(Received for publication, September 20, 1994; and in revised form, December 6, 1994)
From the
Binding of the archaebacterial histone-like protein MC1 to DNA minicircles has been examined by gel retardation and electron microscopy. MC1 preferentially binds to a 207-base pair relaxed DNA minicircle as compared with the linear fragment. Random binding is observed at very low ionic strength, and a slight increase in salt concentration highly favors the formation of a complex that corresponds to the binding of two MC1 molecules per DNA ring. Measurements of dissociation rates show that this complex is remarkably stable, and electron microscopy reveals that it is characterized by two diametrically opposed kinks. These results are discussed in regard to the mechanisms by which MC1 affects DNA structure.
Numerous chromosomal proteins have been found associated with prokaryotic nucleoids. These abundant, small, and usually basic proteins are generally referred to as histone-like proteins. Histone-like proteins compact DNA in vitro and can play essential roles in different cellular processes, but unlike eukaryotic histones, these proteins have not been shown to wrap DNA into stable, regular, and globular structures(1, 2, 3, 4) .
MC1 is
the most abundant chromosomal protein present in various species of Methanosarcinaceae(5) . In the Methanosarcina sp.CHTI 55 strain, MC1 is a 93-amino acid polypeptide, exhibiting
a marked hydrophillic character provided by a large number of basic and
acid residues (24 and 14, respectively) and no hydrophobic
domain(6) . With respect to the characteristic of its primary
and secondary structure, and particularly the distribution of basic
residues and the low helix content, MC1 differs from eukaryotic
histones and from eubacterial and other archaebacterial histone-like
proteins (for a review, see (7) ).
MC1 protein
preferentially binds double-stranded DNA and protects DNA against
thermal denaturation(8, 9) . On linear DNA fragments,
MC1 binds to DNA as a monomer. At a very high protein/DNA ratio, MC1
covers DNA fragments, with a site size of about 11 bp; ()this binding is non-cooperative(10) . It has been
shown by photochemical cross-linking that its carboxyl-terminal region
contains the DNA binding sequence(11) . Although MC1, like all
histone-like proteins, is not expected to exhibit absolute specificity
for one particular sequence, we have recently identified a preferential
binding sequence in an open reading frame of unknown
function(12) .
A quite large DNA conformational change induced by the binding of MC1 has been detected by cyclization experiments on short DNA fragments. Indeed, as for protein HU, MC1 is able to promote the cyclization by T4 DNA ligase of DNA fragments of about 100 bp(13, 14) .
To further analyze the modifications of conformation and/or topological constraints of DNA induced by MC1, we investigated its binding to a 207-bp DNA minicircle. Indeed, DNA minicircles are a simple system providing valuable information on the structure of protein-DNA complexes(15, 16, 17, 18) . We show that a slight increase in salt concentration highly favors the formation of a stable complex that corresponds to the binding of two MC1 molecules per DNA minicircle. Furthermore, using gel retardation, we show that MC1 protein is able to induce at least two levels of DNA compaction on this small DNA ring. These different levels of compaction correspond to different configurations of the minicircles, each bound protein inducing a sharp bend of the DNA, as observed by electron microscopy.
where P(X) is the percentage of complex bearing X-bound protein, and <X> is the average number of bound protein(10) .
Fig. 1A shows the titration of the 207-bp DNA minicircles with increasing protein concentrations in TE buffer. Most of the complexes migrate faster than the free DNA. Only complexes formed with the higher amount of MC1 are not accelerated, and they migrate almost with the free DNA (laneG). MC1 binding should only lead to DNA retardation by increasing the frictional coefficient and by partial charge neutralization. Moreover, this unusual pattern is different from that obtained with a linear fragment, where all complexes migrate less rapidly than free DNA (Fig. 1B and (10) ). Therefore, the accelerated migration of the DNA minicircles in the gel must be due to a drastic DNA conformational change inducing a reduction of the hydrodynamic volume of the minicircles and therefore leading to a compaction of the DNA.
Figure 1: A, titration of the circular 207-bp DNA fragment with MC1 protein. The protein concentrations (nM), from lanesA to G, were 0, 4, 8, 16, 36, 65, and 130, and the DNA concentration was 1.3 nM. Complexes were formed in TE. The positions of the free minicircles (0) and of complexes C1-C12 are schematized. B, titration of the linear 207-bp DNA fragment with MC1 protein. The binding conditions are those described in (13) . Complexes were formed in TE buffer containing 50 mM NaCl. The protein concentrations, from laneH to M, were 0, 5 nM, 36 nM, 0.4 µM, 0.8 µM, and 1.2 µM, and the DNA concentration was 200 nM. Positions of the free DNA fragments (0) and of complexes C1-C8 are schematized.
