BACKGROUND
helicases assume a variety of oligomeric forms from monomers and dimers to larger oligomers such as hexamers and dodecamers .
the oligomeric form of a helicase is often important for its function.
for example, hexameric helicases form ring structures that are thought to encircle single and/or double stranded dna as part of their unwinding function  <cit> .
furthermore, the number of ring constituents is often important for function; methanobacter thermoautotrophicum mcm, which is isolated as hexamers, heptamers or dodecamers, is thought to function as a hexamer  <cit> .
in contrast, some helicases are functional in different oligomeric forms, such as the hepatitis c virus ns <dig> helicase, which functions as a monomer, dimer or larger oligomers  <cit> .
the minichromosome maintenance proteins  <dig> through  <dig>  form a ring-shaped heterohexamer that is the replicative helicase in eukaryotic cells  <cit> .
each of mcm <dig> through  <dig> is essential for the initiation and elongation stages of dna replication  and helicase activity has been detected not only with mcm2- <dig>  but also with complexes containing different combinations of mcm proteins  <cit> , including a heterohexamer comprised of mcm <dig>  mcm <dig> and mcm <dig> that was initially purified from hela cells  <cit> .
the biological significance of these additional helicase complexes is not known.
interestingly, mcm proteins are present in cells at copy numbers that are far in excess of what is expected for two replication forks per active origin  <cit> .
it has been proposed that the "excess" mcm subunits have a role in activating dormant origins during replicative stress in xenopus and human cells  <cit> .
in addition, mcm subunits have been implicated in other cellular processes such as gene expression  <cit> , and maintenance of genomic stability  <cit> .
mcms also interact with chromatin remodeling factors and histones  <cit> .
mcm4/6/ <dig> forms one half of the mcm2- <dig> hexamer ring and may be the catalytic core of the mcm2- <dig> helicase  <cit> .
differences between mcm4/6/ <dig> and mcm2- <dig> are used to investigate mcm2- <dig> mechanisms.
functional mcm4/6/ <dig> has been isolated as both a hexamer and a trimer  <cit> .
electron microscopy studies indicate that mcm4/6/ <dig> hexamers are ring shaped  <cit> .
the functional significance of these observations has not been explored.
here we show that mcm4/6/ <dig> exists in an equilibrium of oligomeric states in which smaller oligomers predominate in the absence of nucleotide.
atp or atpγs and to a lesser extent, adp shifts the equilibrium towards formation of larger oligomers.
use of mcm mutants that are impaired in the function of intersubunit atp sites  indicates that mcm4/6/ <dig> functions as a hexamer.
thus, one of the roles of atp in mcm4/6/ <dig> function is to promote oligomerization required for mcm4/6/ <dig> activity.
RESULTS
reconstitution of mcm4/6/ <dig> by anion exchange chromatography
mcm4/6/ <dig> was reconstituted from individual subunits and isolated by anion exchange chromatograpy.
a portion of each fraction was analyzed by coomassie brilliant blue-stained sds-page to determine the presence of each protein .
in addition, the ability of each fraction to hydrolyze atp or to unwind a synthetic fork substrate was determined .
both atpase activity and dna unwinding activity co-eluted with fractions containing equal ratios of the three proteins.
the lack of activity with individual mcm subunits  and the activity associated with co-elution of all three subunits suggested that an active complex was formed.
we then examined mcm4/6/ <dig> by gel filtration analysis to determine the size of the active complex .
a peak fraction from the mono q elution containing equal ratios of mcm <dig>  mcm <dig> and mcm <dig> was analyzed by gel filtration.
comparison with size standards indicated that the elution of mcm4/6/ <dig> peaked around fraction  <dig>  corresponding to an apparent molecular size of approximately  <dig> kda, close to the predicted molecular size of trimer containing one of each subunit .
consistent with a trimer, densitometric analysis indicated that the peak fractions contained equal ratios of mcm <dig>  mcm <dig> and mcm <dig>  most importantly, it was clear that the peak of mcm4/6/ <dig>  although fairly broad, did not elute as a hexamer .
we were concerned that the dilution of mcm4/6/ <dig> before and during gel filtration may have resulted in the dissociation of mcm4/6/ <dig> hexamers.
consistent with this idea, the mcm4/6/ <dig> elution trails off into later fractions, suggesting a disruption of the complex into single subunits.
thus, gel filtration analysis was repeated at a 5-fold higher protein concentration.
approximately  <dig> nmol each of mcm <dig>  mcm <dig> and mcm <dig> were mixed and then applied to a gel filtration column.
