©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Cytoplasmic Hemimethylated oriC Binding Activity (*)

(Received for publication, July 5, 1995; and in revised form, November 11, 1995)

Jeremy Garwood (§) Masamichi Kohiyama

From the Biochemical Genetics Group, Institut Jacques Monod, Université Paris 7, 2 Place Jussieu, 75251 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using hemimethylated, fully methylated, and unmethylated oligonucleotide probes corresponding to part of the origin of Escherichia coli DNA replication, oriC (+81-136), we have characterized a novel hemimethylated DNA-specific protein binding activity. This activity appears to be located in the cytoplasm rather than in membrane fractions. It has been partially purified and, in DNase footprinting analysis, found to preferentially protect only a subset of the hemimethylated GATC sites present in the minimal oriC. These sites are found adjacent to the DnaA binding box, R1, and overlap the integration host factor binding site. The activity does not correspond to known hemimethylated binding proteins, although in the seqA deletion mutant, there is a 3-fold reduction of the activity. The stage of the cell cycle in synchronized PC2 cultures does not seem to significantly affect the relative levels of this binding activity. A possible role in sequestration of the newly replicated hemimethylated origin is discussed.


INTRODUCTION

The Escherichia coli Dam methyltransferase methylates the adenine residue in its recognition sequence 5`-GATC. When replicated, fully methylated DNA becomes temporarily hemimethylated, providing the cell with a molecular monitor for newly synthesized DNA. As such, the Dam system is potentially a means of ensuring that all replication origins in an individual cell initiate once before any origin can be reinitiated a second time(1, 2) . Dam methylation also plays a major role in cellular repair processes (mismatch-repair) (3) and is involved in the regulation of various promoters.

The clustering of Dam sites and their positional conservation among enterobacterial replication origins suggests that Dam methylation has a role in replication control. The minimal DNA segment that promotes autonomous replication in E. coli has been found to be contained in a segment of 245 base pairs (the minimal oriC from +11 to +256), a sequence containing 11 GATC sites(4, 5) .

Protein factors have been found in membrane-rich extracts that are capable of recognizing the oriC DNA in the hemimethylated rather than the fully methylated or unmethylated state(6, 7, 8, 9) . It has been proposed that such hemimethylated DNA binding activity serves to prevent the Dam methyltransferase from remethylating newly synthesized hemimethylated DNA and that such a ``sequestration'' of the origin of replication in its hemimethylated form delays the reinitiation of chromosome and minichromosome DNA replication, thus playing a temporal regulatory role in the bacterial cell cycle(10, 11, 12, 13, 14) .

Two genes whose proteins appear to be involved in such a hemimethylated DNA sequestration process are seqA(15) and hobH(16) . While it has been proposed that SeqA is involved in a sequestration step that precedes sequestration into the membrane, HobH apparently binds preferentially to the methylated, parental strand at specific sites in the hemimethylated oriC sequence.

Footprinting studies of the oriC region (from -46 to +244) in the hemimethylated state have indicated that there is differential protection by membrane-rich protein extracts of the 11 GATC methylation sites present in this segment, with the strongest binding detected in the two regions covering bases +90-110 and +120-130, sites to which the HobH fusion protein also appears to have higher affinity(16) .

In this study we present a more detailed investigation of the hemimethylated DNA-specific protein interactions with this region, employing an oligonucleotide probe and a gel retardation assay.


EXPERIMENTAL PROCEDURES

Bacterial Strains

PC2 is dnaC leu thy Str(17) . PC2DeltaseqA::tet was a gift from E. Boye. PC2DeltahobH::Tn5 was described previously(16) . PC2DeltaseqADeltahobH was generated by P1 transduction(18) . Sources of other marked deletion or disruption alleles were as follows: mutH::Tn5 (AB1157) and mutS::Tn10 (AB1157) were a gift from M. Radman; dam13::Tn9 (GM 2163) was a gift from M. Marinus; himA::Tn10 (RY826) and hip (himD)::Tn5 (RY827) were a gift from J. Rouvière-Yaniv.

Protein Lysates

Membrane-rich and cytoplasmic fractions were obtained from synchronized cultures of PC2 as described previously (9) . Briefly, chilled cells were resuspended in 10 mM HEPES, pH7.4, 10 mM EDTA and broken by passage through a French pressure cell (25,000 p.s.i.). Any remaining intact cells were removed by low speed centrifugation before layering the lysate over a 60% sucrose shelf onto which total membrane material banded following ultracentrifugation at 45,000 rpm (110,000 times g) for 1 h at 4 °C. Further fractionation of this band was accomplished by centrifugation (35,000 rpm (81,000 times g) for 2.5 h at 4 °C) through a 30-50% (w/w) linear sucrose gradient with a 60% sucrose shelf in HKE buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA). Freeze-thaw lysis entailed resuspending the cells in HE buffer (10 mM HEPES, pH 7.4, 1 mM EDTA) with egg white lysozyme (100 µg/ml or 1 mg/g of cells) and incubating on ice for 1 h before freezing the mixture at -80 °C and lysing the cells by thawing at 37 °C for 5 min. Protein concentrations were determined by the method of Bradford (48) .

