Centromere parC of plasmid R1 is curved
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《核酸研究医学期刊》
Institute for Molecular Biotechnology e.V., Beutenbergstr. 11, D-07745 Jena, Germany, 1 Institute of Evolution, University of Haifa, Haifa 31905, Israel and 2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
* To whom correspondence should be addressed. Tel: +49 3641 656 260; Fax: +49 3641 656 261; Email: diekmann@imb-jena.de
ABSTRACT
The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.
INTRODUCTION
Stable maintenance of bacterial low-copy-number plasmids is ensured by a number of active stabilization loci encoded by the plasmids themselves. These loci can be divided into those that function by the killing of plasmid-free segregants and those that actively segregate plasmid copies to daughter cells at cell division, i.e. partitioning loci (par). All known plasmid par systems operate by determining the intracellular position of their replicon such that, after replication, plasmid copies are rapidly transported to each side of the bacterial septal plane where they remain positioned until cell division takes place. Thus, by actively distributing plasmid molecules to cell progeny, the par systems ensure faithful plasmid inheritance throughout the bacterial population (3–6). In general, partitioning loci encode three elements: centromere-like site(s) in the plasmid DNA, a protein binding to this site and an ATPase. The Escherichia coli plasmid R1 par locus encodes an actin-like ATPase (7,8). The R1 par centromere-like site (parC) contains two sets of five 11 bp direct repeats (iterons) separated by a region containing the par promoter (9,10). The iterons serve as operator sequences to which ParR binds and represses the promoter (10,11). ParR binds to multiple sites in the parC region in a cooperative manner (12). Binding of ParR to parC serves to autoregulate expression of the par genes as well as to form a nucleoprotein complex for partitioning (9,10). The ParM protein interacts with the ParR/parC complex (13) and forms dynamic F-actin-like filaments that are involved in plasmid partitioning (14,15).
In higher eukaryotes, centromere DNA is formed by multi-fold repeats of satellite DNA. Cloned satellite monomers from many organisms (16–28) exhibit anomalously slow migration in polyacrylamide gels indicative of DNA curvature (29–36). Also human centromeric DNA seems to be curved (26). Curved or bendable DNA aids to the tight winding of the DNA around the histone octamer (37–39).
The bacterial par system obviously has centromere and kinetochore function. We thus asked whether the centromeric parC DNA of bacteria is also curved and whether it would share the function of DNA curvature and bending in global structure formation of the kinetochore DNA–protein complex in eukaryotes.
Macroscopic DNA curvature has been associated primarily with the presence of properly phased stretches of adenines . Nelson et al. (41) crystallized a dA6 region and solved the structure. Short (4–9 bp) runs of desoxyadenines repeated with the helix screw produce a global curvature of the DNA double helix . dA-tract-induced DNA curvature can be affected by other sequence elements (49,50) and also non-dAn sequence elements can cause DNA curvature (51–54). Curvature is directly related to the Fourier coefficient of the dinucleotide roll angle at a period equal to the helical repeat (55). One of the most accepted hypotheses suggests that distinctive properties of poly(dA):poly(dT) may be explained by having an unusual ‘B'-form’ structure (32–34) which is sufficiently different from B-form DNA that proteins recognize the difference (56–58). Models of DNA curvature have been proposed (34,36,52,59–63). Numerous examples of naturally occurring DNA curvature have been reported (64–75). Curved DNA has clearly identifiable properties. The curvature measured as retarded gel migration in PAGE is most prominently present at moderate temperatures and decreases at elevated temperatures (31,33,44,76). Ethidium bromide intercalates into the DNA and reduces the differences between B- and B'-form DNA, thereby strongly reducing the migration anomaly (77), in contrast to the behaviour of four-way junctions (78). Wu and Crothers (30) found that a fragment containing the curvature at or near its end migrates most rapidly in polyacrylamide gels whereas fragments in which the curvature is more centrally located migrate more slowly.
Here we show that parC is indeed curved. We determine the value of the migration anomaly in PAGE and measure its properties. We also compare the curvature of this bacterial centromeric DNA with that of eukaryotic centromeres and discuss the potential influence of curvature on centromere function.
MATERIALS AND METHODS
Those parts of the pKG330 plasmid (Figure 1) relevant for curvature analysis (Figure 2A) were verified by sequencing (MWG, Germany). Approximately 10 μg of pKG330 plasmid DNA carrying the parC sequence (see Figure 1) were digested with different restriction endonucleases (New England Biolabs, USA) simultaneously, resulting in different fragments (see Figure 2A) for curvature analysis. According to the time and temperature suggested by the manufacturer (mainly 120 min at 37°C), the DNA was extracted with phenol:chloroform:isoamyl alcohol (15:24:1; Sigma, Germany), precipitated and washed with 80% ethanol, air-dried, re-suspended in 20 μl of 5 mM Tris (pH 8.5) and mixed with 8 μl loading dye solution (60% glycerol, 60 mM EDTA, 0.09% bromphenol blue, 0.09% xylene cyanol FF; MBI Fermentas, Vilnius). The sample (6 μl) was loaded on a native 6% polyacrylamide gel (29:1 acrylamide:bisacrylamide; Bio-Rad, USA). Gels were pre-run for 3 h until current and temperature remained constant. Unless stated otherwise, electrophoreses was carried out in 1 x TBE (90 mM Tris–borate, 2 mM Na-EDTA, pH 8.0) at 150 V (8 mA) for 4 h (migration distance of bromphenol blue 14 cm). The temperature was mainly 24°C (room temperature), or alternatively 4°C (cold room) or 37°C (warm oven). After the runs, the gels were stained for 30 min in an aqueous solution of ethidium bromide (1 mg/l), followed by rinsing in water prior to documentation. The gels were illuminated by a 254 nm UV lamp (Chroma, Brattleboro), documented by a CCD camera system (Polaroid, USA) and gel images printed by a HP Laserjet 2100 M in the size of 26.5 x 18 cm for analysis. The 1 kb plus DNA ladder (Invitrogen, Carlsbad, USA) and the 1 kb DNA ladder (New England Biolabs, USA) served as marker fragments together with the intrinsic plasmid fragments resulting from the restriction digest running in the same lane as the parC fragment. For each gel, the migration of all fragments was determined and calibration curves were plotted using the marker fragments (logarithm of the number of base pairs versus distance migrated). The apparent size in the acrylamide gel relative to the calibration curve was determined for every anomaly migrating fragment.
Figure 1. Plasmid pKG330. Restriction sites and the position of parC are indicated.