We identified the various complexes by their order of appearance on the gel. The first complex (C1) and more explicitly the second one (C2) are distinctly accelerated with respect to the free DNA (Fig. 1A, lanesA-D). Complexes are then regularly retarded with respect to complex C2. The third complex (C3, laneE) thus migrates faster than the fourth one (C4, laneF). Up to 12 types of complexes are visible on the gel. A quantitative analysis of the titration data with the circular and linear fragments is shown in Fig. 2. We scanned the autoradiographies and plotted the relative amount of each complex C1-C4 (expressed in DNA %) against the average number of bound protein per circular or linear DNA fragment. We then compared these experimental values with those calculated assuming a Poisson distribution. In the part of the titration, the relative amount of each complex increases progressively from C1 to C4. The same distribution of the complexes occurs with linear and circular DNA fragments, and it fits that calculated for a random process. Different conclusions can be drawn. First, MC1 binding to the minicircles is a random process under these conditions. No given complex is favored; the binding is both sequence and conformation independent. Furthermore, no cooperativity is detectable: the binding of one MC1 molecule does not seem to enhance the binding of an additional one. Second, in a previous work, we have shown that MC1 is bound to linear DNA fragments as a monomer(10) . The similarity between the appearance of the complexes formed with the linear and circular DNA fragments strongly suggests that, as with a linear fragment, C1 corresponds to the binding of one MC1 monomer, C2, to the binding of two MC1 monomers and so forth.
Figure 2:
Appearance of the complexes with linear
and circular 207-bp DNA fragments during the titration of the DNA by
increasing MC1 concentrations. The % of the complexes C1-C4
(expressed in DNA %) were plotted against the average number of bound
protein per DNA fragment (, DNA minicircles; *, linear DNA
fragments). For the DNA minicircles, the values were obtained from the
scanning of the gel of lanesA-E in Fig. 1A. For the linear DNA, the values were derived
from several retardation gel experiments obtained under the same
conditions as for Fig. 1B. The curves are
calculated for a random process assuming a Poisson
distribution.
Figure 3:
Effect of increasing ionic strength on MC1
binding. Complexes were formed in TE buffer (- slots) and then
adjusted to 25 mM NaCl, 8 mM MgCl (+
slots). The protein concentrations (nM), from lanes 0-5,
were 0, 6, 13, 26, 65, and 130, and the DNA concentration was 1.3
nM.
The differential effects of NaCl and
MgCl on the binding are shown in Fig. 4.
Mg
is more effective than Na
in
shifting from a random binding to complex C2 promotion mode, since
similar results are obtained with either 4 mM MgCl
or 75 mM NaCl.
Figure 4:
Effect of salt addition on the complexes.
Complexes were formed in TE buffer, and then MgCl (A) or NaCl (B) was added to obtain the salt
concentration indicated above each lane. The
concentration of DNA was 1.3 nM, and that of MC1 was 6.8
nM.
Figure 5: Competition between linear and circular DNA fragments for MC1 binding. Equimolar amounts of linear and circular fragments (1.3 nM for each species) were titrated with the following total concentrations (nM) of MC1 in lanes0-4: 0, 13, 26, 65, and 130 nM, respectively. LanesC and L show the mobilities of the complexes with the circular and the linear fragment, respectively. Binding buffer was 80 mM NaCl in TE.
The interaction of MC1 with minicircles was also analyzed by kinetic experiments. The rate of dissociation of preformed complexes was studied by adding unlabeled plasmid DNA. Due to the large excess of plasmid versus DNA minicircles, the majority of any released MC1 protein should bind to this DNA rather than rebind to the minicircles. The dissociation was stopped by loading the samples on the gel, and the DNA distribution was followed up to 5 h (Fig. 6A). The amount of complex C2 decreases, whereas the amount of free DNA increases. Since no increase in complex C1 formation arises from complex C2, it is likely that the two bound MC1 molecules from complex C2 dissociate in the same time. The gel was scanned to measure the amount of bound DNA. The result is given in Fig. 6B, and the deduced half-life is 130 min. When the same experiment was done with a lower MC1 to DNA ratio, that is in conditions where the complex C1 is visible, a half-life of only 48 min was obtained for this complex (not shown). Furthermore, we observed that higher order complexes (C3, C4 . . . ) and all the complexes formed with the linear fragment (C1, C2, C3 . . . ) are immediately dissociated, showing that their half-lives are shorter than 1 min (not shown). These results are consistent with the observations that, first, the complexes C3, C4 . . . are dissociated by a slight salt increase (Fig. 3) and, second, that MC1 preferentially binds to the DNA minicircles (Fig. 5). Clearly, the complex C1 and especially the complex C2 formed on DNA minicircles are remarkably more stable than the other MC1-minicircle complexes and that all the complexes formed with the linear fragment.
