From the Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132-3201
Received for publication, March 17, 2003 , and in revised form, April 23, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The crystal structure of the Pyrococcus furiosus Rad50 (an SMC family member) N-terminal and C-terminal domains has been solved in complex with ATP (16). Solution-phase analysis established that isolated Rad50 N and C termini form a stable heterodimeric NC domain. Two NC domains associate in the presence of ATP to form an active ATPase, and in full-length Rad50 the coiled-coils may help to bring the N and C termini of a single protomer together to form one-half of the four-part active site (20). Hinge-hinge interactions bring the second NC half-site into register to promote the formation of two active sites in trans and sandwich two ATP molecules within the NaCa/NbCb tetramer. Domain analysis suggests that the architecture of the heterodimeric condensin and cohesin SMCs is essentially the same as that predicted for Rad50 and their bacterial SMC counterparts (18, 19).1
As the details of SMC structure have come into focus, speculative models of DNA binding based on their architecture have emerged as well. The "embrace" model was proposed for chromatin binding by the cohesin (19) and depicts two chromatin 30-nm fibers, one from either sister chromatid, as bound between the coils of an Smc1/3 heterodimer. This binding mode is in part consistent with a model based on electron spectroscopic imaging of the Xenopus condensin XCAP-C/E heterodimer (Smc2/4 homologs) in complex with relaxed plasmid DNA (21). Appreciating the resolution limits of this technique, the images appear to show the DNA duplex wrapping twice in a right-handed sense around the NC/NC globular head domains. The image-based model places two gyres of DNA, one from each encircled NC dimer, between the coiled-coils. These and other models of DNA binding await experimental validation, and few mechanistic details of DNA/chromatin binding by either the cohesin or condensin SMCs exist.
To date, the Xenopus complexes are the biochemically best-characterized eukaryotic condensins (9, 12, 22, 23). Two separable SMC-containing complexes have been purified by immuno affinity; they are the five-protein 13 S condensin of XCAP-C, -E, -D2, -G, and -H and the 8 S XCAP-C/E heterodimer. In add-back experiments only the 13 S condensin could rescue in vitro condensation of sperm chromatin in SMC-depleted egg extracts. The 13 S condensin is required for both the establishment and maintenance of chromosome condensation (9), and both activities require ATP and mitotic activation of the complex. Condensin-dependent changes to the geometry of plasmid DNAs were probed in topology-trapping experiments (2224), and two ATP-dependent activities unique to the mitotic 13 S condensin were discovered. At high mole ratios the 13 S condensin induced geometries in circular DNAs, which favored the formation of positive knots in the presence of a type II topoisomerase, and trapped positive supercoils in the presence of a type I topoisomerase (23). The positive loop solenoid model was developed to explain how trapping of positive supercoils in relaxed circular DNA would promote positive knotting in nicked circular substrates. Moreover, these results suggested that the condensin must act to change the global writhe of a DNA substrate rather than inducing changes in DNA twist or locally wrapping DNA. The authors envisaged that the 13 S condensin preferentially traps positive loops by nonplanar bending and sequesters them into a radial solenoidal stack (23).
A detailed understanding of the interaction dynamics of the Smc2/4 complex is essential if we are to correctly interpret the requirement of these proteins in chromosome condensation and segregation. This sort of analysis has been applied only to the study of the Xenopus condensin. In this paper we describe the expression, building, and testing of wild-type and mutant Smc2/4 complexes from the budding yeast and present our findings for the DNA-associated activities of this condensin SMC heterodimer pair.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein Expression and PurificationHigh level expression of
proteins after single or double transformation of BCY123 was performed
essentially as described (37).
Cells were grown in 0.8 liters of selective medium (1.5% glycerol, 1% lactic
acid) and supplemented 1 h before galactose induction with 40 ml of YENB (2%
yeast extract, 4% nutrient broth). Plasmids pJES37 and pJES88 were used for
individual expression of wild-type SMC2-His6 and
SMC2-Strep-tag fusions, and pJES72 and pJES103 were used for
independent expression of the untagged mutant alleles smc2-P and
smc2-Sig, respectively. Untagged wild-type SMC4 was
expressed from pJES19, and intein/CBD fusions of SMC4, wild type, and
smc4 mutants were expressed from the plasmids pSJ50, pJES46, and
pAEH23, respectively. The wild-type Smc2/4 complex was routinely purified from
cells expressing both SMC2 and the SMC4-intein/CBD fusion
under maintenance selection for the plasmids pJES85 (Trp+) and
pSJ50 (Ura+). Because it proved difficult to maintain balanced
expression of smc mutant alleles, the Smc2/4 mutant complexes were
assembled in vitro by co-disruption of cells expressing each protein
separately. Cell harvesting and disruption were carried out as previously
described (37) with the
following changes. Cells were washed 1x in Buffer A containing 25
mM Na-HEPES (pH 8.1), 500 mM NaCl, 10% glycerol, 1
mM EDTA, 1 mM EGTA and then diluted at 1 g of cells/ml
in Buffer B (Buffer A reduced to 0.25 mM EDTA and EGTA) and frozen
at -70 °C. Cell slurry (16 ml) was thawed and spiked with
phenylmethylsulfonyl fluoride (1 mM), leupeptin (0.5 µg/ml),
pepstatin (0.5 µg/ml), and benzamidine (10 mM) before cell
disruption. Cells were broken to 80% completion by glass bead lysis and
diluted in 30 ml of ice-cold Buffer B plus protease inhibitors. Debris was
cleared by centrifugation at 15,000 rpm for 20 min at 0 °C, and soluble
extracts were then exposed to the appropriate affinity resins as described
below. Intein/CBD fusion proteins were purified essentially as described
(33) over chitin resin (New
England Biolabs), washed with 10 volumes of Buffer B containing 0.25% Tween 20
and 5 volumes of Buffer B without detergent, and eluted in Buffer B plus
protease inhibitors containing 40 mM dithiothreitol.