Figure 2. (A) The sequence of parC (bold, 159 bp) and the framing vector sequences (both verified by sequencing) are displayed. The restriction sites within this DNA are indicated. These restrictions sites were used for fragment analysis. The 11 bp iterons are marked by arrows. (B) The mutated parC sequence (H-K) and the parC wild type (Wt) are displayed. The mutated base pairs are indicated by small circles. The mutations introduce two new single cutting sites HindIII and KpnI.
Construction of parC H-K mutant sequence
A mutant sequence of the parC sequence containing 4 bp exchanges was constructed by DNA cloning procedures using standard methods (79). For the introduction of two new single restriction sites, HindIII and KpnI, between the first (parC I) and the second (parC II) 5-fold iterons of parC, the DNA of plasmid pKG330 was amplified by two PCR reactions (Roche, Penzberg, Germany). In the first reaction, we amplified parC I using as upstream primer (parC f) 5'-GTAGTAGGTTGAGGCCGTTGAGCAC-3', which binds 230 bp upstream of parC, and as downstream primer (parC Kpn Hind r) 5'-GGTACCCCATTTCAACCATCAATCAAAGCTT-3', which binds between the two 5-fold iterons and introduces the two new restriction sites. In the second reaction we amplified parC II. The upstream primer (parC Hind Kpn f) 5'-AAGCTTTGATTGATGGTTGAAATGGGGTACC-3', a direct antisense repeat of primer (parC Kpn Hind r) also introduced the HindIII and KpnI sites, whereas the downstream primer (parC Nco r) 5'- CCATGGAAATGTTGAATACTCATACTCTTCC-3' introduced a NcoI site 360 bp downstream of parC. The blunt end PCR products were cloned into pCR 4Blunt-TOPO (Invitrogen, Carlsbad, CA, USA). The resulting vectors pTOPOparC I and pTOPOparC II, carried parC I and parC II respectively. After verification of the base exchanges by sequencing, both vectors were digested by HindIII–NcoI. The 456 bp fragment of pTOPOparC II containing parC II was cloned into the 2839 bp fragment of pTOPOparC I resulting in pTOPOparC H-K. Subsequently pKG330 and pTOPOparC H-K were both digested with BamHI–EcoRI. The 167 bp fragment of pTOPOparC H-K was cloned into the 4120 bp fragment of pKG330 resulting in pKG330 H-K carrying mutant parC with the new restriction sites (parC H-K). The exchange of the four bases was confirmed by sequencing (MWG, Ebersbach, Germany). The base pair exchanges are introduced at the sites 192, 194, 219 and 220 bp (see Figure 2). This mutant DNA was digested and analysed as described above for the wild-type sequence.
Computational calculation of curvature
The prediction of DNA curvature was made by means of our CURVATURE program. This program calculates a three-dimensional path of a DNA molecule and estimates the curvature of the axis path (80). The CURVATURE algorithm is based on the stepwise calculation of geometric transformations according to the set of dinucleotide wedge angles (52,81). A curvature value at a position i corresponds to a curvature of the arc approximating to the predicted DNA path. The DNA curvature was measured in ‘DNA curvature units’ (cu) introduced by Trifonov and Ulanovsky (82) and in ‘degrees per base pair’. Taking 146 bp of DNA wrapping around a histone octamer of 1.65 turns as a basis for conversion, the curvature unit can be related to other measures. For example, having 88.5 bp in the one full nucleosomal turn we find that 1 cu corresponds to 42.7 degrees per helical turn or 4.1 degrees per base pair. The program is available upon request from A.B. at bolshoy@research.haifa.ac.il.
RESULTS
The parC fragment is 159 bp in length and is organized in two regions each containing a 5-fold repeated sequence motif of 11 bp length with a central region of 39 bp containing the par promoter (12). The consensus sequence of the 10 repeats (iterons), dAAAAAAAACCC , contains longer dA-tracts with a repeat length of 11 bp, thus phased slightly distinct from the DNA helix screw . This suggested to us that the parC fragment might potentially be curved and thus might show anomalous gel migration. We analysed the parC fragment in gels under various conditions.
The parC fragment was cloned into the plasmid pKG330 . Those parts of the plasmid relevant for curvature analysis were verified by sequencing and are displayed in Figure 2A. Various DNA fragments were constructed by restriction digest using the endonucleases indicated in Figure 2A. These fragments are listed in Table 1. The migration of these fragments relative to a series of marker fragments in different lanes as well as to plasmid fragments in the same lane was analysed in polyacrylamide (6, 8 and 11%) and agarose gels under different buffer and temperature conditions. A typical polyacrylamide gel is displayed in Figure 3. The apparent size of the fragments was determined relative to normally migrating marker fragments. The quotient ‘apparent divided by known sequence length’ is termed ‘k-factor’ and listed in Table 1 for the different conditions analysed. Several k-factor values were measured repeatedly. We deduce an experimental error of the measurements for the k-factors of ± 0.05.
Table 1. List of 10 analysed fragments with k-factors
Figure 3. Typical 8% PAGE run with 1 x TBE at 25°C. M, marker fragments. Lane 1: fragment 3 (parC; AatII/BanII), lane 2: fragment 3 (parC H-K; AatII/BanII), lane 3: fragment 1 (parC; BspHI), lane 4: fragment 1 (parC H-K; BspHI), lane 6: fragment 4 (parC; HhaI), lane 7: fragment 4 (parC H-K; HhaI), lane 8: fragment 8 (parC; BspHI/EcoRI), lane 9: fragment 7 (parC; BamHI/BstBI). Numbering of the fragments is according to Table 1. The length of the marker fragments in the two lanes M is given at the right side of the gel. The anomaly migrating fragments containing parC are indicated by asterisks. If the anomaly migrating fragments containing parC of lanes 1–4 would migrate normally (i.e. according to their sequence length), their position in the gel would be at the site indicated by a broken line at the left side of the gel.
Most of the plasmid DNA fragments external to the parC, migrated normally in the polyacrylamide gels. In particular, those fragments framing the parC sequence, migrated normally: SalI/BamHI (276 bp length) upstream and EcoRI/BspHI (189 bp length) downstream had k-factors of 0.98 and 1.02, respectively. One fragment, however, which contains the last 120 bp of the origin of replication of the plasmid (HhaI fragment of 270 bp length), migrated slower with a k-factor of 1.09 (8% PAGE, 1x TBE, 25°C). Being curved is a property of plasmid replication origins (73) as well as the SV40 origin (75).