Figure 6: Dissociation of MC1 from DNA minicircles. A, gel retardation assay. The DNA was first incubated in the presence of MC1 for 10 min (lane0). The incubation was continued for the indicated time in the presence of a 50,000 molar excess of unlabeled supercoiled plasmid DNA. Concentration was 170 nM for MC1 and 13 pM for the minicircles. Binding buffer was 80 mM NaCl in TE. B, rate of dissociation of complexes C2. The percentage of complexes C2 (logarithmic scale) was determined densitometrically and plotted versus the incubation time.
Figure 7: Visualization of the complexes by annular dark field electron microscopy in TE buffer. DNA minicircles in the absence of protein (a) and in the presence of MC1 at a protein/DNA minicircle r = 7 (b). Different conformations of the minicircles with sharp DNA kinks can be observed. The bar represents 50 nm.
In 80 mM NaCl, conditions favoring complex C2, the difference between controls (Fig. 8a) and MC1-DNA complexes (Fig. 8, b-h) is far more evident than in TE; most minicircles complexed with MC1 are spindle shaped with two diametrically opposed kinks, thus leading the tapered shape that makes the molecule more compact. No loop induced by protein-protein interactions could be found.
Figure 8: Visualization of the complexes in 80 mM NaCl. DNA minicircles in the presence of MC1 at a protein/DNA minicircle r = 7 (b-h) are compared with the control without protein (a). The most frequent configurations correspond to the complex C2 with two kinks diametrically opposed (b). Typical minicircle configurations are shown at higher magnification. Some DNA minicircles appear in plane (c, f), whereas others appear slightly supercoiled (d, e, h). The bar represents 100 (a, b) and 25 nm (c-h).
The different types of configuration of the minicircles observed reveal different aspects of the kinks. Some indicate a single (Fig. 8c) or two sharp bends (Fig. 8f), the DNA minicircle appearing to be in plane. Others show sharp bends associated with a torsional stress inducing a local twisting of the DNA (Fig. 8, d, e, and h). This torsional stress is always localized around the kink, and only a few figure 8 shapes can be observed (<0.1%). Measurement of the DNA molecules' contour length indicates no significant difference between free and MC1complexed minicircles. This invalidates the hypothesis of a DNA wrapping of more than one turn around MC1. The most typical selected molecules of Fig. 8, c-h, show that a strict discrimination on individual minicircles between C1 and C2 types is not possible, owing to the flexibility of the DNA molecules that present non-discrete shapes so that no strict classification can be made.
In archaebacteria as in eubacteria, the organization of the DNA is due to two classes of proteins: topoisomerases, which induce DNA torsional stress, and histone-like proteins whose role in DNA compaction is less well characterized. In the methanogenes of the Methanosarcinaceae family, the histone-like protein MC1 is both highly conserved and abundant (1 protein/150 bp)(26) . Our aim was to understand the effect of this protein on DNA conformation and whether it can contribute to DNA packaging. In this paper, the interaction between MC1 and a 207-bp relaxed DNA minicircle was investigated. The complexes were first identified at low ionic strength, and binding was shown to be sequence and conformation independent. We then studied binding at higher ionic strength, where the formation of complex C2 was found to be highly favored. Although the stoichiometry of the complexes is not directly measured, the quasi-identical order of appearance of the complexes with the linear and circular fragments (Fig. 2) strongly suggests that, as in the case of linear fragments, the complex C2 arises from the binding of two MC1 monomers per minicircle.