Oligonucleotides and Radioactive Labeling

The oligonucleotides were synthesized using an Applied Biosystems synthesizer model 392 with incorporation of N^6-methyl deoxyadenosine phosphoramidite (Pharmacia Biotech Inc.) at the GATC sites to give the methylated strands. Double-stranded oligonucleotides were generated by hybridizing equal amounts of the complementary single strands in 10 mM Tris, pH 7.5, 1 mM EDTA (heating at 100 °C for 2 min and then leaving the solution to cool to room temperature). These were then radioactively labeled by phosphorylation of the 5`-unphosphorylated ends with T4 polynucleotide kinase, [-P]ATP in 70 mM Tris, pH 7.5, 10 mM MgCl(2), 5 mM dithiothreitol, incubating at 37 °C for 30 min. Unincorporated radioactivity was removed by passing the oligonucleotide through a Sephadex-50 column (Pharmacia).

Gel Retardation Assay

Protein extract was mixed with double-stranded radioactive oligonucleotide (1 times 10M) in binding buffer (6% glycerol, 25 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA) with boiled, sonicated calf thymus DNA as nonspecific competitor (typically 20 µg/ml or 5000-fold molar excess relative to the probe). The final reaction volume was 20 µl. For specific competition experiments, an excess of unlabeled double-stranded oligonucleotide was added prior to the addition of the protein extract. Binding reactions were assembled and incubated at room temperature (25 °C) for 20 min before loading onto native 5% 0.5 times TBE polyacrylamide gels (29:1 acrylamide:bis-acrylamide). Gels were migrated at room temperature at 100 V constant.

Autoradiography and Densitometric Scanning of Hemimethylated Oligonucleotide Binding

Gels were dried and autoradiographed with x-ray film (Fuji RX) preflashed with the Amersham Sensitizer Flash unit according to the manufacturer's instructions, such that the film's exposure could be quantified as a linear function of the amount of radioactivity. The autoradiographs were scanned using an Apple One Scanner (Apple) and the Ofoto program (Light Source Computer Images Inc.). The densitometric analysis of the scans was accomplished using the program NIH Image (Wayne Rasband, National Institutes of Health). The total bound hemimethylated oligonucleotide in complexes 1 and 2 was measured under binding conditions in which only these complexes were formed with the probe (i.e. 1 times 10M probe in the presence of 5000-fold nonspecific competitor DNA). Preliminary experiments indicated that variations in the levels of total bound oligonucleotide were directly proportional to the amount of extract added. Fractions were measured at equivalent subsaturating protein concentrations (25 µg/ml), and results between experiments were standardized by measurement of the corresponding areas on control lanes without extract and with 25 µg/ml of membrane extract.

Protein Purification

Bacteria were lysed by freeze-thaw lysis in HE buffer (10 mM HEPES, pH 7.4, 1 mM EDTA), and the debris was removed by low speed centrifugation. NaCl was added to a final concentration of 350 mM, and the lysate was loaded onto a BioRex A-70 (Bio-Rad) column pre-equilibrated in HE/350 mM NaCl. After the column was washed with 10 column volumes, it was then stepped with HE/800 mM NaCl. The resulting eluate was diluted to 200 mM NaCl with HE and then loaded onto and eluted from a column of SP-Sepharose (Pharmacia) with a 200-600 mM NaCl gradient (10 column volumes), using a Waters 650 Advanced Protein Purification System (fast protein liquid chromatography). The protein composition was analyzed by electrophoresis in a 15% SDS-polyacrylamide gel, staining with Coomassie Blue.

DNase Footprinting

The DNase footprinting reaction and the preparation of the hemimethylated and methylated oriC probes were performed as described previously(16) . Briefly, peak 1 protein was preincubated with the probe for 10 min at 37 °C before a limited digestion of the protein-DNA mixture by DNase. This was then deproteinized, and the digested DNA preparations were separated by electrophoresis through 8% denaturing (7 M urea) polyacrylamide gels.


RESULTS

Characterization of Hemimethylated DNA-specific Binding Activity

The protection of hemimethylated oriC from DNase digestion was observed from around 150 µg/ml of membrane extract(16) . Employing a similar concentration of this membrane extract in a gel retardation assay with the hemimethylated oriC probe, we observed a single major complex, which upon reduction of the membrane concentration (to 25 µg/ml) could be resolved into six or seven retarded complexes (see Fig. 2a).