Smc2-His6 was purified over nickel nitrilotriacetic-agarose
(Qiagen, Chatsworth, CA), washed in Buffer B, and eluted in the same buffer by
imidazole challenge. Strep-tag fusion proteins were captured on Strep-Tactin
resin, washed with Buffer B, and eluted by competition with 2 mM
desthiobiotin.
Irrespective of which affinity approach was used in the first step, the
second purification step for Smc2p, Smc4p, and Smc2/4 was ion exchange over
Q-Sepharose. Proteins used in ATPase assays were purified using
phosphocellulose (P11, Whatman; 0.25 ml of resin/mg of protein). For proteins
used in other analyses, high concentration fractions eluting from Q-Sepharose
(2 mg/ml; 300 µl) were separated by size over a column of high
resolution (Amersham Biosciences high resolution) Superose 6 (1.0 x 30
cm; exclusion limit
4 x 107 Da) in B:300 (Buffer B with
300 mM NaCl) running at a flow rate of 0.5 ml/min (4 °C). The
column was calibrated in the same buffer using 20 S proteasome (1.2 MDa, a
gift from the laboratory of C. P. Hill, University of Utah) in addition to
thyroglobulin (669 kDa), apoferritin (440 kDa), catalase (240 kDa), IgG (158
kDa), and BSA2 (67
kDa). The column void volume was established with blue dextran >2 MDa.
Protein concentrations were determined using the Bio-Rad protein assay reagent
(Bradford) by measurement of absorbance at 280 nm (
280 =
112,430 M -1 cm-1 for Smc2/4
(38)) and/or by comparison of
Coomassie-stained BSA in SDS-polyacrylamide gels. Proteins were frozen in
liquid N2 and stored at -70 °C.
Sedimentation Equilibrium AnalysisEquilibrium sedimentation
was performed after size separation of the wild-type Smc2/4 in buffer
containing 10 mM Na-HEPES (pH 7.6), 300 mM NaCl, and 0.1
mM EDTA. The inclusion of EDTA was necessary for Smc2/4 stability,
and the absorbance (A235) of this buffer was still
relatively low (0.04 absorbance units). Analytical ultracentrifugation
experiments were performed at 20 °C using a Beckman XL-A centrifuge
equipped with absorbance optics. Sedimentation was performed at 5000, 6500,
7000, and 7500 rpm, and the data were collected at three wavelengths (360,
280, and 235 nm) as a function of radial distance by averaging 10 absorbance
measurements at steps of 0.001 cm. Scans were begun 12 h after the start of
the run or increase in speed, and successive scans were repeated every 4 h
thereafter. Equilibrium was judged to have been reached when the overlay of UV
traces from two or more successive scans showed no variation across the radial
distribution. Radial absorbance data were plotted using
XLGraph,3 and the data
in the optical window were selected using
Xlaedit.4 Scans were
analyzed individually and globally using the winNONLIN3
4 version of the
fitting program NONLIN (39).
Equilibrium absorbance distributions were first fit to an ideal,
non-dissociating single species model in which the absorbance at a particular
radial distance Ar equals
Ar0 exp[(r2
- r02)] + Y, where
Ar0 is the absorbance at the reference
radius r0. The effective reduced molecular weight sigma
(
) is defined as M1(1 -
)]
2/2RT, where M1 is
the molecular weight of the smallest species,
is the partial specific
volume of the protein (ml/g),
is the density of the solvent (g/ml),
is the angular velocity (radians/s), and R and T are
the universal gas constant and absolute temperature, respectively. The
base-line offset term Y serves as a normalization factor for
Ar0 values. The program SEDNTERP
(version 1.01)5 was
used to derive estimates of the partial specific volume for proteins based on
amino acid composition (0.7401 ml/mg for Smc2p and 0.7353 ml/mg for Smc4p) and
the density of the sedimentation solvent (1.0194 g/ml) at 20 °C with data
from the International Critical Tables and standard references
(40). The equilibrium
association constants obtained for fits to a reversible Smc2p plus Smc4p
heterodimerization model, assuming a theoretical average monomer
Mr of 148,059, were high and variable, well outside a
meaningful range of values, suggesting that the Smc2/4 complex behaves
essentially as a non-dissociating complex. Hence, the Smc2/4 complex was
treated as a monomer for all subsequent fits. Smc2/4 association and other
equilibrium models were fit to the general expression
Ar = Ar0
exp[
(r2 - r02)] +
An, where the absorbance of each n-mer
species, An, equals
KnAr0n
exp[n
(r2 - r02)] +
Y, and Kn is the equilibrium association
constant (in absorbance units) for the formation of the nth species
(second, third, fourth, etc.). The best fits were obtained using a monomer
dimer equilibrium model, in which the monomer molecular weight was
equal to that of the Smc2/4 heterodimer. The
value for the smallest
species in association models was calculated (
calc) using
the theoretical heterodimer Mr based on the sequence for
Smc2/4 (296,118 g/mol), with
av = 0.7377 ml/g taken as the
average of the
estimates for Smc2p and Smc4p. Calculated association
constants were converted to molar units using the equation
Ka =
(KaAbs-1(1.2 cm x
)n-1/(n)
(41). The theoretical native
molar extinction coefficients at 280 nm for Smc2p (56,730 M
-1 cm-1) and Smc4p (55,700 M-1
cm-1) were converted to values appropriate for 235 nm using the
235/280 conversion ratio 4.05 to yield the Smc2/4 extinction coefficient
235 = 463,442 M- 1
cm-1. Base-line offset terms (Y) were floated, and
was then globally fit to a single species model without compensatory
adjustment for non-ideality. More complex models were fit globally to a common
association constant by holding
calc for the theoretical
Smc2/4 heterodimeric Mr and n constant. The fit
was judged to be in good agreement with the model if separate
Kn values for each data set converged with the
global Kn. For a more complete description of
fitting strategies using NONLIN see Hansen et al.