The migration anomaly in polyacrylamide gels hardly varies for the salt and buffer conditions analysed here (Table 1). For parC framed by 120 and 191 bp on either ends (fragment 1; BspHI digest), at 25°C in 1x TBE (i.e. 90 mM Tris–borate, 2 mM Na-EDTA, pH 8.0) we measured a k-factor of 1.71 in 8% PAGE. This changed to 1.76 at 0.5x TBE and 1.68 at physiological conditions (10 mM MgCl2, 170 mM NaCl in the presence of 1x TBE). Other parC containing fragments confirmed this invariance (fragments 3, 4 and 7; see Table 1); e.g. for fragment 7 the k-factor was 1.52 at 1x TBE, which changed to 1.49 at 0.5x TBE as well as physiological conditions.
On the other hand, the migration anomaly depends on the polyacrylamide concentration (Table 1). For fragment 1 (BspHI digest) the k-factor increased from 1.59 at 6% acrylamide concentration to 1.71 at 8% and 1.94 at 11% (see Figure 4B). Correspondingly, for fragment 4 (HhaI digest) the k-factor increased from 1.44 at 6% to 1.59 at 8% and 1.82 at 11% (see Figure 4B). In the measured polyacrylamide concentration range, the k-factor increase is linear.
Figure 4. (A) Temperature dependence of the k-factors of fragments 2 and 6 (see Table 1). Fragment 2 contains framing vector sequences and shows a larger k-factor while fragment 6 is parC, almost without framing vector sequences. (B) Dependence of the k-factors of fragments 1 and 4 (see Table 1) on polyacrylamide concentration.
We observed a strong decrease of the migration anomaly with increasing temperature (Table 1). For fragment 1, the k-factor decreased from 2.45 at 4°C to 1.71 at 25°C and 1.27 at 50°C (see Figure 4A). For parC nearly without framing sequences (6 and 2 bp, BamHI/EcoRI double digest; fragment 6), the k-factor decreased from 1.53 at 4°C to 1.29 at 25°C and 1.03 at 50°C (see Figure 4A). Extrapolation suggests that the k-factor decreases to 1.00 (normal migration) at a temperature of 60–70°C (with a slightly smaller value for fragment 6), as observed for other curved sequences (31,33,44,76).
For controls we measured the migration anomaly of several parC containing fragments in agarose gels as well as in the presence of ethidium bromide in the gel buffer. In 2% agarose gels, the migration anomaly of the analysed fragments 1–6 was normal (k-factors 1.00; see Table 1). In the presence of 1 mg/l ethidium bromide, the migration anomaly was considerably reduced . For most analysed fragments, the migration became normal (k-factors 1.00; see Table 1) in the presence of 3 mg/l ethidium bromide. This behaviour is known for curved DNA fragments (28,77).
By the exchange of four bases between the two 5-fold repeated iterons within the parC sequence (at the sites 192, 194, 219 and 220 bp; see Figure 2A), the parC sequence dAATAATCATTGATTGATGGTTGAAATGGGGTAAACTTGA is modified to dAATAAGCTTTGATTGATGGTTGAAATGGGGTACCCTTGA (see Figure 2B) introducing two new single cutting restriction sites. When analysing this modified parC sequence, we measured a considerably reduced migration anomaly. For fragment 1 (BspHI digest) the k-factor was reduced from 1.71 to 1.50 (same length of DNA; 8% PAGE at 1x TBE at 25°C). A similar reduction was observed for fragments 2–6 (see Table 1). The temperature dependence of the migration anomaly of this modified parC sequence was measured to be similar in nature; however, the slope of the k-factor versus temperature plot was slightly reduced (data not shown). As for the unmodified parC sequence, extrapolation suggests that the k-factor decreases to 1.00 (normal migration) at a temperature of 60°C for the modified sequence as well. The dependence of the migration anomaly on the polyacrylamide concentration was similar to the unmodified parC at 6 and 8% (same slope of k-factor versus percentage of acrylamide; data not shown); however, in contrast to the unmodified parC, hardly increased further for 11%. Changing the salt and buffer conditions had little or no effect on the migration anomaly (compare fragments 1, 3 and 4 in Table 1), as for the unmodified sequence. In agarose gels as well as in the presence of 3 mg/l ethidium bromide, the modified parC migrated normally.
These base pair exchanges create two new single cutting sites within the parC sequence, KpnI and HindIII, which allow studying the first and the second 5-fold repeated iterons separately. Using the newly introduced restriction sites, we constructed additional fragments (see Table 1) the migration of which was analysed in gels as well. DNA fragments containing the first 5-fold repeated iteron sequence with framing sequences at the 5'-end only showed rather small migration anomaly. When the up-stream HindIII site was used with framing sequences of 41 (HhaI/HindIII double digest) and 120 bp (BspHI/HindIII double digest) lengths, k-factors of 1.10 and 1.04 were measured, respectively (at 1x TBE at 25°C in 8% PAGE; data not shown). In parallel, when the down-stream KpnI site is used with framing sequences of 41 (HhaI/KpnI double digest), 120 (BspHI/KpnI double digest) and 292 bp (SalI/KpnI double digest) lengths, k-factors of 1.10, 1.04 and 1.09 were measured, respectively (at 1x TBE at 25°C in 8% PAGE; data not shown). Fragments containing the second 5-fold repeated iteron sequence with framing sequences at the 3'-end only showed larger migration anomaly compared to the fragments containing the first parC part, however still small compared to the migration anomaly of the full parC. Partly this might be due to the positioning of the curved sequence within the analysed fragment at the 5' and 3'end, respectively. Maximal migration anomaly is observed when the curved sequence is located in the middle of the fragment (30). When the up-stream HindIII site was used with 3'-framing sequences of 191 (HindIII/BspHI double digest) and 246 bp (HindIII/HhaI double digest) lengths, k-factors of 1.15 and 1.19 were measured, respectively (at 1x TBE at 25°C in 8% PAGE; data not shown). In parallel, when the down-stream KpnI site was used with framing sequences of 191 (KpnI/BspHI double digest), 246 (KpnI/HhaI double digest) and 894 bp (KpnI/PstI double digest) lengths, k-factors of 1.16, 1.18 and 1.07 were measured, respectively (at 1x TBE at 25°C in 8% PAGE; data not shown).
Using the newly introduced restriction sites, we re-cloned the parC 5-fold repeated iterons. We constructed vectors in which either the second or the first part of parC was deleted. We thus could analyse fragments in which either the first (fragment 9, SpeI/SnaBI double digest) or the last (fragment 10, AatII/BsrGI double digest, see Table 1) 5-fold repeated iteron sequence was positioned in the middle of the fragment. In 8% PAGE in 1x TBE at 25°C both fragments showed the same migration anomaly which increased to about the same extent at low temperatures (4°C, see Table 1).