a portion of each fraction was analyzed by coomassie-stained sds-page as well as examined for dna unwinding and atp hydrolysis .
the resulting complex was active and the peak of activity co-eluted with the peak containing all three proteins and eluted earlier from the gel filtration column than the individual proteins .
together, these observations suggest that mcm4/6/ <dig> formed and is active for dna unwinding and atp hydrolysis.
the peak elution of mcm4/6/ <dig>  corresponds to a size of approximately  <dig> kda, the predicted size of a trimer.
thus, even at the increased protein concentration used for this experiment, the peak of active mcm4/6/ <dig> eluted at a position inconsistent with a hexamer.
adenine nucleotides affect the oligomerization of mcm4/6/7
the oligomerization of helicases is often affected by the nucleotides to which they bind  <cit> .
to test whether atp promotes oligomerization of s. cerevisiae mcm4/6/ <dig>  we examined the effects of atp, atpγs and adp on the oligomerization of mcm4/6/ <dig> using gel filtration.
for these studies we used mcm4/6/ <dig> pre-formed in the absence of atp.
for comparison, the elution of mcm4/6/ <dig> from the gel filtration column in the absence of nucleotides from figure  <dig> is shown .
addition of atp to both the column buffer and the protein applied to the column promoted an increase in the apparent size of mcm4/6/ <dig> .
the elution of mcm4/6/ <dig> with atp is fairly broad, however the elution profile has shifted into earlier fractions than in the absence of nucleotide, suggesting an increase in size.
to examine whether atp hydrolysis by mcm4/6/ <dig> affects its oligomeric state, the effects of atpγs and adp were examined.
atpγs also supported elution of mcm4/6/ <dig> at an earlier volume than in the absence of nucleotide .
it should be noted that atpγs was only added to the protein and not to the column buffer in this experiment.
for comparison, we performed an experiment in which atp was only added to the protein and not the column buffer.
under these conditions, the peak of mcm4/6/ <dig> elution was similar to the elution in the absence of nucleotide .
these results suggest that while atp binding by mcm4/6/ <dig> promotes further oligomerization, production of adp has a destabilizing influence on mcm4/6/ <dig> oligomers.
to determine whether adp supports a shift in mcm4/6/ <dig> elution volume, we examined the elution profile of mcm4/6/ <dig> with adp present.
when mcm4/6/ <dig> + adp was analyzed through the gel filtration column equilibrated in nucleotide-free buffer, elution of mcm4/6/ <dig> closely resembled that of nucleotide free protein .
when adp was added to both the protein and the column equilibration buffer, there was a subtly and reproducibly different elution volume .
the intermediate size observed with adp likely results from an equilibrium between different forms of mcm4/6/ <dig> that exchange more rapidly than they are separated through the gel filtration resin.
indeed, the ratio of mcm <dig> to mcm4/ <dig> remains approximately 1: <dig> in the fractions from the adp experiment, consistent with hexamer and trimer and not other intermediate size complexes .
an exchange between different-sized complexes may also explain the relatively broad elution profile of mcm4/6/ <dig>  consistent with an equilibrium, the effect of atp on mcm4/6/ <dig> was reversible; removal of atp from mcm4/6/ <dig> hexamers and the buffer resulted in a return to elution of mcm4/6/ <dig> in the later fractions .
our observations are consistent with the idea that binding of atp or atpγs has a greater stabilizing effect on mcm4/6/ <dig> oligomerization than adp.
an alternative explanation is that atpγs is bound better than atp or adp by mcm4/6/ <dig> and hence atpγs more strongly promotes oligomerization.
we discounted this possibility with the observation that adp and atpγs inhibit atp hydrolysis by mcm4/6/ <dig> equally , suggesting that adp and atpγs are bound equally but have different effects on oligomerization.
these experiments suggested that atp binding promotes oligomerization of mcm4/6/ <dig> and that atp hydrolysis has a destabilizing influence.
chemical crosslinking of mcm4/6/7
to further explore the idea that atp promotes oligomerization of mcm4/6/ <dig>  we performed chemical crosslinking on mcm4/6/ <dig> with glutaraldehyde.
when mcm4/6/ <dig>  was treated with glutaraldehyde in the absence of nucleotide and then analyzed on  <dig> % acrylamide sds-phosphate gels, a portion of mcm4/6/ <dig> was found in three diffuse bands, labeled "i", "ii" and "iii" in figure 5a.
initially, we thought that band i corresponded to intramolecular crosslinked monomers, band ii corresponded to dimers  and band iii corresponded to trimers  as that is most consistent with the migration of size standards on sds-page.