The fact that MC1 binding increases the electrophoretic mobility of DNA shows that MC1 compacts the DNA minicircles. This compaction is particularly significant for the complex C2. It is likely that only two levels of DNA compaction could be induced, since the binding of an additional MC1 molecule to complex C2 regularly retards the DNA migration. Similar behaviors of DNA minicircles on retardation gels have been previously reported for two different protein-induced DNA conformational changes. First, in the case of the Lac and Ara repressors, when the simultaneous binding of one protein to two DNA sites induces the DNA molecule to form a loop(16, 27) , and second, in the case of chromatin, where DNA wraps around proteins(15) . Visualization of MC1-DNA complexes by EM excludes these two possibilities and shows that a tight DNA bending is responsible for the compaction of the minicircles. It is likely that MC1 preferentially binds to the minicircles since the DNA is already curved. For a 207-bp minicircle, the mean bending is 1.7° per base pair. One can therefore postulate that since MC1 binding bends the DNA, a prior bending of the double helix must decrease the energy required to change the DNA conformation and consequently would favor protein binding.
One of the most important results of this study is that the complex C2 is highly favored with minicircles and not with linear fragments(10) . This difference probably results from the topological constraints existing on circular DNA and most particularly on short minicircles. It has been proposed that bent DNA converts Twist (Tw) into Writhe (Wr) in closed DNA. In the case of two intrinsic bends in phase and diametrically opposite, each bend should induce a local discontinuity of the twist and a conversion of this Tw into Wr(28) . The characteristics of the local variations of DNA conformation depend upon the contribution of the bending in the plane, resulting from modifications of the wedge angle and the torsional contribution that results from a modification of twist angles. The different kink configurations selected in Fig. 8(c-h) suggest that MC1 induces both a bending and a torsional stress in the DNA. We propose therefore that the binding of the first MC1 molecule induces at the opposite side of the minicircle a configuration that favors the binding of the second MC1 molecule required to obtain a stable complex C2. This configuration probably corresponds to an increase of the local curvature and/or a modification of the torsional constraints of the DNA. Furthermore, the complex C2 is probably so tapered and stable that the binding of additional MC1 molecules is energetically unfavored.
At least three
non-exclusive factors could explain that the complex C2 is only favored
in the presence of salt (at least 4 mM MgCl or 75
mM NaCl). First, at low ionic strength, the binding constants
are very large as demonstrated by fluorescence experiments (8) and, consequently, the lifetimes of the various complexes
may be too long to allow the adjustment of equilibrium. Indeed, we
observed that the half-lives of all of the complexes formed in TE
buffer are longer than 4 h (not shown). Second, the conformation of the
protein may change since we observed that the CD spectra of the protein
in 10 mM Tris and in 10 mM Tris plus 1 mM MgCl
are different. (
)Finally, a decrease
in the DNA chain stiffness can be expected. The DNA stiffness can be
explained, at least for a large part, by electrostatic repulsions
between negative charges of the phosphates. When the ionic strength is
increased, phosphate charges are effectively neutralized, and DNA
flexibility increases. Reports in agreement with this point show that
the persistence length of DNA strongly decreases upon addition of NaCl
in the range of 0-0.1 M NaCl in the absence of
Mg
(29, 30) .
We previously showed that upon polymerization, MC1 binding favors cyclization of short linear DNA fragments by T4 DNA ligase. During this process, negatively supercoiled minicircles are formed. With a 207-bp DNA fragment, a -1 and -2 variation in the linking number of the DNA was observed, depending of the amount of MC1 complexed to the fragment(14) . Different mechanisms can be responsible for a reduction of the linking number of a circular DNA by a protein in the presence of ligase or topoisomerase. EM visualization of the complexes allows us to exclude two of those mechanisms: DNA wrapping around MC1 as in the case of HU (1, 31) and DNA looping as in the case of HMG proteins(32) . Since MC1 protects DNA against thermal denaturation(9) , a local strand separation is unlikely, and we can postulate that an untwisting of the double helix is certainly responsible for the reduction of the linking number of the DNA. In addition, this hypothesis is consistent with the configurations of the MC1 minicircle complexes shown in Fig. 8, d, e, and h, where a local twisting of the DNA can be seen.
Bacterial chromatin is considered to be a dynamic structure(1, 2) where histone-like proteins dissociate rapidly from DNA(31, 33) . Our results show that, depending on DNA conformation, remarkably stable complexes can be formed with histone-like proteins. In the same way, it has been recently shown that HU binds strongly to particular DNA structures containing sharp angles(34, 35) .
The fact that MC1 on the one hand can compact DNA and, on the other hand, can bind DNA non-randomly, is consistent with the importance of MC1 as a factor of chromosomal DNA organization. An attractive hypothesis would be that our results reveal a kind of phasing of MC1 binding to DNA. Such a phasing would not be due to signals on DNA but to the dramatic DNA conformational changes induced by the protein binding.