Predicted DNA curvature
The software CURVATURE (80) was applied to calculate the trajectories of the wild-type as well as the mutant parC sequence. The juxtaposed calculated DNA paths are presented in Figure 5A. The wedge model (36) with the angles of Bolshoy et al. (52) and Kabsch et al. (81) predicts the planarity of the fragment. Compared to wild type, the modified parC sequence displayed a considerably reduced gel mobility. CURVATURE identified changes in the DNA trajectory of the modified parC fragment due to the four base exchanges: the modified fragment is less curved than the original parC fragment. The two base pair exchanges at 219 and 220 bp result in a curvature reduction at this site of 17 degrees (see Figure 5A). Interestingly, both fragments are rather planar although the iteron repeat length is 11 bp (see Figure 2A), not perfectly matching the helical repeat (10.5 bp). However, the distance between the two sets of iterons (39 bp) is slightly less than an integral number of helical turns so that both effects might compensate. The end-to-end distances for the fragments are 43.9 nm and 39.8 nm, the smaller distance corresponding to the more curved wild-type fragment. The CURVATURE program can be used also to map the DNA curvature along the sequence. In Figure 5B we show the juxtaposed curvature maps of the mutant and wild-type fragments with the window- size of 21 bp. The program predicts that the first pair of modifications (at positions 192 and 194, see Figure 2) should not influence the gel retardation, while the replacement of the dinucleotide dAA by the dinucleotide dCC at positions 219 and 220 (see Figure 2) is predicted to cause a dramatic reduction of the gel migration anomaly.
Figure 5. Juxtaposition of predicted DNA curvature for wild-type and modified sequences parC. The wild type and modified sequences were applied to the software CURVATURE (80) with the specified parameters (52,81). Both fragments are 167 bp long. The modifications were made at positions 77, 79, 104 and 105 relative to this fragment length (corresponding to sites 192, 194, 219 and 220 in the numbering frame of Figure 2). (A) Two-dimensional representation of the DNA paths. Two projections of the DNA paths are presented on a plane of the maximal observed curvature (a) and on a reciprocal plane (b). To display the degree of curvature of a fragment, the calculated coordinates of the phosphates and base pair centers are projected on that plane, for which the mean square distance from all the points is minimal. The line connecting the base pair centers presents the helical axes of the original (wild type: green continued by red) and modified fragment (mutant: green). The phosphates are shown in light and dark blue (mutant) switching to light brown and green for the wild type. The second projection (right) shows the planarity of the fragments. The two base pair exchanges at 219 and 220 bp result in a curvature reduction at this site of 17 degrees. (B) DNA curvature maps versus sequence with window-size of 21 bp. The overall DNA shapes are characterized curvatures of a sliding arc of 21 bp of length approximating to the paths of the axes of the given DNA fragments. The curvature map (given in ‘cu units’ on the left axis and ‘degrees per bp’ on the right axis) for wild-type parC is plotted as a solid line and for the mutant by a dashed line. The two vertical dotted lines indicate the positions of the base pair modifications.
DISCUSSION
parC is the centromere of the E.coli plasmid R1. Here we analysed the intrinsic sequence-directed curvature of parC both by its migration anomaly in polyacrylamide gels and by applying the wedge-model based software CURVATURE. The experimentally observed gel migration anomaly indicated as ‘k-factor’ (i.e. an apparent fragment length in the gel divided by correct sequence length) of the parC sequence is 1.7 (see Table 1) and thus smaller than that of another naturally occurring strongly curved DNA, the whole kinetoplast DNA fragment (29,31), which displayed k-factors even >4. However, parC (159 bp) is much shorter than the kinetoplast fragment (414 bp). When the parC curvature is compared with that of parts of the kinetoplast fragment of similar length (e.g. fragments of the lengths 187 and 262 bp) , similar k-factors were obtained indicating a similar degree of curvature. The parC curvature is also similar to that of constructed fragments of similar length with 8-fold repeats of the sequence dGACAAAACTC within framing non-curved sequences (32,33). Thus, for a naturally occurring sequence, the parC curvature is large.
The properties of the parC curvature agree with those of other curved dA-tract sequences. Just like parC, the 187 bp kinetoplast fragment as well as the constructed fragment showed hardly any dependency of the k-factor on NaCl or MgCl2 concentration within the measured range (see Table 1) (31,33). Also the temperature dependence of the k-factors of these DNA fragments is very similar, extrapolating to 60°C for normal migration (k-factor = 1.00) in all three cases (31,33). Furthermore, these fragments behave normally in agarose gels and show a strongly reduced migration anomaly in the presence of ethidium bromide (see Table 1). This similarity in properties of their gel migration anomaly indicates that the phenomenon is of the same origin.
parC contains two regions of 5-fold repeated iterons separated by 39 bp (see Figure 2A). We modified 4 bp within this intermediate sequence creating two new single cutting sites (see Figure 2B). This modified sequence of identical length showed considerably smaller curvature measured in PAGE (see Table 1) and calculated by CURVATURE (see Figure 5A). This indicates that the two repeat regions are not independent curved parts of parC but that the overall parC curvature is a property of the whole fragment. The intermediate sequence contributes not only by its number of base pairs (which also determines the phasing relation between the first and the second 5-fold repeat) but also to a considerable amount by its sequence.
The two new cutting sites allowed us to analyse the curvature of the first and the second repeat independently. When analysing the first repeat without 3'-framing sequences and the second repeat without 5'-framing sequences, we measured a smaller curvature for the first repeat. However, when placing the repeats in the centre of fragments with non-curved framing sequences, we found similar k-factors for both (Table 1). This discrepancy might be explained by the different arrangement of curved sequences within the repeats, maybe placing curved elements in the first case more to the end of the fragment where their influence on the migration anomaly is reduced (30,31).
Curvature seems to be a general phenomenon of eukaryotic centromeric DNA. Mostly, the degree of curvature is small (k-factors of up to 1.20) and only in some cases the k-factors are larger (26). All 16 centromere sequences of Saccharomyces cerevisiae show migration anomaly in polyacrylamide gels (27,28); the largest k-factor value was measured for CEN14 as 1.24. The curvature of most eukaryotic centromere DNA as well as of the 16 yeast sequences is considerably smaller than the curvature of the bacterial centromeric sequence parC analysed here.
Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate whether curvature is a general property of centromeres. The centromeres of higher eukaryotes are organised as multi-fold repeats of -satellite DNA, many of these repeats showing curvature (26). In many eukaryotes, the repeat lengths of the satellite DNA are similar to the repeat lengths of nucleosomes in chromatin or to multimers of this length (26). The satellite curvature might thus serve as a nucleosome positioning signal. Experiments reconstituting nucleosomes onto satellite DNAs confirmed that view (86,87). The curvature of the parC sequence found here might support the multiple parR binding to parC resulting in a globular folded shape of the kinetochore-like DNA protein complex also in bacteria, may be even similar in size and nature to a nucleosome.