however, when the products were analyzed by gel filtration, the elution volume was more consistent with a mixture of trimers and dimers rather than a mixture of monomers and dimers , suggesting that band i corresponds to dimer and band ii corresponds to trimers.
presumably, intramolecular crosslinks prevent complete unfolding of the proteins in sds and result in a more compact protein with increased mobility through sds-page.
notably, a hexamer-sized band was not detected in the absence of nucleotide.
in contrast, when  <dig> mm atp, atpγs or adp was incubated with the same preparation of mcm4/6/ <dig> before treatment with glutaraldehyde, the majority of the protein shifts up to a band that migrates just above the  <dig> kda marker on the sds-phosphate gel, consistent with the nucleotides supporting the formation of larger oligomers .
when the crosslinking reaction in the presence of atp was analyzed by gel filtration , mcm4/6/ <dig> eluted in two overlapping peaks .
the larger peak corresponded to a elution volume consistent with a hexamer whereas the second peak corresponded to a trimer-sized peak.
sds-phosphate gels of the gel filtration fractions confirmed that crosslinked hexamers  were present in the first peak and bands i, ii and iii were present in the second peak .
addition of  <dig>  mm or lower atp, or  <dig> mm amp did not support crosslinking of mcm4/6/ <dig> into the hexamer-sized band .
use of antibodies against mcm <dig> and mcm <dig> as well as radiolabeled mcm <dig> indicate that the crosslinked bands contain mcm <dig>  mcm <dig> and mcm <dig> .
these observations are consistent with the idea that binding of adenine nucleotides to mcm4/6/ <dig> promotes its oligomerization.
in contrast to the gel filtration experiments in figure  <dig>  no differences were observed between atp, atpγs and adp with the crosslinking.
it is important to note that crosslinking disrupts equilibria and in the experiments shown here, hexamers are trapped by crosslinking.
a similar disruption of equilibria by crosslinking has been observed previously  <cit> .
thus, we conclude that under the conditions utilized here, mcm4/6/ <dig> exists in an equilibrium of different oligomers.
in the absence of nucleotide, smaller oligomers predominate whereas in the presence of atp or atpγs, the equilibrium shifts towards larger oligomers, likely hexamers.
mcm4/6/ <dig> functions as a hexamer
the strongest evidence to date that mcm4/6/ <dig> functions as a hexamer is the finding that mcm <dig>  which inhibits dna unwinding by mcm4/6/ <dig> disrupts mcm4/6/ <dig> hexamers to form an inactive mcm2/4/6/ <dig> tetramer  <cit> .
with the observation that mcm <dig> required nucleotide to inhibit mcm4/6/ <dig> but not to interact with it  <cit>  as well as the observation here that active mcm4/6/ <dig> does not elute from gel filtration columns as a hexamer, the question of whether mcm4/6/ <dig> functions as a hexamer needs to be readdressed.
we used mutational analysis, making use of the observations that atp sites are formed at the interface of mcm subunits and residues from both subunits are required for activity  <cit> .
specifically, one subunit contributes an arginine residue  for catalysis of atp bound by the neighbouring subunit  <cit> .
mutation of the arginine fingers has been previously shown to interfere with atp hydrolysis but not subunit interaction of mcm proteins  <cit> , thus mutations in the arginine fingers are ideally suited to test the number of subunit interfaces required for full activity of mcm4/6/ <dig> .
if mcm4/6/ <dig> is a trimer, mutation of only two of the three arginine fingers to alanine will affect mcm4/6/ <dig> activity.
the third arginine  is not expected to affect activity since it is not engaged in an atp site.
in contrast, if mcm4/6/ <dig> is a hexamer then mutation of each of the arginine fingers would affect function.
the arginine finger mutants mcm4r701a, mcm6r708a and mcm7r593a were reconstituted with the corresponding wild-type proteins to yield mcm4ra/6/ <dig>  mcm4/6ra/ <dig> and mcm4/6/7ra, respectively .
next, the atpase and dna unwinding activities of the mutant complexes were compared to wild-type complex using the peak fractions from each of the columns.
we first examined our results to determine whether the mutant complexes had formed.
we noted that the elution of the mutant mcm4/6/ <dig> complexes from the gel filtration column was closely similar to that of wild-type complex and differed from the individual proteins .
in addition, atpase activity was detected with the mutant complexes, albeit at a lower rate than wild-type complex .
this lower activity did not appear to be due to contaminating activity from pair-wise combinations of mcm proteins.