(42) or Laue and co-workers
(43).
ATPase AssaysSteady state ATPase assays were performed in a
coupled ATP-regenerating system or by TLC essentially as described
(44,
45). Coupled assays were
performed in 0.5 ml of binding, knotting, and supercoiling buffer (BKS: 10
mM Na-HEPES (pH 7.6), 50 mM NaCl, 10% glycerol, and 1
mM EDTA) supplemented with 100 µg/ml BSA, 3 mM
MgCl2, 1mM dithiothreitol, 505000
µM ATP, and 150 nM wild-type Smc2/4 complex (300
nM ATPase active sites) and coupling reagents phosphoenol pyruvate,
NADH, and protein kinase/lactate dehydrogenase at the concentrations described
(44). For TLC assays, 25-µl
reactions were performed in BKS supplemented with 100 µg/ml BSA, 3
mM MgCl2,1mM dithiothreitol, 1.5
mM ATP, 0.125 µl[-32P]ATP (8 nM),
and 300 nM wild-type or mutant Smc2/4 complex (600 nM
ATP active sites). Assays done in the presence of DNA were performed at a
base-pair concentration of 50 µM (167 bp per Smc2/4). Other
details are found in the figure legends.
DNA Substrate Preparation, Binding Reactions, and
ElectrophoresisTopoisomerase I was purified from raw wheat germ
(WG topo I) as described (46)
and stored at -70 °C at 400 µg/ml. The various plasmids used in
analyses, pBSKS(+) (Stratagene, 2961 bp), pRS424
(47) (5616 bp), or pJES7
(14,700 bp), were relaxed at 23 °C with WG topo I, singly nicked in the
presence of ethidium bromide with DNase I as described
(48), or linearized by
restriction digestion. Circular pBSKS(+) was radioactively labeled by nick
translation in the presence of [-32P]dCTP using standard
procedures. The plasmid pBSKS(+) was linearized with XmaI and
end-labeled by incorporation of [
-32P]dCTP with T4 DNA
polymerase. Radiolabeled DNA was quantitated by ethidium bromide staining in
agarose gels by comparison to non-labeled DNA standards. All binding reactions
(50 µl) contained 1 mM ATP unless indicated otherwise and either
10 or 100 ng of the various topologic forms of DNA. Other details can be found
in the figure legends. Reaction mixtures were separated at ambient temperature
in 0.8% agarose gels in 1x TAE at 3 V/cm for 1218 h. To prevent
the loss of Smc2/4-DNA-bound species that could not enter the gel, samples
were embedded in the wells with molten agarose before gel submersion. Molten
agarose (20 µlat55 °Cof 1% low melting temp agarose in 1x TAE
plus 0.01% bromphenol blue) was rapidly mixed with each 50-µl reaction and
quickly transferred to dry wells. Gels were stained with ethidium bromide for
image-capture under UV illumination or vacuum-dried for autoradiography.
Knotting and Supercoiling AssaysAll topology trapping and DNA binding assays were performed in complete BKS, BKS containing 100 µg/ml BSA (fraction V, Sigma), 1 mM dithiothreitol, and 2.5 mM MgCl2. Phage T4 topoisomerase II was kindly provided by Wai-Mun Huang (University of Utah), and yeast topoisomerase II was purified as described previously (34). Supercoiling reactions (50 µl) were performed by combining 37 µl of complete BKS, with or without 0.5 µl of 100 mM ATP (1 mM final), 100 ng of supercoiled pRS424 (0.55 nM plasmid) or relaxed pBSKS(+) (1 nM plasmid), and variable amounts of Smc2/4 (10 µl in Buffer B:300) as detailed in Fig. 4. This mixture was incubated at 23 °C for 15 min, and 100 ng of WG topo I was then added for an additional 30 min to relax the DNA. Reactions were quenched with 20 µl of stop solution (50 mM Tris-HCl (pH 7.5), 1% SDS, 10 mM EDTA, and 100 µg/ml proteinase K) and placed at 42 °C for 1 h to digest proteins. DNA was recovered by ethanol precipitation after phenol:chloroform extraction, separated on 0.8% TAE-agarose gels (with or without 0.2 µg/ml chloroquine), and recorded after ethidium bromide staining. Knotting reactions were performed under the same conditions except that nicked pRS424 was used, and incubation in the presence of 100 ng of T4 or yeast topo II was limited to 15 min.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Assembly and Purification of the Smc2/4 ComplexWhen Smc2p and Smc4-Int/CBD were co-expressed, a stable 1:1 complex of the proteins could be purified based on the Smc4p affinity tag (Fig. 1B). All highly purified preparations of wild-type Smc2/4 used in the present study were obtained in this manner. One of the co-purifying proteins near 70 kDa, present in SMC preparations at variable levels (see Fig. 2B), was confirmed by antibody staining to be Hsp70p (data not shown).