ACKNOWLEDGEMENTS
We thank S. Pfeiffer and S. Hosid for excellent technical support.
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* To whom correspondence should be addressed. Tel: +49 3641 656 260; Fax: +49 3641 656 261; Email: diekmann@imb-jena.de
ABSTRACT
The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.
INTRODUCTION
Stable maintenance of bacterial low-copy-number plasmids is ensured by a number of active stabilization loci encoded by the plasmids themselves. These loci can be divided into those that function by the killing of plasmid-free segregants and those that actively segregate plasmid copies to daughter cells at cell division, i.e. partitioning loci (par). All known plasmid par systems operate by determining the intracellular position of their replicon such that, after replication, plasmid copies are rapidly transported to each side of the bacterial septal plane where they remain positioned until cell division takes place. Thus, by actively distributing plasmid molecules to cell progeny, the par systems ensure faithful plasmid inheritance throughout the bacterial population (3–6). In general, partitioning loci encode three elements: centromere-like site(s) in the plasmid DNA, a protein binding to this site and an ATPase. The Escherichia coli plasmid R1 par locus encodes an actin-like ATPase (7,8). The R1 par centromere-like site (parC) contains two sets of five 11 bp direct repeats (iterons) separated by a region containing the par promoter (9,10). The iterons serve as operator sequences to which ParR binds and represses the promoter (10,11). ParR binds to multiple sites in the parC region in a cooperative manner (12). Binding of ParR to parC serves to autoregulate expression of the par genes as well as to form a nucleoprotein complex for partitioning (9,10). The ParM protein interacts with the ParR/parC complex (13) and forms dynamic F-actin-like filaments that are involved in plasmid partitioning (14,15).
In higher eukaryotes, centromere DNA is formed by multi-fold repeats of satellite DNA. Cloned satellite monomers from many organisms (16–28) exhibit anomalously slow migration in polyacrylamide gels indicative of DNA curvature (29–36). Also human centromeric DNA seems to be curved (26). Curved or bendable DNA aids to the tight winding of the DNA around the histone octamer (37–39).
The bacterial par system obviously has centromere and kinetochore function. We thus asked whether the centromeric parC DNA of bacteria is also curved and whether it would share the function of DNA curvature and bending in global structure formation of the kinetochore DNA–protein complex in eukaryotes.
Macroscopic DNA curvature has been associated primarily with the presence of properly phased stretches of adenines . Nelson et al. (41) crystallized a dA6 region and solved the structure. Short (4–9 bp) runs of desoxyadenines repeated with the helix screw produce a global curvature of the DNA double helix . dA-tract-induced DNA curvature can be affected by other sequence elements (49,50) and also non-dAn sequence elements can cause DNA curvature (51–54). Curvature is directly related to the Fourier coefficient of the dinucleotide roll angle at a period equal to the helical repeat (55). One of the most accepted hypotheses suggests that distinctive properties of poly(dA):poly(dT) may be explained by having an unusual ‘B'-form’ structure (32–34) which is sufficiently different from B-form DNA that proteins recognize the difference (56–58). Models of DNA curvature have been proposed (34,36,52,59–63). Numerous examples of naturally occurring DNA curvature have been reported (64–75). Curved DNA has clearly identifiable properties. The curvature measured as retarded gel migration in PAGE is most prominently present at moderate temperatures and decreases at elevated temperatures (31,33,44,76). Ethidium bromide intercalates into the DNA and reduces the differences between B- and B'-form DNA, thereby strongly reducing the migration anomaly (77), in contrast to the behaviour of four-way junctions (78). Wu and Crothers (30) found that a fragment containing the curvature at or near its end migrates most rapidly in polyacrylamide gels whereas fragments in which the curvature is more centrally located migrate more slowly.
Here we show that parC is indeed curved. We determine the value of the migration anomaly in PAGE and measure its properties. We also compare the curvature of this bacterial centromeric DNA with that of eukaryotic centromeres and discuss the potential influence of curvature on centromere function.
MATERIALS AND METHODS
Those parts of the pKG330 plasmid (Figure 1) relevant for curvature analysis (Figure 2A) were verified by sequencing (MWG, Germany). Approximately 10 μg of pKG330 plasmid DNA carrying the parC sequence (see Figure 1) were digested with different restriction endonucleases (New England Biolabs, USA) simultaneously, resulting in different fragments (see Figure 2A) for curvature analysis. According to the time and temperature suggested by the manufacturer (mainly 120 min at 37°C), the DNA was extracted with phenol:chloroform:isoamyl alcohol (15:24:1; Sigma, Germany), precipitated and washed with 80% ethanol, air-dried, re-suspended in 20 μl of 5 mM Tris (pH 8.5) and mixed with 8 μl loading dye solution (60% glycerol, 60 mM EDTA, 0.09% bromphenol blue, 0.09% xylene cyanol FF; MBI Fermentas, Vilnius). The sample (6 μl) was loaded on a native 6% polyacrylamide gel (29:1 acrylamide:bisacrylamide; Bio-Rad, USA). Gels were pre-run for 3 h until current and temperature remained constant. Unless stated otherwise, electrophoreses was carried out in 1 x TBE (90 mM Tris–borate, 2 mM Na-EDTA, pH 8.0) at 150 V (8 mA) for 4 h (migration distance of bromphenol blue 14 cm). The temperature was mainly 24°C (room temperature), or alternatively 4°C (cold room) or 37°C (warm oven). After the runs, the gels were stained for 30 min in an aqueous solution of ethidium bromide (1 mg/l), followed by rinsing in water prior to documentation. The gels were illuminated by a 254 nm UV lamp (Chroma, Brattleboro), documented by a CCD camera system (Polaroid, USA) and gel images printed by a HP Laserjet 2100 M in the size of 26.5 x 18 cm for analysis. The 1 kb plus DNA ladder (Invitrogen, Carlsbad, USA) and the 1 kb DNA ladder (New England Biolabs, USA) served as marker fragments together with the intrinsic plasmid fragments resulting from the restriction digest running in the same lane as the parC fragment. For each gel, the migration of all fragments was determined and calibration curves were plotted using the marker fragments (logarithm of the number of base pairs versus distance migrated). The apparent size in the acrylamide gel relative to the calibration curve was determined for every anomaly migrating fragment.
Figure 1. Plasmid pKG330. Restriction sites and the position of parC are indicated.
Figure 2. (A) The sequence of parC (bold, 159 bp) and the framing vector sequences (both verified by sequencing) are displayed. The restriction sites within this DNA are indicated. These restrictions sites were used for fragment analysis. The 11 bp iterons are marked by arrows. (B) The mutated parC sequence (H-K) and the parC wild type (Wt) are displayed. The mutated base pairs are indicated by small circles. The mutations introduce two new single cutting sites HindIII and KpnI.