our conclusion is based on the observations that the pairwise combinations of mcm <dig> and  <dig> and mcm <dig> and  <dig> have little or no atpase activity compared to mcm4/6/7; and that the extent to which mutation of mcm <dig> and mcm <dig> affect atp hydrolysis differs between the pair-wise complexes  and mcm4/6/ <dig> complexes .
we conclude from these results that the mutant mcm4/6/ <dig> complexes had formed.
with the assurance that the mutant complexes had formed, we then examined the results to determine whether mcm4/6/ <dig> functions as a trimer.
dna unwinding above background was not detected with mcm4ra/6/ <dig>  mcm4/6ra/ <dig> or mcm4/6/7ra, even at a concentration of protein at which wild-type complex unwinds approximately 70% of the substrate .
atp hydrolysis was about 35-50% of that by non-mutant complex .
these results support the idea that mcm4/6/ <dig> functions as a hexamer since all three arginine fingers and hence the formation of interfaces between each of the proteins is required for full function of mcm4/6/ <dig>
mcm4/6/ <dig> binds dna as a hexamer
if mcm4/6/ <dig> functions as a hexamer, then it should bind to dna as a hexamer.
to test this idea, we crosslinked mcm4/6/ <dig> bound to single stranded dna using glutaraldehyde .
to ensure single stranded dna, we used polydeoxythymidine  as the substrate.
mcm4/6/ <dig> was prepared in the absence of atp and then incubated with dna in the presence and absence of atp.
after crosslinking, the products were analyzed on a native gel along with size markers.
in the presence of mcm4/6/ <dig> and crosslinker, the dna migrated just below the thyroglobulin marker , consistent with the binding of hexamer to the dna .
staining of the gel with colloidal coomassie blue detected a single band at the same position as the dna .
furthermore, the migration of crosslinked mcm4/6/ <dig> through the gel is the same in the presence and absence of dna .
the hexamer-sized band is protein dependent  and is dependent on atp .
these experiments suggest that mcm4/6/ <dig> binds to single stranded dna as a hexamer.
thus, the accumulated evidence in this study suggests that mcm4/6/ <dig> functions as a hexamer and that formation of the hexamer is supported by atp.
discussion
we propose that mcm4/6/ <dig> exists in an equilibrium of oligomers that is shifted towards larger oligomers by atp binding.
mcm4/6/ <dig> that elutes from the gel filtration column as small oligomers, probably trimers, appears to be active.
this is in seeming contrast to the mutagenic analysis indicating that all three types of atp sites are required for mcm4/6/ <dig> function.
it should be noted that the assays of mcm4/6/ <dig> function required the addition of atp and this likely accounts for the activity of the smaller oligomers; atp shifts the equilibrium towards larger oligomers, likely hexamers thus forming the active complex.
these findings have implications for the use of mcm4/6/ <dig> as a probe for mcm2- <dig> mechanisms.
mcm4/6/ <dig> oligomerization
we suggest that mcm4/6/ <dig> oligomerizes into hexamers in the presence of atp.
densitometry on the sds-page gels of the fractions from the gel filtration columns in the presence of atp and atpγs indicate that there are equal ratios of mcm <dig>  mcm <dig> and mcm <dig> and thus are consistent with hexamers, but not other sized complexes that would be expected to contain extra copies of one or two of the subunits.
furthermore, the hexameric form of mcm4/6/ <dig> is the form that is most frequently reported in other studies  <cit> .
here, the elution profiles from the gel filtration columns are relatively broad suggesting that there are different sized species.
we suggest that mcm4/6/ <dig> exists in an equilibrium of different sized complexes that exchanges more rapidly than they are separated by gel filtration.
we carried out additional biophysical measurements to assist in determining the size of the mcm4/6/ <dig> complex in the presence and absence of adenine nucleotides such as analytical ultracentrifugation and dynamic light scattering.
while neither of these techniques was useful in determining a size, they both indicated that the size of mcm4/6/ <dig> increases when atp was added .
given the opposing influences of shape on gel filtration and sedimentation equilibrium ultracentrifugation as well as the dramatic increase in size in the cross-linking experiments, we concluded that the changes in elution in the gel filtration experiments dependent on atp are not solely due to conformational changes rather than changes in oligomeric forms.
the observation of smaller oligomers in the absence of nucleotide is consistent with studies on mammalian mcm4/6/ <dig> expressed in insect cells where trimers have been observed under certain conditions  <cit> , but contrasts with the observation of only hexamers with budding and fission yeast mcm4/6/ <dig>  <cit> .