|
Smc2p and Smc4p were purified individually, and we found that each protein in isolation was more prone to nonspecific absorption, degradation, or aggregation than the Smc2/4 complex. Smc4p was particularly sensitive to degradation, a phenomenon that may be similar in nature to the time-dependent cleavage of the Schizosaccharomyces pombe Smc4p homolog, Cut3, previously observed (49). Smc2p, although resistant to degradation, tended to aggregate.
We assessed whether isolated Smc2p or Smc4p could assemble into stable homodimers by performing affinity separation with mixtures of differentially size-tagged Smc2p or Smc4p (i.e. tags large enough to detect size shifts on SDS-gels). No untagged-Smc2p co-purified with the tagged Smc2p; the same was true for Smc4p (data not presented). These pull-down results do not prove a lack of self-association, but under these conditions, SMC homodimerization appears minimal.
When purified Smc2p and Smc4p were mixed, they assembled rapidly in vitro to form stable 1:1 complexes. Fig. 1C displays the results of one such a mixing experiment that employed Smc2-His6 and untagged Smc4p. This, in combination with the results of affinity purification presented in Fig. 1B, showed that placement of a single affinity tag at the C terminus of Smc2p or Smc4p permits isolation of the Smc2/4 complex. Assembly was independent of nucleotide, and Smc2 and -4 assembled after purification behaved similarly to in vivo assembled (co-expressed) Smc2/4 (i.e. stability, solution behavior, DNA binding, and ATP hydrolysis).
Solution Properties of the Smc2/4 ComplexIn vivo
assembled Smc2/4 complex was subjected to a number of analyses to determine
the oligomeric state of the 1:1 hetero-complex. Size separation of the SMC
complex over Superose 6 (Fig.
2A) showed that Smc2/4 (296 kDa) could distribute across
a range of exclusion volumes predicted for globular proteins of 200 kDa
to 1.2 MDa. This column has a very large exclusion limit, and the largest
Smc2/4 species eluting in the UV trace shown in
Fig. 2A were within
the separation range of the column. The broadly eluting distribution could
represent the fractionation of stable "shape conformers" of
individual heterodimeric Smc2/4 complexes with identical mass
(50) or could arise by
oligomerization of the Smc2/4 complexes. To discriminate between these two
possibilities, proteins eluting at different positions in the distribution
were chosen for shape-independent mass determination by sedimentation
equilibrium. We found that the data from the protein complex in fraction 2
(Fig. 2, A and
B) did not fit any scheme for a single molecular weight
species, suggesting that the larger species from the gel filtration column
were not simply shape conformers of an Smc2/4 heterodimer. We noted that the
Smc2/4 complex was also prone to aggregation, and the species in fraction 2
may have represented an oligomeric mixture. This tendency, coupled with the
low absorbance of Smc2/4 at 280 nm, restricted the concentration range that
could be used and prevented further analysis of fraction 2. For fraction 8
(Fig. 2, A and
B) we performed two independent sedimentation experiments
and globally fit the data from each (Experiment 1, 9 data sets, 3 speeds, and
three concentrations; Experiment 2, 6 data sets, two speeds, and three
concentrations) to models describing single species and association behavior.
The data from both experiments were also combined (15 data sets) and fit
globally. Neither of these data sets was adequately described by a single
Smc2/4 species or by models involving dissociation of the heterodimer.
However, the data were fit well by a monomer-dimer equilibrium model (2(2/4)
(2/4)2), where the monomer molecular mass was equal to
that of the Smc2/4 heterodimer. Examples of fits obtained for this model are
presented in Fig. 2C,
for which the estimated Kd was 3.2 ± 1.3
µM. Residuals were small and random, indicating a close
correspondence between the experimental data and the absorbance distributions
predicted by the association model. Good agreement was also obtained for the
fitted molecular size (295 ± 23 kDa) when its value was held constant
during fitting ln Kd to a monomer-dimer
equilibrium. Moreover, association constants obtained by simultaneous fitting
of separate ln Ka values for individual data sets
converged with the common association constant obtained by global fitting.