Construction of parC H-K mutant sequence
A mutant sequence of the parC sequence containing 4 bp exchanges was constructed by DNA cloning procedures using standard methods (79). For the introduction of two new single restriction sites, HindIII and KpnI, between the first (parC I) and the second (parC II) 5-fold iterons of parC, the DNA of plasmid pKG330 was amplified by two PCR reactions (Roche, Penzberg, Germany). In the first reaction, we amplified parC I using as upstream primer (parC f) 5'-GTAGTAGGTTGAGGCCGTTGAGCAC-3', which binds 230 bp upstream of parC, and as downstream primer (parC Kpn Hind r) 5'-GGTACCCCATTTCAACCATCAATCAAAGCTT-3', which binds between the two 5-fold iterons and introduces the two new restriction sites. In the second reaction we amplified parC II. The upstream primer (parC Hind Kpn f) 5'-AAGCTTTGATTGATGGTTGAAATGGGGTACC-3', a direct antisense repeat of primer (parC Kpn Hind r) also introduced the HindIII and KpnI sites, whereas the downstream primer (parC Nco r) 5'- CCATGGAAATGTTGAATACTCATACTCTTCC-3' introduced a NcoI site 360 bp downstream of parC. The blunt end PCR products were cloned into pCR 4Blunt-TOPO (Invitrogen, Carlsbad, CA, USA). The resulting vectors pTOPOparC I and pTOPOparC II, carried parC I and parC II respectively. After verification of the base exchanges by sequencing, both vectors were digested by HindIII–NcoI. The 456 bp fragment of pTOPOparC II containing parC II was cloned into the 2839 bp fragment of pTOPOparC I resulting in pTOPOparC H-K. Subsequently pKG330 and pTOPOparC H-K were both digested with BamHI–EcoRI. The 167 bp fragment of pTOPOparC H-K was cloned into the 4120 bp fragment of pKG330 resulting in pKG330 H-K carrying mutant parC with the new restriction sites (parC H-K). The exchange of the four bases was confirmed by sequencing (MWG, Ebersbach, Germany). The base pair exchanges are introduced at the sites 192, 194, 219 and 220 bp (see Figure 2). This mutant DNA was digested and analysed as described above for the wild-type sequence.
Computational calculation of curvature
The prediction of DNA curvature was made by means of our CURVATURE program. This program calculates a three-dimensional path of a DNA molecule and estimates the curvature of the axis path (80). The CURVATURE algorithm is based on the stepwise calculation of geometric transformations according to the set of dinucleotide wedge angles (52,81). A curvature value at a position i corresponds to a curvature of the arc approximating to the predicted DNA path. The DNA curvature was measured in ‘DNA curvature units’ (cu) introduced by Trifonov and Ulanovsky (82) and in ‘degrees per base pair’. Taking 146 bp of DNA wrapping around a histone octamer of 1.65 turns as a basis for conversion, the curvature unit can be related to other measures. For example, having 88.5 bp in the one full nucleosomal turn we find that 1 cu corresponds to 42.7 degrees per helical turn or 4.1 degrees per base pair. The program is available upon request from A.B. at bolshoy@research.haifa.ac.il.
RESULTS
The parC fragment is 159 bp in length and is organized in two regions each containing a 5-fold repeated sequence motif of 11 bp length with a central region of 39 bp containing the par promoter (12). The consensus sequence of the 10 repeats (iterons), dAAAAAAAACCC , contains longer dA-tracts with a repeat length of 11 bp, thus phased slightly distinct from the DNA helix screw . This suggested to us that the parC fragment might potentially be curved and thus might show anomalous gel migration. We analysed the parC fragment in gels under various conditions.
The parC fragment was cloned into the plasmid pKG330 . Those parts of the plasmid relevant for curvature analysis were verified by sequencing and are displayed in Figure 2A. Various DNA fragments were constructed by restriction digest using the endonucleases indicated in Figure 2A. These fragments are listed in Table 1. The migration of these fragments relative to a series of marker fragments in different lanes as well as to plasmid fragments in the same lane was analysed in polyacrylamide (6, 8 and 11%) and agarose gels under different buffer and temperature conditions. A typical polyacrylamide gel is displayed in Figure 3. The apparent size of the fragments was determined relative to normally migrating marker fragments. The quotient ‘apparent divided by known sequence length’ is termed ‘k-factor’ and listed in Table 1 for the different conditions analysed. Several k-factor values were measured repeatedly. We deduce an experimental error of the measurements for the k-factors of ± 0.05.
Table 1. List of 10 analysed fragments with k-factors
Figure 3. Typical 8% PAGE run with 1 x TBE at 25°C. M, marker fragments. Lane 1: fragment 3 (parC; AatII/BanII), lane 2: fragment 3 (parC H-K; AatII/BanII), lane 3: fragment 1 (parC; BspHI), lane 4: fragment 1 (parC H-K; BspHI), lane 6: fragment 4 (parC; HhaI), lane 7: fragment 4 (parC H-K; HhaI), lane 8: fragment 8 (parC; BspHI/EcoRI), lane 9: fragment 7 (parC; BamHI/BstBI). Numbering of the fragments is according to Table 1. The length of the marker fragments in the two lanes M is given at the right side of the gel. The anomaly migrating fragments containing parC are indicated by asterisks. If the anomaly migrating fragments containing parC of lanes 1–4 would migrate normally (i.e. according to their sequence length), their position in the gel would be at the site indicated by a broken line at the left side of the gel.
Most of the plasmid DNA fragments external to the parC, migrated normally in the polyacrylamide gels. In particular, those fragments framing the parC sequence, migrated normally: SalI/BamHI (276 bp length) upstream and EcoRI/BspHI (189 bp length) downstream had k-factors of 0.98 and 1.02, respectively. One fragment, however, which contains the last 120 bp of the origin of replication of the plasmid (HhaI fragment of 270 bp length), migrated slower with a k-factor of 1.09 (8% PAGE, 1x TBE, 25°C). Being curved is a property of plasmid replication origins (73) as well as the SV40 origin (75).
The migration anomaly in polyacrylamide gels hardly varies for the salt and buffer conditions analysed here (Table 1). For parC framed by 120 and 191 bp on either ends (fragment 1; BspHI digest), at 25°C in 1x TBE (i.e. 90 mM Tris–borate, 2 mM Na-EDTA, pH 8.0) we measured a k-factor of 1.71 in 8% PAGE. This changed to 1.76 at 0.5x TBE and 1.68 at physiological conditions (10 mM MgCl2, 170 mM NaCl in the presence of 1x TBE). Other parC containing fragments confirmed this invariance (fragments 3, 4 and 7; see Table 1); e.g. for fragment 7 the k-factor was 1.52 at 1x TBE, which changed to 1.49 at 0.5x TBE as well as physiological conditions.