the strongest contrast is the atp-dependence for oligomerization found in this study and the isolation of budding yeast mcm4/6/ <dig> hexamers by the scwacha group  <cit> .
there are many differences in the preparation of these complexes and a single factor that contributes to the differing oligomeric states cannot be identified.
however, it is interesting to note that there are also differences in activity between mcm4/6/ <dig> isolated as a hexamer versus mcm4/6/ <dig> isolated as a trimer.
specifically, mcm4/6/ <dig> isolated as a trimer binds to circular single stranded dna  <cit> .
in contrast, with mcm4/6/ <dig> isolated as a hexamer, single stranded circular dna was unable to compete mcm4/6/ <dig> away from linear oligonucleotides  <cit> .
consistent with this contrast, we have found that binding of mcm4/6/ <dig> to circular single stranded dna is inhibited by first incubating mcm4/6/ <dig> in atp or atpγs, which promotes hexamerization .
one explanation of these findings is that mcm4/6/ <dig> hexamers are unable to bind to circular single stranded dna because dna is unable to access its binding sites in the interior of the ring.
a similar effect has been observed with other helicases  <cit> .
note that crosslinking experiments suggest that mcm4/6/ <dig> bound to dna is a hexamer .
thus, it is the formation of hexamer before binding to dna and not formation of the hexamer per se that is inhibitory.
consistent with the lower dna binding when incubated in atp before dna, mcm4/6/ <dig> reconstituted in the presence of atp also had lower dna unwinding activity .
in addition to hexamers, there are also instances of mcm complexes as double hexamers.
based on its dna unwinding behaviour at "y"-shaped forks, s. pombe mcm4/6/ <dig> is proposed to be more processive as a double hexamer.
more recently, it was shown that s. cerevisiae mcm2- <dig> assembles on origins as a double hexamer  <cit> .
these observations are similar to double hexamers proposed for viral  <cit>  and bacterial  <cit>  replicative helicases.
formation of double hexamers of mcm complexes likely requires the presence of dna and/or origin assembly factors required for dna replication initiation.
atp and the oligomerization of helicases
nucleotides affect the oligomerization of other helicases such as bacteriophage t <dig> gene 4a  <cit> , e. coli dnab  <cit> , bacillus subtilus dnac  <cit>  and sv <dig> tag  <cit> .
similar to mcm proteins, the atp sites of the aforementioned proteins are located at the interface of subunits and thus are ideally located to affect oligomerization.
however, unlike mcms these helicases are comprised of several copies of the same subunit.
thus, subunit interfaces differ only by the nucleotide binding state of the subunits involved.
conversely, mcm4/6/ <dig> not only has nucleotide-defined differences at its subunit interfaces, but also differences in interfaces defined by subunit composition .
our model predicts that mcm4/6/ <dig> trimers would oligomerize into hexamers through interaction between mcm <dig> and mcm <dig>  the predicted interaction between mcm <dig> and mcm <dig> may be the weak link in formation of mcm4/6/ <dig> hexamers.
in our previous studies, we did not detect a pair-wise physical interaction between mcm <dig> and mcm <dig> under conditions where interactions of mcm <dig> with mcm <dig> or mcm <dig> were detected  <cit> .
in another study, co-expression of s. cerevisiae mcm <dig> and mcm <dig> in insect cells did not yield a stable complex  <cit> .
furthermore, we were unable to detect interaction between mcm <dig> and mcm <dig> in the presence of atp and glutaraldehyde , although a weak atpase activity was noted when mcm <dig> and mcm <dig> were combined .
in contrast, a synthetic lethal interaction between temperature sensitive alleles of genes encoding the s. pombe homologues of mcm <dig>  and mcm <dig>  as well as a weak physical interaction between the human proteins have been reported  <cit> .
it may be that a robust interaction between mcm <dig> and mcm <dig> requires mcm <dig> in budding yeast.
alternatively, the relative weakness of a mcm6-mcm <dig> interaction may be compensated for by the presence of two mcm6- <dig> interfaces in the mcm4/6/ <dig> hexamer.
mcm4/6/ <dig> atp site utilization
mutation of the arginine fingers in mcm <dig>  mcm <dig> or mcm <dig> resulted in complexes that were unable to unwind dna and had reduced atpase activity.
these observations suggested to us that mcm4/6/ <dig> does not function as a trimer.
in addition, the effects of the arginine finger mutation on atp hydrolysis by mcm4/6/ <dig> are not what one would expect if each of the atp sites functioned independently of the others and contributed equally to atp hydrolysis by the complex .