ATPase Activity of Wild-type and Mutant SMC ProteinsAs members of the ABC family of ATPases, the SMC proteins are predicted to hydrolyze ATP. Although a number of results for the eukaryotic heterodimeric complexes (13, 22, 51) and prokaryotic homodimeric complexes have been reported (18, 52, 53), no steady state parameters have yet been established for any SMC-containing complex. We examined the ATPase activity of the wild-type Smc2/4 complex and found that it obeyed simple Michaelis-Menten kinetics. Hydrolysis rates determined under initial velocity conditions using a range of ATP concentrations (505000 µM) were fit to a kcat of 0.21 ± 0.005 s-1 and a Km of 420 ± 30 µM (Fig. 3A). These data provide the first steady state parameters describing ATP hydrolysis by an SMC protein from any species.
|
We also determined that the relatively low level of ATPase activity displayed by the Smc2/4 complex is in fact intrinsic to the heterodimer and not due to a contaminating source. ATPase activity could be reconstituted when separately purified Smc2p and Smc4p, neither of which could hydrolyze ATP on its own (see Fig. 3), were mixed at a 1:1 stoichiometry. The ATPase activity of the in vitro assembled complex equaled that of the complex assembled in vivo (Fig. 3B). In contrast, when the Smc4-P-loop mutant or the ABC Signature motif mutant Smc4-Sig was mixed with wild-type Smc2p, no ATPase activity was recovered. These results confirm that the condensin Smc2/4 sub-complex can hydrolyze ATP and, furthermore, that targeted inhibitory mutations in the ATPase domain of one SMC can act in trans to inhibit hydrolysis activity of the opposite ABC catalytic site. As expected, the Smc2-P/4-P double P-loop mutant complex could not hydrolyze ATP (Fig. 3B) but retained its ability to bind DNA as well as wild-type Smc2/4 (data not shown). Finally, neither linear (sheared salmon sperm) nor a mixture of supercoiled and relaxed plasmid DNA (pRS424) affected the ATPase activity of in vitro or in vivo assembled Smc2/4 complex (Fig. 3B) under conditions that favored DNA binding.
Binding Induced Geometry Change; DNA Knotting and Supercoiling AssaysWe next tested whether exposure to the Smc2/4 complex could introduce geometric changes in closed circular DNA. For historical reference, topologic trapping experiments utilizing topoisomerases and naked plasmid DNA provided the first biochemical readout of the effects of DNA binding and ATP hydrolysis by the mitotically active Xenopus 13 S condensin complex (2224). Knotting reactions utilize nicked circular DNA substrates that should not accumulate twist to detect changes in net writhe as reflected by formation of knots after reaction with a type II topoisomerase. Supercoiling assays utilize a relaxed plasmid substrate and a type I topoisomerase to trap compensatory changes in twist and writhe generated upon binding and/or energy-dependent reconfiguration of the DNA by the condensin.
DNA templates were relaxed after exposure to a high mole ratio of Smc2/4:DNA (64:1), in the presence of ATP. SMC-dependent geometry changes were assessed after gel separation of reaction products and are presented in Fig. 4, A and B. The Smc2/4 complex isolated from asynchronous yeast cultures induced the formation of a broad distribution of DNA knots in the presence of either phage T4 or yeast topo II (Fig. 4A). Knot formation was strictly dependent on both topo II (lane 1) and the Smc2/4 complex (lane 2). Smc2/4-dependent knots increased in direct proportion to increases in the Smc2/4:plasmid ratio up to 256:1 (the highest ratio tested), and no knotting was detected at mole ratios below 32:1 (data not shown). These results demonstrate that the Smc2/4 complex can induce topo II-dependent DNA knotting in the absence of the Ycs4p-Ycg1p-Brn1p regulatory complex and without mitotic activation. In the supercoiling assays only minor changes in net writhe were detected after exposure of DNA substrates to the Smc2/4 complex (Fig. 4B, lane 3) and topo I.
To further analyze this lack of supercoiling, we tested for concentration-dependent positive supercoiling using the smaller 2.96-kb relaxed plasmid pBSKS(+) as a template (Fig. 4C). The products of the reactions were resolved in the presence of 0.2 µg/ml chloroquine to allow the magnitude and direction of changes in superhelicity to be assessed. A slight positive shift in the resultant topoisomer distribution was observed after simultaneous plasmid exposure to Smc2/4 and topo I (lane 5, 128:1). A minor ATP dependence was also seen (compare lanes 5 and 6), but this ATP effect on positive supercoiling was not reproducible. As a control, the relaxation activity of WG topo I was shown not to be limited by plasmid binding at the high mole ratios of Smc2/4:plasmid used in this study (see Fig. 4C, lane 8). Additionally, supercoiling reactions performed at the highest mole ratios without topo I caused no changes in the topoisomer distribution of relaxed or supercoiled DNA substrates (Fig. 4C, lanes 7 and 10), indicating that Smc2/4 preparations were free of topoisomerase or nuclease contamination.
Stoichiometry and Stability of DNA Binding by the 2/4 ComplexFig. 5 presents the results of a series of gel shift experiments that summarize the DNA binding studies of the Smc2/4 complex under a number of conditions. Earliest attempts to resolve DNA-bound species electrophoretically failed due to their lack of mobility in agarose gels (see Fig. 5D). The formation of low mobility complexes after binding of Smc2/4 to DNA appears to be a general characteristic of many SMCs (16, 22, 51, 52, 54, 55). This limitation likely results from one or more of the following factors: 1) the large size of multiple Smc2/4 complexes bound to the same plasmid, 2) the even larger size of networks formed by intermolecular protein-DNA cross-linking, 3) the precipitation of protein-DNA aggregates by exposure to low salt electrophoresis buffer, and 4) the neutralization of DNA charge at high protein occupancy. The molten agarose/cosolidification loading protocol described in the procedures was adopted to avoid buffer-induced precipitation and facilitated entry of Smc2/4-bound species into the gel.