On the other hand, the migration anomaly depends on the polyacrylamide concentration (Table 1). For fragment 1 (BspHI digest) the k-factor increased from 1.59 at 6% acrylamide concentration to 1.71 at 8% and 1.94 at 11% (see Figure 4B). Correspondingly, for fragment 4 (HhaI digest) the k-factor increased from 1.44 at 6% to 1.59 at 8% and 1.82 at 11% (see Figure 4B). In the measured polyacrylamide concentration range, the k-factor increase is linear.
Figure 4. (A) Temperature dependence of the k-factors of fragments 2 and 6 (see Table 1). Fragment 2 contains framing vector sequences and shows a larger k-factor while fragment 6 is parC, almost without framing vector sequences. (B) Dependence of the k-factors of fragments 1 and 4 (see Table 1) on polyacrylamide concentration.
We observed a strong decrease of the migration anomaly with increasing temperature (Table 1). For fragment 1, the k-factor decreased from 2.45 at 4°C to 1.71 at 25°C and 1.27 at 50°C (see Figure 4A). For parC nearly without framing sequences (6 and 2 bp, BamHI/EcoRI double digest; fragment 6), the k-factor decreased from 1.53 at 4°C to 1.29 at 25°C and 1.03 at 50°C (see Figure 4A). Extrapolation suggests that the k-factor decreases to 1.00 (normal migration) at a temperature of 60–70°C (with a slightly smaller value for fragment 6), as observed for other curved sequences (31,33,44,76).
For controls we measured the migration anomaly of several parC containing fragments in agarose gels as well as in the presence of ethidium bromide in the gel buffer. In 2% agarose gels, the migration anomaly of the analysed fragments 1–6 was normal (k-factors 1.00; see Table 1). In the presence of 1 mg/l ethidium bromide, the migration anomaly was considerably reduced . For most analysed fragments, the migration became normal (k-factors 1.00; see Table 1) in the presence of 3 mg/l ethidium bromide. This behaviour is known for curved DNA fragments (28,77).
By the exchange of four bases between the two 5-fold repeated iterons within the parC sequence (at the sites 192, 194, 219 and 220 bp; see Figure 2A), the parC sequence dAATAATCATTGATTGATGGTTGAAATGGGGTAAACTTGA is modified to dAATAAGCTTTGATTGATGGTTGAAATGGGGTACCCTTGA (see Figure 2B) introducing two new single cutting restriction sites. When analysing this modified parC sequence, we measured a considerably reduced migration anomaly. For fragment 1 (BspHI digest) the k-factor was reduced from 1.71 to 1.50 (same length of DNA; 8% PAGE at 1x TBE at 25°C). A similar reduction was observed for fragments 2–6 (see Table 1). The temperature dependence of the migration anomaly of this modified parC sequence was measured to be similar in nature; however, the slope of the k-factor versus temperature plot was slightly reduced (data not shown). As for the unmodified parC sequence, extrapolation suggests that the k-factor decreases to 1.00 (normal migration) at a temperature of 60°C for the modified sequence as well. The dependence of the migration anomaly on the polyacrylamide concentration was similar to the unmodified parC at 6 and 8% (same slope of k-factor versus percentage of acrylamide; data not shown); however, in contrast to the unmodified parC, hardly increased further for 11%. Changing the salt and buffer conditions had little or no effect on the migration anomaly (compare fragments 1, 3 and 4 in Table 1), as for the unmodified sequence. In agarose gels as well as in the presence of 3 mg/l ethidium bromide, the modified parC migrated normally.
These base pair exchanges create two new single cutting sites within the parC sequence, KpnI and HindIII, which allow studying the first and the second 5-fold repeated iterons separately. Using the newly introduced restriction sites, we constructed additional fragments (see Table 1) the migration of which was analysed in gels as well. DNA fragments containing the first 5-fold repeated iteron sequence with framing sequences at the 5'-end only showed rather small migration anomaly. When the up-stream HindIII site was used with framing sequences of 41 (HhaI/HindIII double digest) and 120 bp (BspHI/HindIII double digest) lengths, k-factors of 1.10 and 1.04 were measured, respectively (at 1x TBE at 25°C in 8% PAGE; data not shown). In parallel, when the down-stream KpnI site is used with framing sequences of 41 (HhaI/KpnI double digest), 120 (BspHI/KpnI double digest) and 292 bp (SalI/KpnI double digest) lengths, k-factors of 1.10, 1.04 and 1.09 were measured, respectively (at 1x TBE at 25°C in 8% PAGE; data not shown). Fragments containing the second 5-fold repeated iteron sequence with framing sequences at the 3'-end only showed larger migration anomaly compared to the fragments containing the first parC part, however still small compared to the migration anomaly of the full parC. Partly this might be due to the positioning of the curved sequence within the analysed fragment at the 5' and 3'end, respectively. Maximal migration anomaly is observed when the curved sequence is located in the middle of the fragment (30). When the up-stream HindIII site was used with 3'-framing sequences of 191 (HindIII/BspHI double digest) and 246 bp (HindIII/HhaI double digest) lengths, k-factors of 1.15 and 1.19 were measured, respectively (at 1x TBE at 25°C in 8% PAGE; data not shown). In parallel, when the down-stream KpnI site was used with framing sequences of 191 (KpnI/BspHI double digest), 246 (KpnI/HhaI double digest) and 894 bp (KpnI/PstI double digest) lengths, k-factors of 1.16, 1.18 and 1.07 were measured, respectively (at 1x TBE at 25°C in 8% PAGE; data not shown).
Using the newly introduced restriction sites, we re-cloned the parC 5-fold repeated iterons. We constructed vectors in which either the second or the first part of parC was deleted. We thus could analyse fragments in which either the first (fragment 9, SpeI/SnaBI double digest) or the last (fragment 10, AatII/BsrGI double digest, see Table 1) 5-fold repeated iteron sequence was positioned in the middle of the fragment. In 8% PAGE in 1x TBE at 25°C both fragments showed the same migration anomaly which increased to about the same extent at low temperatures (4°C, see Table 1).