rather, larger decreases in hydrolysis were observed, in keeping with the idea that atp binding and/or hydrolysis at one site affects the activity of other subunits in the helicase ring.
a similar effect has been observed in other helicases, including mcm2- <dig>  <cit> .
indeed, mcm complexes provide an excellent system to study this phenomenon since mutation of one or two subunits with defined positions in the helicase ring is more easily achieved in a heterohexamer than a homohexamer.
the contribution that each atp site makes to the activity of the complex appears to be roughly equal .
this observation contrasts to the studies of you and colleagues on mouse mcm4/6/ <dig>  which indicated that each atp site made different contributions to atp hydrolysis, dna binding and dna unwinding  <cit> .
the mouse study used mutations in the walker a and b motifs, which in some proteins affects atp binding as well as hydrolysis and this may account for the differences.
mcm4/6/ <dig> within mcm2-7
one of the motivations for studying mcm4/6/ <dig> is to discern differences between mcm4/6/ <dig> and mcm2- <dig> that may yield clues to the mechanism of mcm2- <dig>  all of the subunits are essential for dna unwinding in vivo  <cit> .
in contrast, mcm4/6/ <dig> is sufficient for dna unwinding in vitro and mcm <dig> or mcm3/ <dig> inhibit dna unwinding  <cit> .
these observations have led to the suggestion that mcm <dig>   <dig> and  <dig> may play essential regulatory roles within mcm2- <dig>  recent mutational studies are consistent with that idea  <cit> .
however, the proposal that only a subset of mcm2- <dig> subunits participates directly in dna unwinding must be reconciled with studies on homohexameric helicases in which all subunits participate directly in the molecular mechanisms required for dna translocation and dna unwinding  <cit> .
our conclusion that the active form of mcm4/6/ <dig> is the hexamer and not a trimer is a first step towards addressing this issue.
still to be addressed are the questions of whether the requirement of a hexameric mcm4/6/ <dig> is due to all six subunits participating directly in dna unwinding or to formation of the ring, which tethers the helicase to dna and/or forms a steric block for dna unwinding.
CONCLUSIONS
we conclude that s. cerevisiae mcm4/6/ <dig> exists in an equilibrium of different oligomers in solution.
when atp, atpγs or adp is added to mcm4/6/ <dig>  the equilibrium shifts towards formation of larger oligomers.
finally, mutation of a residue important for the function of intersubunit atp sites indicates that the three types of atp site interfaces are required for full activity, consistent with mcm4/6/ <dig> functioning as a hexamer.

 helicases assume a variety of oligomeric forms from monomers and dimers to larger oligomers such as hexamers and dodecamers. the oligomeric form of a helicase is often important for its function. for example, hexameric helicases form ring structures that are thought to encircle single and/or double stranded dna as part of their unwinding function. furthermore, the number of ring constituents is often important for function; methanobacter thermoautotrophicum mcm, which is isolated as hexamers, heptamers or dodecamers, is thought to function as a hexamer. in contrast, some helicases are functional in different oligomeric forms, such as the hepatitis c virus ns helicase, which functions as a monomer, dimer or larger oligomers. the minichromosome maintenance proteins through form a ring-shaped heterohexamer that is the replicative helicase in eukaryotic cells. each of mcm through is essential for the initiation and elongation stages of dna replication and helicase activity has been detected not only with mcm2- but also with complexes containing different combinations of mcm proteins, including a heterohexamer comprised of mcm mcm and mcm that was initially purified from hela cells. the biological significance of these additional helicase complexes is not known. interestingly, mcm proteins are present in cells at copy numbers that are far in excess of what is expected for two replication forks per active origin. it has been proposed that the "excess" mcm subunits have a role in activating dormant origins during replicative stress in xenopus and human cells. in addition, mcm subunits have been implicated in other cellular processes such as gene expression, and maintenance of genomic stability. mcms also interact with chromatin remodeling factors and histones. mcm4/6/ forms one half of the mcm2- hexamer ring and may be the catalytic core of the mcm2- helicase. differences between mcm4/6/ and mcm2- are used to investigate mcm2- mechanisms. functional mcm4/6/ has been isolated as both a hexamer and a trimer. electron microscopy studies indicate that mcm4/6/ hexamers are ring shaped. the functional significance of these observations has not been explored. here we show that mcm4/6/ exists in an equilibrium of oligomeric states in which smaller oligomers predominate in the absence of nucleotide. atp or atpγs and to a lesser extent, adp shifts the equilibrium towards formation of larger oligomers. use of mcm mutants that are impaired in the function of intersubunit atp sites indicates that mcm4/6/ functions as a hexamer. thus, one of the roles of atp in mcm4/6/ function is to promote oligomerization required for mcm4/6/ activity.  