Figure 2: Gel retardations demonstrating the hemimethylated binding activity of a membrane extract. a, binding of a hemimethylated oriC fragment (-46 to +244) (1 times 10M) with 0, 25, and 100 µg/ml membrane extract in the presence of 50 µg/ml calf thymus DNA (3000-fold excess). b, binding interactions of the membrane extract (100 µg/ml) in the presence of 20 µg/ml calf thymus DNA (600-fold excess) with the labeled oligonucleotide (1 times 10M). The first six lanes are with the hemimethylated (5`-3`) probe; ct, without extract; A, nonspecific competitor only; B, C, D, and E, plus specific competition with 100-fold excess unlabeled oligo; B, hemimethylated (5`-3`); C, unmethylated; D, hemimethylated (3`-5`); E, fully methylated. Next three lanes with the unmethylated probe: ct`, without extract; F, nonspecific competitor only; G, plus 100-fold excess unmethylated oligonucleotide; H, fully methylated probe with extract and nonspecific competitor only. The three complexes formed with the hemimethylated probe are indicated by the numbered arrows on the left, while the arrow on the right indicates the complex formed with the unmethylated and fully methylated probes. c, binding of the hemimethylated oligonucleotide probe (5`-3`) (1 times 10M) in the presence of 5000-fold excess calf thymus DNA (20 µg/ml) with increasing concentrations of membrane extract. A, 1; B, 3; C, 6; D, 12.5; E, 30; F, 60; G, 125 µg/ml.



In order to simplify our analysis of the protein-DNA interactions with the hemimethylated oriC, we decided to employ probes corresponding to just the most strongly protected region. Hence we synthesized oligonucleotides for the DNA sequence from +81 to +136 of the replication origin that possess either unmethylated or N^6-methyl adenine residues at the three GATC positions located at 93, 107, and 125 (Fig. 1). By combining the resulting four single-stranded oligonucleotides in their complementary hybrid forms we could readily obtain double-stranded 56-mers in the unmethylated, fully methylated, and two hemimethylated states. With these probes we conducted a series of gel retardation experiments, seeking to elucidate further the methylation state-sensitive nature of the protein-DNA interactions with this region.


Figure 1: Structural organization of the E. coli oriC region and the sequence of the oligonucleotides used in this study. In the upper part, the oriC region from +1 to +365 is represented with the relative orientations of the GATC methylation sites shown as filled boxes above the line and protein binding sites for DnaA, IciA, IHF, FIS, and ROB indicated as empty boxes below the line (based on (47) ). In the lower part are shown the sequences of the four single-stranded oligonucleotides (the upper two are oriented 5` to 3`, left to right; the lower two are shown 3`-5`). ^mA are the methylated adenine residues. The four complementary double-stranded forms are designated on the right.



In our initial studies with the oligonucleotide probes, employing a concentration of 1 times 10M in the binding reaction, with 100 µg/ml of membrane extract and a 600-fold excess of nonspecific DNA, we observed the formation of three complexes with the hemimethylated oligonucleotides but only a single complex with both the unmethylated and fully methylated probes (Fig. 2b). Specific competition with a 100-fold excess of each of the oligonucleotides under these conditions reveals that complexes 1 and 2, which are only formed with the hemimethylated probes, are also only competed by these hemimethylated forms, while complex 3, which co-migrates with that formed with the unmethylated and fully methylated probes is competed by all four oligonucleotides (Fig. 2b), suggesting that complexes 1 and 2 are only specific for the hemimethylated state of this sequence, while complex 3 is formed by a specific interaction with this sequence, which is not dependent upon the methylation status of the three GATC sites that it contains.

We further found that the hemimethylated DNA-specific complexes, 1 and 2, were of a much higher affinity than complex 3 since we could effectively prevent the formation of complex 3 by increasing the stringency of the binding conditions. Thus, through lowering the probe concentration (to 1 times 10M) and increasing the relative excess of nonspecific DNA (to 5000-fold), only the complexes 1 and 2 were formed. In addition, under these conditions, we found that by varying the extract concentration over a large range, we observed the formation of complex 1 at a lower protein concentration than complex 2, suggesting that the former results from a stronger protein-DNA interaction (Fig. 2c).

In order to further characterize the specificity of the protein interactions in these two retardation complexes we tested the effect upon the formation of the complexes of competition with an increasing excess of each of the unlabeled oligonucleotides present in the reaction mixture prior to the addition of the protein extract, finding that whereas both complexes were strongly competed by as little as a 10-fold excess of either of the hemimethylated oligonucleotides, neither of them was significantly competed by even a 1000-fold excess of either the unmethylated or fully methylated probes (Fig. 3).