|
The salt stability and electrophoretic mobility of Smc2/4-DNA bound species were examined in binding reactions employing a 2961-bp linear or nicked circular DNA at varying degrees of Smc2/4 binding saturation. Fig. 5, A and B, demonstrate that Smc2/4-DNA-bound species began to resist salt disruption as the level of protein:DNA increased. We tested mole ratios that bracketed those used in knotting and supercoiling reactions and found that DNA binding at a mole ratio of 16:1 could induce a complete shift of both linear and circular DNA. These shifted species were disrupted by modest salt concentrations of between 100 and 300 mM NaCl (Fig. 5, A and B); however, as the mole ratio of Smc2/4:DNA in binding reactions was increased (64:1 linear; 256:1 circular), the sensitivity to salt challenge disappeared, and the resistant species remained stable to 0.5 M NaCl challenge. Salt-refractory complexes appeared to form at a lower mole ratio for linear DNA than for circular DNA. This result was not anticipated, and it might have been reasonable to expect that proteins bound to linear DNA having free ends would dissociate more readily than from those bound to closed circular DNA. Despite this paradox, it is clear that DNA binding of Smc2/4 at protein:DNA mole ratios less than or equal to 16:1 is reversible.
The DNA binding experiments in Fig. 5, A and B, were performed in the presence of 1 mM ATP to mimic knotting and supercoiling reaction conditions. It has been suggested that DNA binding by the SMCs may be modulated by ATP binding/hydrolysis (16, 52, 53, 55). To test the effect of nucleotide on DNA binding, reactions were performed in the absence of nucleotide (No Nuc) or the presence of ATP, ADP + sodium orthovanadate (Na2VO4), ATP + Na2VO4, or the non-hydrolyzable analog AMP-PNP (Fig. 5C). In these experiments, the Smc2/4 complex was exposed to each nucleotide or nucleotide analog for 30 min at 30 °C before adding nicked circular DNA. We found no apparent nucleotide-based differences in the degree or magnitude of gel shifts for the bound species formed.
To determine the effect of template size on binding, two plasmids of
different lengths (3 and 14.7 kb) were compared in parallel reactions
(Fig. 5D). Binding
titrations were performed by holding plasmid concentrations constant at 2
nM while varying the concentration of the Smc2/4 complex. Bound
species were resolved, and the concentrations at which 100% of the DNA shifted
were compared. In these experiments the gels were loaded without molten
agarose (see "Experimental Procedures"), and consequently the
bound species failed to enter the gel. These results demonstrate that binding
occurs over a very narrow range of protein concentrations that correlates with
the Smc2/4 to base pair ratio, not with the Smc2/4 to plasmid ratio. The point
at which substrate DNA was shifted completely occurred at an Smc2/4:bp ratio
of between 1:50 and 1:100 for both plasmids. This corresponds to an
Smc2/4:plasmid ratio of between 64:1 and 128:1 for the small template and
256:1 and 512:1 for the large template
(Fig. 5D, see the
arrows above the gels). Estimates of the percent of total
protein bound were made by differential centrifugation of the protein-DNA
complexes (18,
52,
53). By this measure we
estimated that 50% of the total protein in these assays was bound to the
DNA. Moreover, we noted that the percent protein bound increased with
increasing mole ratios of Smc2/4:plasmid.
DNA Competition StudiesThe DNA knotting and supercoiling activities ascribed to the Xenopus condensin (13, 22, 23) and the yeast Smc2/4 complex presented in this work are manifest only at high protein to DNA ratios. Therefore, we tested whether the salt-stable species assembled at higher molar ratios of protein to DNA (Fig. 5, A and B) remained dynamic and reversible or became static protein-DNA aggregates. Fig. 6A shows the results of DNA challenge experiments in which Smc2/4 pre-bound to 32P-labeled nicked circular DNA at a mole ratio of 128:1 was challenged with unlabeled DNA of an equivalent length (linear, nicked circular, or supercoiled). It is important to note that a single Smc2/4-32P-labeled DNA master mix was used for the entire set of reactions shown; the only difference between lanes is the form and concentration of the competitor DNA added. Both unlabeled linear and supercoiled DNA efficiently displaced Smc2/4-bound DNA species (Fig. 6A; lanes 26 and 1718) as judged by the reappearance of free, labeled DNA. In contrast, nicked circular DNA was not an efficient competitor and failed to cause release of labeled DNA at the highest concentrations used (lanes 912). These results demonstrate two facts about DNA binding by the Smc2/4 complex: 1) Smc2/4-DNA complexes formed at high mole ratios remain dynamic and reversible, and 2) competitor DNAs with different geometries appear to engage the Smc2/4 complex in fundamentally different ways.