reconstitution of mcm4/6/ by anion exchange chromatography mcm4/6/ was reconstituted from individual subunits and isolated by anion exchange chromatograpy. a portion of each fraction was analyzed by coomassie brilliant blue-stained sds-page to determine the presence of each protein. in addition, the ability of each fraction to hydrolyze atp or to unwind a synthetic fork substrate was determined. both atpase activity and dna unwinding activity co-eluted with fractions containing equal ratios of the three proteins. the lack of activity with individual mcm subunits and the activity associated with co-elution of all three subunits suggested that an active complex was formed. we then examined mcm4/6/ by gel filtration analysis to determine the size of the active complex. a peak fraction from the mono q elution containing equal ratios of mcm mcm and mcm was analyzed by gel filtration. comparison with size standards indicated that the elution of mcm4/6/ peaked around fraction corresponding to an apparent molecular size of approximately kda, close to the predicted molecular size of trimer containing one of each subunit. consistent with a trimer, densitometric analysis indicated that the peak fractions contained equal ratios of mcm mcm and mcm most importantly, it was clear that the peak of mcm4/6/ although fairly broad, did not elute as a hexamer. we were concerned that the dilution of mcm4/6/ before and during gel filtration may have resulted in the dissociation of mcm4/6/ hexamers. consistent with this idea, the mcm4/6/ elution trails off into later fractions, suggesting a disruption of the complex into single subunits. thus, gel filtration analysis was repeated at a 5-fold higher protein concentration. approximately nmol each of mcm mcm and mcm were mixed and then applied to a gel filtration column. a portion of each fraction was analyzed by coomassie-stained sds-page as well as examined for dna unwinding and atp hydrolysis. the resulting complex was active and the peak of activity co-eluted with the peak containing all three proteins and eluted earlier from the gel filtration column than the individual proteins. together, these observations suggest that mcm4/6/ formed and is active for dna unwinding and atp hydrolysis. the peak elution of mcm4/6/ corresponds to a size of approximately kda, the predicted size of a trimer. thus, even at the increased protein concentration used for this experiment, the peak of active mcm4/6/ eluted at a position inconsistent with a hexamer. adenine nucleotides affect the oligomerization of mcm4/6/7 the oligomerization of helicases is often affected by the nucleotides to which they bind. to test whether atp promotes oligomerization of s. cerevisiae mcm4/6/ we examined the effects of atp, atpγs and adp on the oligomerization of mcm4/6/ using gel filtration. for these studies we used mcm4/6/ pre-formed in the absence of atp. for comparison, the elution of mcm4/6/ from the gel filtration column in the absence of nucleotides from figure is shown. addition of atp to both the column buffer and the protein applied to the column promoted an increase in the apparent size of mcm4/6/. the elution of mcm4/6/ with atp is fairly broad, however the elution profile has shifted into earlier fractions than in the absence of nucleotide, suggesting an increase in size. to examine whether atp hydrolysis by mcm4/6/ affects its oligomeric state, the effects of atpγs and adp were examined. atpγs also supported elution of mcm4/6/ at an earlier volume than in the absence of nucleotide. it should be noted that atpγs was only added to the protein and not to the column buffer in this experiment. for comparison, we performed an experiment in which atp was only added to the protein and not the column buffer. under these conditions, the peak of mcm4/6/ elution was similar to the elution in the absence of nucleotide. these results suggest that while atp binding by mcm4/6/ promotes further oligomerization, production of adp has a destabilizing influence on mcm4/6/ oligomers. to determine whether adp supports a shift in mcm4/6/ elution volume, we examined the elution profile of mcm4/6/ with adp present. when mcm4/6/ + adp was analyzed through the gel filtration column equilibrated in nucleotide-free buffer, elution of mcm4/6/ closely resembled that of nucleotide free protein. when adp was added to both the protein and the column equilibration buffer, there was a subtly and reproducibly different elution volume. the intermediate size observed with adp likely results from an equilibrium between different forms of mcm4/6/ that exchange more rapidly than they are separated through the gel filtration resin. indeed, the ratio of mcm to mcm4/ remains approximately 1: in the fractions from the adp experiment, consistent with hexamer and trimer and not other intermediate size complexes. an exchange between different-sized complexes