Figure 3: Specific competition of hemimethylated oligonucleotide binding. a, gel retardation showing competition of the retarded complexes formed between the hemimethylated (5`-3`) oligonucleotide (1 times 10M) binding with membrane extract (25 µg/ml) in the presence of 5000-fold excess nonspecific competitor DNA. ct, no extract; v. HM 5`-3`, 0-, 10-, 50-, 100-, and 250-fold increasing excess of unlabeled hemimethylated oligonucleotide; v. NM, 100-, 250-, 500-, and 1000-fold versus increasing excess of unlabeled unmethylated oligonucleotide; v. HM 3`-5`, versus increasing excess of unlabeled hemimethylated (3`-5`); and v. MM, fully methylated oligonucleotide. b, graph of the densitometric analysis of competition shown in Fig. 3a).



There are a number of possible explanations for the presence of two complexes with a specificity for the hemimethylated probes. They might arise as a result of two completely independent interactions with the oligonucleotide by two different protein species. Alternatively, there may be protein components common to the two complexes, or indeed both complexes may be due to multiple binding interactions by the same species. It is possible that the formation of complex 2 is related to complex 1 since we do not observe its formation independently of the latter despite extensive fractionation of the bacterial lysate (see below). As such it might result from cooperative interactions between proteins bound at different sites on the oligonucleotide. With three GATC sites available in this probe for hemimethylated specific binding, the formation of a third complex might be predicted. However, although DNase footprinting indicates that all three sites are protected at extract concentrations that give both retarded complexes in mobility shift assays, we do not observe a third complex. Perhaps one of the two complexes involves an interaction at more than one GATC site; for example, complex 1 might represent two bound sites, while complex 2 represents additional binding at the remaining GATC.

In order to determine if these complexes arose from interactions with more than one protein species, we assayed the activity in different lysate fractions as a preliminary step to its purification.

Distribution of This Hemimethylated Specific Oligonucleotide Binding Activity in the Bacterial Cell

Previous studies have indicated that there is a hemimethylated oriC-specific binding activity associated with the bacterial membrane(6, 8, 9, 16, 19, 20) , and since we have characterized such an activity in a membrane extract with our oligonucleotides, we sought to confirm the relative localization of our activity within the bacterial membrane. Several techniques are available for the fractionation and isolation of the membranous components of the cell envelope of Gram-negative bacteria, almost all based on density gradient centrifugation of disrupted cells (21, 22) .

Many studies of oriC membrane-associated interactions have employed such a preparative procedure, fractionating the lysates obtained after passage through a French pressure cell by ultracentrifugation of sucrose gradients(6, 8, 9, 16, 19, 20) . Origin DNA has been found in membrane fractions that were prepared in this manner, but its presence seems to vary according to the initiation potential of the cell as shown by the use of synchronized cultures of initiation mutants(19, 23) . Hence, these studies have also employed synchronized cultures of the thermosensitive dnaC mutant, PC2(17) , in which, at the nonpermissive growth temperature (>37 °C), new rounds of initiation of DNA replication are blocked while those that have begun continue until terminated; by bringing a culture of PC2 back to the permissive temperature (<36 °C) after 1 h at the nonpermissive temperature there is a synchronized reinitiation of all origins in the culture(24) .

To localize the distribution of the hemimethylated binding activity further, we assayed it in fractions obtained from the initial centrifugation of the lysate over a 60% sucrose bed, which results in the banding of the total membrane material on the bed(21) .

All of the cultures assayed gave roughly the same levels of specific binding for the hemimethylated oligonucleotide probe (Fig. 4a). In addition, we found that, although the distribution of the total hemimethylated binding activity in the first sedimentation gradient was skewed toward the total membrane fraction, its levels were only around twice those found in the cytoplasmic fractions taken from the top and middle of the gradient, representing around a quarter of the total activity in the initial unfractionated lysate (Fig. 4b). Further fractionation of the total membrane into outer, inner, and intermembrane fractions using a 30-60% sucrose gradient did not result in any further enriched peak of activity for either complex (data not shown).


Figure 4: Histograms showing the hemimethylated oligonucleotide binding activity of lysate fractions from synchronized and asynchronous cultures of PC2. a, the specific activity of the initial crude lysates and of the fractions of equal volume resulting from ultracentrifugation of the initial lysates over a 60% sucrose bed. Results are given for the first, third, and fifth fractions, the last containing all of the banded total crude membrane. The specific activity is defined as the amount of total bound hemimethylated oligonucleotide (complexes 1 and 2) found for each fraction standardized for differences in protein concentration. 0, 5, 10, 20, 30, and 40, time in minutes after the transfer of the synchronized PC2 culture to the permissive growth temperature (30 °C) from the nonpermissive temperature (42 °C); E and S, exponential and stationary phase asynchronous PC2 cultures grown at 30 °C. b, the distribution between the fractions of the step gradient of the total hemimethylated oligonucleotide binding activity measured in the initial lysates. The cultures are as in panel a.