Predicted DNA curvature
The software CURVATURE (80) was applied to calculate the trajectories of the wild-type as well as the mutant parC sequence. The juxtaposed calculated DNA paths are presented in Figure 5A. The wedge model (36) with the angles of Bolshoy et al. (52) and Kabsch et al. (81) predicts the planarity of the fragment. Compared to wild type, the modified parC sequence displayed a considerably reduced gel mobility. CURVATURE identified changes in the DNA trajectory of the modified parC fragment due to the four base exchanges: the modified fragment is less curved than the original parC fragment. The two base pair exchanges at 219 and 220 bp result in a curvature reduction at this site of 17 degrees (see Figure 5A). Interestingly, both fragments are rather planar although the iteron repeat length is 11 bp (see Figure 2A), not perfectly matching the helical repeat (10.5 bp). However, the distance between the two sets of iterons (39 bp) is slightly less than an integral number of helical turns so that both effects might compensate. The end-to-end distances for the fragments are 43.9 nm and 39.8 nm, the smaller distance corresponding to the more curved wild-type fragment. The CURVATURE program can be used also to map the DNA curvature along the sequence. In Figure 5B we show the juxtaposed curvature maps of the mutant and wild-type fragments with the window- size of 21 bp. The program predicts that the first pair of modifications (at positions 192 and 194, see Figure 2) should not influence the gel retardation, while the replacement of the dinucleotide dAA by the dinucleotide dCC at positions 219 and 220 (see Figure 2) is predicted to cause a dramatic reduction of the gel migration anomaly.
Figure 5. Juxtaposition of predicted DNA curvature for wild-type and modified sequences parC. The wild type and modified sequences were applied to the software CURVATURE (80) with the specified parameters (52,81). Both fragments are 167 bp long. The modifications were made at positions 77, 79, 104 and 105 relative to this fragment length (corresponding to sites 192, 194, 219 and 220 in the numbering frame of Figure 2). (A) Two-dimensional representation of the DNA paths. Two projections of the DNA paths are presented on a plane of the maximal observed curvature (a) and on a reciprocal plane (b). To display the degree of curvature of a fragment, the calculated coordinates of the phosphates and base pair centers are projected on that plane, for which the mean square distance from all the points is minimal. The line connecting the base pair centers presents the helical axes of the original (wild type: green continued by red) and modified fragment (mutant: green). The phosphates are shown in light and dark blue (mutant) switching to light brown and green for the wild type. The second projection (right) shows the planarity of the fragments. The two base pair exchanges at 219 and 220 bp result in a curvature reduction at this site of 17 degrees. (B) DNA curvature maps versus sequence with window-size of 21 bp. The overall DNA shapes are characterized curvatures of a sliding arc of 21 bp of length approximating to the paths of the axes of the given DNA fragments. The curvature map (given in ‘cu units’ on the left axis and ‘degrees per bp’ on the right axis) for wild-type parC is plotted as a solid line and for the mutant by a dashed line. The two vertical dotted lines indicate the positions of the base pair modifications.
DISCUSSION
parC is the centromere of the E.coli plasmid R1. Here we analysed the intrinsic sequence-directed curvature of parC both by its migration anomaly in polyacrylamide gels and by applying the wedge-model based software CURVATURE. The experimentally observed gel migration anomaly indicated as ‘k-factor’ (i.e. an apparent fragment length in the gel divided by correct sequence length) of the parC sequence is 1.7 (see Table 1) and thus smaller than that of another naturally occurring strongly curved DNA, the whole kinetoplast DNA fragment (29,31), which displayed k-factors even >4. However, parC (159 bp) is much shorter than the kinetoplast fragment (414 bp). When the parC curvature is compared with that of parts of the kinetoplast fragment of similar length (e.g. fragments of the lengths 187 and 262 bp) , similar k-factors were obtained indicating a similar degree of curvature. The parC curvature is also similar to that of constructed fragments of similar length with 8-fold repeats of the sequence dGACAAAACTC within framing non-curved sequences (32,33). Thus, for a naturally occurring sequence, the parC curvature is large.
The properties of the parC curvature agree with those of other curved dA-tract sequences. Just like parC, the 187 bp kinetoplast fragment as well as the constructed fragment showed hardly any dependency of the k-factor on NaCl or MgCl2 concentration within the measured range (see Table 1) (31,33). Also the temperature dependence of the k-factors of these DNA fragments is very similar, extrapolating to 60°C for normal migration (k-factor = 1.00) in all three cases (31,33). Furthermore, these fragments behave normally in agarose gels and show a strongly reduced migration anomaly in the presence of ethidium bromide (see Table 1). This similarity in properties of their gel migration anomaly indicates that the phenomenon is of the same origin.
parC contains two regions of 5-fold repeated iterons separated by 39 bp (see Figure 2A). We modified 4 bp within this intermediate sequence creating two new single cutting sites (see Figure 2B). This modified sequence of identical length showed considerably smaller curvature measured in PAGE (see Table 1) and calculated by CURVATURE (see Figure 5A). This indicates that the two repeat regions are not independent curved parts of parC but that the overall parC curvature is a property of the whole fragment. The intermediate sequence contributes not only by its number of base pairs (which also determines the phasing relation between the first and the second 5-fold repeat) but also to a considerable amount by its sequence.
The two new cutting sites allowed us to analyse the curvature of the first and the second repeat independently. When analysing the first repeat without 3'-framing sequences and the second repeat without 5'-framing sequences, we measured a smaller curvature for the first repeat. However, when placing the repeats in the centre of fragments with non-curved framing sequences, we found similar k-factors for both (Table 1). This discrepancy might be explained by the different arrangement of curved sequences within the repeats, maybe placing curved elements in the first case more to the end of the fragment where their influence on the migration anomaly is reduced (30,31).
Curvature seems to be a general phenomenon of eukaryotic centromeric DNA. Mostly, the degree of curvature is small (k-factors of up to 1.20) and only in some cases the k-factors are larger (26). All 16 centromere sequences of Saccharomyces cerevisiae show migration anomaly in polyacrylamide gels (27,28); the largest k-factor value was measured for CEN14 as 1.24. The curvature of most eukaryotic centromere DNA as well as of the 16 yeast sequences is considerably smaller than the curvature of the bacterial centromeric sequence parC analysed here.
Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate whether curvature is a general property of centromeres. The centromeres of higher eukaryotes are organised as multi-fold repeats of -satellite DNA, many of these repeats showing curvature (26). In many eukaryotes, the repeat lengths of the satellite DNA are similar to the repeat lengths of nucleosomes in chromatin or to multimers of this length (26). The satellite curvature might thus serve as a nucleosome positioning signal. Experiments reconstituting nucleosomes onto satellite DNAs confirmed that view (86,87). The curvature of the parC sequence found here might support the multiple parR binding to parC resulting in a globular folded shape of the kinetochore-like DNA protein complex also in bacteria, may be even similar in size and nature to a nucleosome.
ACKNOWLEDGEMENTS
We thank S. Pfeiffer and S. Hosid for excellent technical support.
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