|
The models in Fig. 6B depict DNA binding by "coil-confinement" as envisaged for the Smc2/4 complex. These models bear resemblance to the embrace model proposed for sister chromatid cohesion by the cohesin SMCs (19). The two binding modes, "threading" and "entrapment," depict two ways in which a single DNA duplex could potentially gain access to the internal space bounded by the coiled-coils of a single Smc2/4 complex. According to these models, one would predict that binding of an open circular DNA duplex (nicked or relaxed plasmid) would occur preferentially by entrapment and rarely by threading. This prediction of the coil-confinement model was used to develop the "threading-displacement" and "first-in-last-out" mechanisms illustrated in Fig. 7 to explain how DNAs of equal length but differing shape/topologies could vary in their ability to act as cold DNA competitors.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Self-association of Smc2/4
HeterodimersSedimentation equilibrium results showed that Smc2/4
heterodimers interact reversibly. The free energy change for Smc2/4
self-association (G2/4-(2/4)2) is within a
physiologically moderate range (
8 kcal/mole). Absorbance interference
prevented analysis of the effects of ATP binding/hydrolysis on the
heterodimer-heterotetramer transition. However, inclusion of Mg-ATP did not
alter the sedimentation profiles of Smc2/4 through sucrose gradients or the
elution profiles from sizing columns (data not presented). These results raise
the possibility that interaction between SMC pairs could occur in
vivo and that perhaps these integral condensin proteins could contribute
to a framework essential for the proper management of chromatin and/or the
maintenance of chromosome structure. For proteins that may have a scaffolding
or architectural role in chromosomes, it is useful to know that they have the
intrinsic ability to self-associate at relevant concentrations.
Hydrolysis of ATP by the Smc2/4 ComplexBecause of the large number of potentially contaminating ATPases in cells, it is difficult to prove that a low level of ATPase activity in a protein preparation is intrinsic to the protein of interest. We have proven that the Smc2/4 complex is an ATPase by mixing the separately purified Smc2p and Smc4p. Although before mixing there is essentially no ATPase activity, an equimolar combination of the proteins increased this activity by at least 6-fold. This ATPase activity is independent of, and not stimulated by double-stranded DNA.
A mutation in the P-loop or the signature motif of only one of the SMCs abolishes the ATPase activity of the complex, and each of these mutations fails to rescue deletions of the respective SMC gene. Based upon the Rad50 structure (16) one might predict that mutation of a single P-loop or signature motif might not affect the second active site. Nevertheless, the failure of wild-type/mutant Smc2/4 heterodimers to hydrolyze ATP suggests that the ATPase mechanism may involve active site cooperation. Mutation of one ATPase active site of P-glycoprotein (Pgp), another member of the ABC family, was found to have a similar effect (56), and this enzyme is proposed to operate by alternating ATP hydrolysis between two active sites (57, 58). A similar mechanism has been predicted for other ABC ATPases including the cystic fibrosis transmembrane conductance factor, the histidine permease HisP, and the maltose transporter MalK, as reviewed by Holland et al. (59). The results presented here are consistent with a similar ATPase mechanism for Smc2/4.
Smc2/4 Promotes Knotting without Appreciably Changing WritheThe Smc2/4 complex promotes knotting of circular DNA in the presence of topo II but does not cause significant trapping of positive supercoils in the presence of topo I. Topoisomer distributions were shifted only slightly in the presence of Smc2/4:DNA at ratios that promoted robust knotting (Fig. 4C). Unlike the activated Xenopus 13 S condensin, which showed a strong correlation between knotting and the level of positive supercoiling (22, 24), DNA knotting promoted by the Smc2/4 complex does not appear to introduce significant superhelical strain. The chirality of the knots formed in the presence of Smc2/4 has been analyzed, and the results will be presented elsewhere.6 We conclude that knotting and supercoiling can represent two distinct and separable activities of the condensin. Although both Smc2/4 and the Xenopus 13 S condensin induce shape changes in DNA, their exact geometries clearly differ.
DNA Binding by the Smc2/4 ComplexGel shift analyses were performed using a 3-kb circular or linear DNA to examine the stability and reversibility of Smc2/4-DNA bound species as a function of protein:DNA ratio. Reversibility testing of DNA-bound species was necessary since Smc2/4-dependent DNA knotting, supercoiling, and re-annealing are manifest only at high protein:DNA plasmid ratios (Refs. 22 and 49 and this study). Smc2/4-DNA bound species resisted salt disruption as the ratio of Smc2/4:DNA in binding reactions was increased. Mole ratios that were too low to support efficient knotting (i.e. 16:1) remained sensitive to salt disruption; however, the knotting-proficient ratio of 64:1 caused DNA-bound species to be more salt-resistant (compare 16:1 and 64:1 ratios in Fig. 5B). This correlation between DNA knotting and salt stability may suggest that both DNA binding and interactions between heterodimers participate in the formation of knotting geometries.
The presence or type of nucleotide did not affect binding of Smc2/4 to circular DNA (see Fig. 5C). The crystal structure of the P. furiosus Rad50 catalytic domain revealed that binding of a non-hydrolyzable ATP analogue, AMP-PNP, trapped the NC head domains into a (NC)2 tetrameric configuration (16). Thus, one might expect that binding of AMP-PNP or ADP + orthovanadate would lock the ATPase domains of Smc2/4 in a closed state and block access of circular DNA to the inter-coil space (Fig. 7A). However, neither nucleotide prevented binding of circular DNA. These results indicate that either 1) these nucleotides do not lock the ATPase domains, 2) binding does not occur by coil-confinement, or 3) open circular DNA is capable of threading between the coils.
Apparent Cooperativity in Smc2/4 DNA BindingThe
interaction of Smc2/4 with DNA is more complex than expected. No binding to
plasmid DNA was detected until the Smc2/4 to base pair ratio reached
1:100. This ratio held over a wide range of protein/DNA concentrations
and plasmid lengths. Binding occurred in an all-or-nothing fashion, and
additional protein caused the shifted species to migrate even more slowly.