To test the possibility that lysing the bacteria under high pressure with the French press had dissociated the activity from the membrane structure, we compared this lysis procedure with the less physically disruptive ``freeze-thaw'' technique. However, lysate fractions obtained by these methods from identical cultures exhibited a similar level and distribution of the hemimethylated binding activity (data not shown).

This apparent absence of any large, or majority, peak of specific binding for the hemimethylated probe in the total, crude membrane fraction relative to the other lysate fractions would seem to contrast with the result expected for an activity that is directly associated with the membrane components.

Possible Candidates for the Hemimethylated Binding Activity

There are a number of known proteins that might be responsible for the hemimethylated binding activity we observe. These include the Dam methylase, which recognizes and subsequently methylates the hemimethylated GATC sequences(2) , or MutH, one of the proteins involved in methyl-directed mismatch repair(25) , which also recognizes the hemimethylated GATC sequence. Yet lysates prepared from deletion mutants for these genes retained the same activity as the wild-type or PC2 lysates (data not shown). We also tested integration host factor (IHF) (^1)mutants (both himA and himD(hip)) since this protein has a characterized binding site in the midst of the oligonucleotide sequence (+98-123)(26) , which contains the middle of the three GATC sequences, but once more we found wild-type activity levels (data not shown).

There are also eight of nine residues of the DnaA box, R1, present in the probe's sequence (+80-88)(27, 28, 29) . However, since this well defined consensus sequence is at an extremity of the oligonucleotide and incomplete we would not expect it to be involved in this binding interaction, which, besides, is apparently associated with modification of the GATC sequences, the closest lying at +93-96. In addition, the binding of DnaA protein to oriC in vitro has already been shown to occur irrespective of the methylation state of the origin (20) , and both in vivo(30) and in vitro DNase footprinting studies with DnaA protein (27, 31, 32) clearly show strong protection at the R1 box and not in the immediately flanking sequence. This is the opposite of the pattern observed with the hemimethylated oriC in which the R1 box is not protected despite strong footprinting of adjacent regions ((16) , Fig. 7). Nevertheless we tried to ``supershift'' our hemimethylated specific complexes by using an anti-DnaA polyclonal antiserum (33, 34) in our binding reaction, which at 1:100 dilution has already been shown to completely inhibit DnaA protein-dependent in vitro oriC plasmid replication(33) . However, although we added large amounts of the antiserum (up to 1:20 dilution) to the reaction mixture prior to addition of the probe, we did not observe either a diminution or a shifting of the hemimethylated complexes, nor did the use of this antiserum inhibit the DNase footprinting pattern observed with the hemimethylated minimal oriC (data not shown). Thus, it seems unlikely that the DnaA protein is present in these complexes.


Figure 7: Protection from DNase digestion by peak 1 protein of the oriC probe (-46 to +244) in the hemimethylated (HM) and fully methylated (M) state. The first three lanes contain 0, 100, and 210 ng of peak 1 protein with the hemimethylated probe (5`-3`), the last three lanes contain 0, 100, and 210 ng of peak 1 with the methylated probe. The positions of the GATC methylation sites are indicated by arrowheads. The black bar shows the IHF binding sequence (+98-123), and the DnaA R1 box is indicated by an empty oblong (+80-88). The three cross-hatched boxes show the location of the principal protected regions.



More likely candidates are the proteins HobH (16) and SeqA(15) , both of which have been reported as being associated with hemimethylated oriC binding activity. We have prepared lysates from deletion mutants for both of these genes individually and in combination. Surprisingly, the absence of the hobH gene does not seem to have any discernible effect upon the hemimethylated binding activity, whether in the cytoplasmic or membrane-enriched fractions (data not shown).

However, in the DeltaseqA mutant the specific activity for both of the hemimethylated complexes is significantly lower than that observed with PC2 alone (Fig. 5a), neither complex being formed until higher extract concentrations, although the relative migration of the two retarded complexes is the same as in the wild type and complex 2 is still formed after complex 1. In addition, although the levels of the two complexes are only around a third of that found in the wild-type fractions, their relative distribution in both synchronized and nonsynchronized cultures is similar to that observed in the wild type (Fig. 5b).


