents reinitiation of newly replicated, hemimethylated origins. SeqA also seems to inhibit initiation of fully methylated origins, thus contributing to the regulation of chromosomal replication. The SeqA protein was found to bind to two sites in the left part of the origin, near the AT-rich region where strand separation takes place during initiation of replication. The same binding sites seemed to be preferred irrespective of whether the origin was in the newly replicated (hemimethylated) state or not. In addition to binding specifically to groups of GATC sites, the SeqA protein was capable of interacting non-specifically with negatively supercoiled DNA, restraining the supercoils in a fashion similar to that seen with histone-like protein HU. The restraint of supercoils by SeqA was, in contrast to that of HU, cooperative.     

Arrangement of Dam methylation sites (GATC) in the Escherichia coli chromosome.

The occurrence of GATC (Dam-recognition) sites in available E. coli DNA sequences (representing about 2% of the chromosome) has been determined by a simple numerical analysis. Our approach was to analyze the nucleotide composition of nine large sequenced DNA stretches ("cantles") in order to identify patterns of GATC distribution and to rationalize such patterns in biological/structural terms. The following observations were made: (i) In addition to oriC, GATC-rich regions are present in numerous locations. (ii) There is a wide variation in GATC frequency both between and within DNA cantles which led to the identification of a void-cluster pattern of GATC arrangement. The distance between two GATCs was never greater than 2 kb. (iii) GATC sites are found more frequently in translated regions than (in decreasing order) non-coding or non-translated regions. In particular, rRNA and tRNA encoding genes exhibit the lowest GATC content.     

Replication fork arrest at relocated replication terminators on the Bacillus subtilis chromosome.

The replication terminus region of the Bacillus subtilis chromosome, comprising TerI and TerII plus the rtp gene (referred to as the terC region) was relocated to serC (257 degrees) and cym (10 degrees) on the anticlockwise- and clockwise-replicating segments of the chromosome, respectively. In both cases, it was found that only the orientation of the terC region that placed TerI in opposition to the approaching replication fork was functional in fork arrest. When TerII was opposed to the approaching fork, it was nonfunctional. These findings confirm and extend earlier work which involved relocations to only the clockwise-replicating segment, at metD (100 degrees) and pyr (139 degrees). In the present work, it was further shown that in the strain in which TerII was opposed to an approaching fork at metD, overproduction of the replication terminator protein (RTP) enabled TerII to function as an arrest site. Thus, chromosomal TerII is nonfunctional in arrest in vivo because of a limiting level of RTP. Marker frequency analysis showed that TerI at both cym and metD caused only transient arrest of a replication fork. Arrest appeared to be more severe in the latter situation and caused the two forks to meet at approximately 145 degrees (just outside or on the edge of the replication fork trap). The minimum pause time erected by TerI at metD was calculated to be approximately 40% of the time taken to complete a round of replication. This significant pause at metD caused the cells to become elongated, indicating that cell division was delayed. Further work is needed to establish the immediate cause of the delay in division.     

Mechanisms of replication fork restart in Escherichia coli

Replication of the genome is crucial for the accurate transmission of genetic information. It has become clear over the last decade that the orderly progression of replication forks in both prokaryotes and eukaryotes is disrupted with high frequency by encounters with various obstacles either on or in the template strands. Survival of the organism then becomes dependent on both removal of the obstruction and resumption of replication. This latter point is particularly important in bacteria, where the number of replication forks per genome is nominally only two. Replication restart in Escherichia coli is accomplished by the action of the restart primosomal proteins, which use both recombination intermediates and stalled replication forks as substrates for loading new replication forks. These reactions have been reconstituted with purified recombination and replication proteins.     

Aluminium toxicity and binding to Escherichia coli

The toxicity and binding of aluminium to Escherichia coli has been studied. Inhibition of growth by aluminium nitrate was markedly dependent on pH; growth in medium buffered to pH 5.4 was more sensitive to 0.9 mM or 2.25 mM aluminium than was growth at pH 6.6–6.8. In medium buffered with 2-(N-morpholino)ethanesulphonic acid (MES), aluminium toxicity was enhanced by omission of iron from the medium or by use of exponential phase starter cultures. Analysis of bound aluminium by atomic absorption spectroscopy showed that aluminium was bound intracellularly at one type of site with a K _m of 0.4 mM and a capacity of 0.13 mol (g dry wt)^-1. In contrast, binding of aluminium at the cell surface occurred at two or more sites with evidence of cooperativity. Addition of aluminium nitrate to a weakly buffered cell suspension caused acidification of the medium attributable to displacement of protons from cell surfaces by metal cations. It is concluded that aluminium toxicity is related to pH-dependent speciation [with Al(H₂O) ₆ ³⁺ probably being the active species] and chelation of aluminium in the medium. Aluminium transport to intracellular binding sites may involve Fe(III) transport pathways.     

SeqA limits DnaA activity in replication from oriC in Escherichia coli

A mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been Isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation In this gene, named seqA1, has also been isolated. Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2-containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild-type seqA gene on a low-copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39°C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles. The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold). The introduction of minichromosomes or a low-copy-number plasmid carrying five DnaA-boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild-type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.     

Zinc binding by the methylation signaling domain of the Escherichia coli Ada protein.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.