These observations suggest that stable DNA binding begins at a critical level
of Smc2/4 saturation along the DNA lattice (nucleation), after which the
loading density of Smc2/4 increases in proportion to the protein:DNA ratio.
The free energy of Smc2/4 self-association may stabilize the nucleation. When
DNA-bound Smc2/4 complexes are forced into close proximity with each other,
protein oligomerization may be favored. This would bring distant segments of
DNA together and could lead to a knotting-proficient geometry. Atomic force
images of the S. pombe condensin assembled with DNA at high protein
ratios showed the formation of clustered condensin-DNA aggregates
(51), and it is possible that
these aggregates represent reversible species relevant to in vitro
condensin function.
Reversibility of DNA BindingBecause knotting-proficient Smc2/4-DNA species resisted salt challenge, excess unlabeled DNA was used to drive apart the complexes to show that they indeed remained reversible. Both linear and supercoiled DNA readily dissociated labeled, circular DNA prebound to Smc2/4, demonstrating that Smc2/4-DNA bound complexes formed at ratios as high as 128:1 are reversible (Fig. 6A; lanes 26 and 1718). Under the same conditions, open circular DNA could not displace prebound circular DNA (Fig. 6A, lanes 812). This was not expected, since the only difference between these competitor DNAs was essentially their shape.
We propose that this shape-dependent DNA competition can be explained by topologic trapping of DNA between the Smc2/4 coiled-coils. This explanation is consistent with the unique geometry of SMC pairs (19, 50) and the recent findings for DNA binding by isolated domains of the Bacillus SMC (18). Bacillus SMC deleted for both the N- and C-terminal globular domains forms dimers capable of binding DNA (18). Mutations that disrupt dimerization of this construct block DNA binding, and dimers of just the hinge regions also do not bind DNA. These results suggest that DNA binding requires both coiled-coils in correct proximity to the hinge dimer and is consistent with DNA binding in the cleft formed by the coiled-coil of a folded SMC pair. The cohesin SMCs have been proposed to embrace DNA segments (19), and this general mode of DNA binding can be used to explain the present results.
DNA Competition Supports a Mechanism of Gated DNA Binding and ReleaseFig. 7A illustrates how capture of an open circular DNA could occur by gated-entrapment. This model predicts that interaction of the globular NC (ATPase) domains is transient, and that reversible NC-NC interaction could form a "gate" through which DNA duplexes diffuse or traverse by active transport. Operating in this fashion, the Smc2/4 complex would mimic gated ligand transport promoted by other ATPases of the ABC transporter family (59). Gate dynamics, possibly mediated by ATP binding/hydrolysis, could lead to transient trapping of a DNA duplex in the Smc2/4 inter-coil space. DNA could also bind by threading between the coils (Fig. 6B), but only linear or supercoiled DNA is envisioned to do so easily.
Based on these ideas two mechanisms of shape-dependent competition become apparent (Fig. 7, B and C). Threading-displacement can be promoted by linear or supercoiled DNA (colored gray), which may insert itself between the coils of Smc2/4 to displace prebound DNA circles (colored red). Open circular DNA would not be expected to readily thread between the coils and would, thus, have to enter between the head domains. The first-in-last-out mechanism explains how unlabeled circular DNA (gray) could sterically inhibit dissociation of prebound DNA circles (red).
The mechanisms presented are based on the prediction that plectonemically
interwound branches of supercoiled DNA would thread more efficiently than
nicked circular DNA. This is consistent with experimentally derived physical
measurements of the average superhelical radius of interwound plasmids
(60). For a 3.5-kb plasmid
with a specific linking difference close to that of a plasmid directly
isolated from Escherichia coli, the average radius was found to be
4 nm, nearly 14 times less than that of an open circular plasmid of the
same length (57.5 nm). The threading-displacement mechanism can be tested
since the competitive potential of circular DNA should decrease as the
superhelical radius increases (i.e. as the linking difference nears
zero).
![]() |
FOOTNOTES |
---|
Supported in part by National Institutes of Health Training Grant
5T32GM07464-24.
To whom correspondence should be addressed: Dept. of Biochemistry, University
of Utah Shool of Medicine, 20 North, 1900 East, Salt Lake City, UT 84132-3201.
E-mail:
Janet.Lindsley{at}hsc.utah.edu.
1 J. E. Stray, J. E. Lindsley, S. Gradia, and J. Berger, unpublished
results.
2 The abbreviations used are: BSA, bovine serum albumin; WG topo I,
topoisomerase I from raw wheat germ; kb, kilobase(s); BKS, binding, knotting,
and supercoiling buffer; AMP-PNP, adenosine 5'-(,
-imino)
triphosphate; TAE, Tris acetate EDTA.
3 XLGraph was written by John Philo (available at
www.cauma.uthsa.edu).
4 Xlaedit and winNONLIN3 were written by J. Lary and D. A. Yphantis
(available at
www.cauma.uthsa.edu).
5 SEDNTERP (version 1.01) was written by D. T. Hayes, T. M. Laue, and J.
Philo (available at
www.cauma.uthsa.edu).
6 J. E. Stray and J. E. Lindsley, manuscript in preparation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|