Figure 5: Histograms showing the total hemimethylated oligonucleotide binding activities of lysate fractions from synchronized and asynchronous cultures of PC2DeltaseqA.a, a comparison of the specific activities of the initial lysates and the step gradient fractions of cultures of PC2DeltaseqA and PC2. The bacterial cultures were grown, harvested, lysed, and assayed side by side. The solid and cross-hatched bars represent the average of eight measurements for each fraction, the top bars represent the standard deviation. Cross-hatched bars, PC2DeltaseqA.; filled bars, PC2. b, a comparison of the distribution between the fractions of the step gradient of the total hemimethylated oligonucleotide binding activity measured in the initial PC2DeltaseqA and PC2 lysates. The blocks are as in panel a.



A further comparison of the lysates from DeltaseqA and the double mutant, DeltaseqADeltahobH, did not reveal any further differences in the levels of hemimethylated binding; hence, it would seem that the HobH protein is not responsible for the hemimethylated binding activity observed under these conditions for this particular oligonucleotide sequence, nor does its absence seem to have any indirect effect upon the levels of this activity.

Partial Purification of the Hemimethylated Oligonucleotide Binding Activity

In the absence of a major enrichment of the binding activity in membrane fractions, we continued with a chromatographic separation of the total lysate, testing a range of chromatographic resins (data not shown), before finding that both hemimethylated specific complexes were of a basic nature having affinity for cation exchange resins. By using a combination of a weak cation exchange column (Biorex A70) and a stronger resin (SP-Sepharose) we obtained a peak of enriched activity, peak 1, which on analysis by SDS-polyacrylamide gel electrophoresis contains three principal protein species at around 95, 90, and 20 kDa ( Fig. 6and Table 1).


Figure 6: Partial purification of the hemimethylated oligonucleotide binding activity. a, 15% SDS-polyacrylamide gel showing the protein composition of the initial bacterial lysate (L) (13 µg), the 0.8 M NaCl eluate from the BioRex A70 column (B) (2.5 µg), and the concentrated combined active elution fractions from the SP-Sepharose column corresponding to peak 1 (SP1) (9 µg). M, molecular mass markers, 106, 80, 49.5, 32.5, 27.5, and 18.5 kDa. b, graph of the absorbance at 280 nm of the elution fractions from the SP-Sepharose column. Bar indicates fractions containing hemimethylated oligonucleotide-specific binding activity assayed by gel retardation with the hemimethylated oligonucleotide probe; dashed line indicates NaCl elution gradient (0.2-0.6 M). c, gel retardation showing the hemimethylated oligonucleotide (5`-3`; at 1 times 10M) binding activity of increasing concentrations of peak 1: A, 0; B, 0.15; C, 0.8; D, 5; E, 15; F, 50 µg/ml; lanes G and H, 5 µg/ml, with specific competition by 50-fold excess hemimethylated and 300-fold excess methylated oligonucleotides, respectively. All lanes with 5000-fold excess nonspecific competitor DNA.





Peak 1 gives the same pattern of hemimethylated oligonucleotide binding as that observed with cruder lysate fractions, a single complex forming at lower protein concentrations and a more retarded second complex appearing as the concentration rises (Fig. 6c). Hence it seems that the active species responsible for both of the hemimethylated specific complexes are co-purifying in the same peak, a result that suggests that both complexes may result from differential binding interactions by the same protein species present in this fraction.

Similarly, an in vitro DNase footprinting analysis of the entire minimal oriC sequence using peak 1 clearly shows that, at the same protein concentration, there is protection of the hemimethylated but not of the fully methylated probe (Fig. 7). The resulting protection pattern is similar to that observed previously with much higher concentrations of membrane extracts (2.5-7.5 µg)(16) , there being principally two regions protected, between +90 and +110, and +120 and +130, corresponding to the sequence of our oligonucleotide probe. Three GATC hemimethylated sites are covered by these footprints, at +93-96, 107-110, and 125-128. Of the remaining GATC sites in the minimal oriC region only that at +67-70 seems to be protected by peak 1 at this concentration. Hence there is clearly a preferential affinity for only a subset of the hemimethylated GATC sequences present in oriC. Thus, it seems probable that the recognition sequence for the protein-DNA interactions is larger than simply the hemimethylated GATC site and that there is additional recognition of the flanking sequences. It is interesting to note in this respect that the DnaA R1 box (+80-88), which is not protected by peak 1, occurs adjacent to the largest footprint, while the IHF binding site from +98 to 123 is partially protected by the footprints at either end of it.

Relating the footprinting data to the gel retardation results, we suggest that the two retarded complexes formed with the hemimethylated oligonucleotide probe during gel retardation are probably due to successive interactions at the hemimethylated sites, the less retarded complex resulting from interaction at both +93-96 and 107-110, while the more retarded complex forms with additional protein binding at +125-128.


DISCUSSION

The association of the bacterial chromosome with the bacterial membrane was originally proposed as a possible mechanism for the physical segregation of the newly replicated chromosomes and the associated cellular division(35) . Since the replicon hypothesis was first advanced there have been many experimental studies that have sought to find evidence for such an interaction. While many of these studies have been dismissed (36) as showing interactions of a nonspecific nature that have arisen during the process of bacterial lysis and fractionation, there has been a body of evidence presented that indicates that the origin of chromosome replication has an interaction of a more specific nature with the membrane, an observation that has been refined to the origin in the newly replicated, hemimethylated state(6, 9, 16, 20) .

Replication initiation in E. coli is a very precisely controlled event, the initiation process responding sensitively and dramatically to a small incremental change in cell physiology(37) . However, each copy of oriC present in the bacteria will only undergo one round of initiation per cell cycle. While the timing of initiation can be varied by changing the level of DnaA initiator protein, additional factors remain to be discovered(38) . It has been proposed that the control of a single round of initiation per cell cycle is a consequence of the combined effects of two processes(11, 12, 39) . In the first, the initiation potential would increase to a high level at which it would be sufficient to trigger efficient and synchronous initiation at all available origins by virtue of one or more highly cooperative interactions. In the second, further unwanted rounds of initiation would be prevented by a blocking mechanism. Sequestration is such a mechanism that would exclude secondary initiation events at a time in the cell cycle when the initiation potential is high, by maintaining the freshly replicated origins in their hemimethylated state(9, 11, 15) . A similar sequestration of the dnaA locus, located close to oriC, has also been proposed, which, by blocking transcription of the initiator protein, could facilitate a drop in the initiation potential immediately after replication initiation(11, 40) .

In this study, we have chosen a region of the origin that, in the hemimethylated state, is the most protected by membrane-rich extracts from DNase digestion in footprinting studies. While previous investigations have employed relatively large DNA fragments to analyze the methylation-sensitive DNA-protein interactions of the origin with membrane-rich extracts, we have sought to obtain more precise information about the nature of such interactions using a smaller oriC probe, which is readily prepared in the various possible methylation states.

We have found that there is indeed a hemimethylated DNA-specific activity for our probe in membrane-rich extracts. However, such activity is not apparently limited to such membrane extracts (whether inner, outer, or intermembrane), nor does it seem to be dependent upon the synchronization state of the bacterial culture. Thus, whether the activity that we present in this article plays a role in a membrane-associated sequestration of the origin is open to question.

Although our oligonucleotide probe covers only 3 of the 11 GATC sites in oriC, a footprinting study of the entire oriC region suggests that this is the part of oriC with the highest affinity for the hemimethylated DNA-specific binding activity that we have characterized(16) . There may be other hemimethylated oriC-specific binding activities in E. coli, whether membrane-associated or cytoplasmic, but these are not apparently as strong in our lysates under the conditions tested. It is possible that alternative activities have a lower affinity for oriC or that, despite an affinity that is as strong as that presented here, their relative concentration is much lower in crude extracts. It is also possible that there is a requirement for additional factors such as divalent cations. Previous studies have employed the technique of filter binding to detect protein interactions with their larger oriC fragments(6, 7, 9, 20) . As shown in Fig. 2, the binding pattern with the entire oriC region is quite complicated, and it is not easy to distinguish the different binding complexes using gel retardation, let alone employing filter binding. In addition, we found much higher nuclease levels in cytoplasmic as opposed to enriched membrane fractions. In the presence of Mg, the DNA probe was rapidly degraded when testing crude lysates, degradation that might not have been as readily detected with filter binding in which the loss of the radioactive probe might be interpreted as an absence of DNA binding activity.

There are clearly protein factors in the bacterial cell that are capable of binding with high affinity to the hemimethylated origin DNA, although the number and the relative concentration and binding affinities of such factors are still not clear. We have partially purified the hemimethylated oriC binding activity that we have characterized, and footprinting studies with this active fraction indicate that there is a preferential interaction with only three or four of the available hemimethylated sites in oriC. The location of these sites relative to the DnaA box, R1, and the IHF binding sequence coincides with a potential blocking of the initiation of DNA replication. DnaA is the key protein in the initiation of bacterial DNA replication, binding with high affinity to the four DnaA boxes in oriC(27) . Interaction of 10-20 DnaA monomers at these boxes results in the formation of the ``initial complex''(41) , the DnaA protein subsequently mediating a local unwinding reaction in an AT-rich part of oriC that results in the conversion of the initial complex into the ``open complex''(42) . IHF is implicated in this process, changing the DNA structure by binding at its single oriC binding site. One dimer of IHF is sufficient to induce a strong bend, while mutation of the IHF binding site inactivates oriC(43) . IHF seems to help DnaA to unwind oriC(44) , allowing DnaA-mediated strand opening at temperatures between 21 and 37 °C (DnaA can function alone at 37 °C and above)(45) . Hence by preferentially binding the hemimethylated sequences in this region of the origin, reinitiation by DnaA might be inhibited until some mechanism of desequestration succeeded in removing the hemimethylated oriC binding factor, allowing the Dam methylase to remethylate the region. Since the hemimethylated oriC binding factor does not have a high affinity for the fully methylated sequence it would not intervene again until a subsequent reinitiation and formation of a newly replicated, hemimethylated origin.

Studies of the duration of the hemimethylated state suggest that, while oriC and the dnaA promoter remain hemimethylated for between and of a generation before becoming rapidly remethylated (6-10 min)(9, 11) , other regions of the chromosome analyzed so far are remethylated within 1-2 min(5, 15) . Our observation of a reduction in our hemimethylated oriC binding activity in the seqA deletion mutant suggests that the absence of the SeqA protein is having an effect upon it, whether direct or indirect. However, while it is possible that SeqA is binding directly, either on its own or in a larger protein complex, with the hemimethylated DNA, the observed change in our hemimethylated binding activity is apparently quantitative rather than qualitative, since we observed both complexes 1 and 2 in PC2DeltaseqA lysates and, upon partial purification with cation exchange chromatography, we obtained the same peak of binding activity as PC2 with the same apparent protein composition (data not shown). Hence the association of SeqA with this activity is more likely to be of an indirect nature, for example, in regulating its levels.

SeqA has been presented as playing a part in sequestration, also serving as a negative modulator of the primary initiation process(15) . It has been suggested that SeqA might negatively affect initiation by inhibiting the expression or activity of DnaA. Another study (46) also suggests that SeqA plays a role in negative modulation of DnaA activity independent of the methylation status of the origin, and hence in a manner not directly related to oriC sequestration. It found that the level of DnaA protein was increased 2-fold in the seqA mutant, which was overinitiating replication at oriC. This abnormally greater frequency of replication initiation could result from either an impairment of sequestration of the hemimethylated origin and/or a defect in the regulation of initiation itself. Based on these results it was proposed that the role of SeqA is to limit the activity level of DnaA(15, 46) . However, our observation in DeltaseqA lysates of a 3-fold reduction in the hemimethylated oriC binding activity suggests that there is also an impairment in the sequestration mechanism of this mutant, and hence it is possible that the observed changes in DnaA levels might reflect the consequences of such a diminished sequestration operating at the level of the dnaA promoter, as previously suggested (11, 40) , rather than upon the DnaA protein directly.

The observation of a difference in the in vivo remethylation kinetics of hemimethylated oriC between synchronized PC2 and PC2DeltaseqA cultures (15) might also be interpreted in the light of our observation of a reduction in the levels of hemimethylated binding activity in this mutant. While both cultures in that study gave a peak of hemimethylated oriC at around 9 min after reinitiation of the culture, the peak in PC2 was higher and had a longer duration either side of this peak (±7 min), whereas in DeltaseqA, the peak had a reduced size and duration (±3 min). Thus, although there appears to be an impairment of a hemimethylated oriC sequestration mechanism in the absence of seqA, there is nevertheless a maintenance, albeit reduced, of the recently replicated origin in its hemimethylated state. The hemimethylated oriC binding activity that we present here is similarly reduced but not abolished in DeltaseqA and hence may account for the persistence in this mutant of the hemimethylated origin.

In conclusion, we present in this article a novel hemimethylated oriC-specific binding activity that can be observed at similar levels in both cytoplasmic and membrane fractions. This activity is particularly associated with a region adjacent to the strong DnaA binding site, R1, and covers part of the binding site for IHF, a protein that has been shown can help DnaA to unwind the origin during initiation. This observed hemimethylated oriC binding activity could play a role in the proposed sequestration process that has been presented as a means of blocking the reinitiation of DNA replication at the origin. However, this activity does not appear to correspond to either SeqA or HobH, two proteins previously implicated in sequestration. Nevertheless, in the absence of the seqA gene, we observe a strong reduction in the level, but not the qualitative nature, of this hemimethylated oriC binding activity.

We are currently completing our purification of the active species responsible for the hemimethylated oriC binding activity with the intention of identifying the corresponding gene(s).


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by European Community Grant CHRX-CT92-0010 (to M. K.) under the auspices of the Human Capital Mobility Program. To whom correspondence should be addressed: EMBL Gastehaus, Im Eichwald, 69126 Heidelberg, Germany. Tel.: 49-6221-548311; Fax: 49-6221-387512.

(^1)
The abbreviation used is: IHF, integration host factor.


ACKNOWLEDGEMENTS

We thank Renée Kern for the DNase footprinting analysis and Philippe Lecomte, Danièle Joseleau-Petit, and Erik Boye for helpful discussions.


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