may also explain the relatively broad elution profile of mcm4/6/ consistent with an equilibrium, the effect of atp on mcm4/6/ was reversible; removal of atp from mcm4/6/ hexamers and the buffer resulted in a return to elution of mcm4/6/ in the later fractions. our observations are consistent with the idea that binding of atp or atpγs has a greater stabilizing effect on mcm4/6/ oligomerization than adp. an alternative explanation is that atpγs is bound better than atp or adp by mcm4/6/ and hence atpγs more strongly promotes oligomerization. we discounted this possibility with the observation that adp and atpγs inhibit atp hydrolysis by mcm4/6/ equally, suggesting that adp and atpγs are bound equally but have different effects on oligomerization. these experiments suggested that atp binding promotes oligomerization of mcm4/6/ and that atp hydrolysis has a destabilizing influence. chemical crosslinking of mcm4/6/7 to further explore the idea that atp promotes oligomerization of mcm4/6/ we performed chemical crosslinking on mcm4/6/ with glutaraldehyde. when mcm4/6/ was treated with glutaraldehyde in the absence of nucleotide and then analyzed on % acrylamide sds-phosphate gels, a portion of mcm4/6/ was found in three diffuse bands, labeled "i", "ii" and "iii" in figure 5a. initially, we thought that band i corresponded to intramolecular crosslinked monomers, band ii corresponded to dimers and band iii corresponded to trimers as that is most consistent with the migration of size standards on sds-page. however, when the products were analyzed by gel filtration, the elution volume was more consistent with a mixture of trimers and dimers rather than a mixture of monomers and dimers, suggesting that band i corresponds to dimer and band ii corresponds to trimers. presumably, intramolecular crosslinks prevent complete unfolding of the proteins in sds and result in a more compact protein with increased mobility through sds-page. notably, a hexamer-sized band was not detected in the absence of nucleotide. in contrast, when mm atp, atpγs or adp was incubated with the same preparation of mcm4/6/ before treatment with glutaraldehyde, the majority of the protein shifts up to a band that migrates just above the kda marker on the sds-phosphate gel, consistent with the nucleotides supporting the formation of larger oligomers. when the crosslinking reaction in the presence of atp was analyzed by gel filtration, mcm4/6/ eluted in two overlapping peaks. the larger peak corresponded to a elution volume consistent with a hexamer whereas the second peak corresponded to a trimer-sized peak. sds-phosphate gels of the gel filtration fractions confirmed that crosslinked hexamers were present in the first peak and bands i, ii and iii were present in the second peak. addition of mm or lower atp, or mm amp did not support crosslinking of mcm4/6/ into the hexamer-sized band. use of antibodies against mcm and mcm as well as radiolabeled mcm indicate that the crosslinked bands contain mcm mcm and mcm. these observations are consistent with the idea that binding of adenine nucleotides to mcm4/6/ promotes its oligomerization. in contrast to the gel filtration experiments in figure no differences were observed between atp, atpγs and adp with the crosslinking. it is important to note that crosslinking disrupts equilibria and in the experiments shown here, hexamers are trapped by crosslinking. a similar disruption of equilibria by crosslinking has been observed previously. thus, we conclude that under the conditions utilized here, mcm4/6/ exists in an equilibrium of different oligomers. in the absence of nucleotide, smaller oligomers predominate whereas in the presence of atp or atpγs, the equilibrium shifts towards larger oligomers, likely hexamers. mcm4/6/ functions as a hexamer the strongest evidence to date that mcm4/6/ functions as a hexamer is the finding that mcm which inhibits dna unwinding by mcm4/6/ disrupts mcm4/6/ hexamers to form an inactive mcm2/4/6/ tetramer. with the observation that mcm required nucleotide to inhibit mcm4/6/ but not to interact with it as well as the observation here that active mcm4/6/ does not elute from gel filtration columns as a hexamer, the question of whether mcm4/6/ functions as a hexamer needs to be readdressed. we used mutational analysis, making use of the observations that atp sites are formed at the interface of mcm subunits and residues from both subunits are required for activity. specifically, one subunit contributes an arginine residue for catalysis of atp bound by the neighbouring subunit. mutation of the arginine fingers has been previously shown to interfere with atp hydrolysis but not subunit interaction of mcm proteins, thus mutations in the arginine fingers are ideally suited to test the number of subunit interfaces required for full activity of mcm4/6/. if mcm4/6/ is a trimer, mutation of only two of the three arginine fingers to alanine will affect mcm4/6/ activity. the third arginine is not expected to affect activity since it is not engaged in an atp site. in contrast, if mcm4/6/ is a hexamer then mutation of each of the arginine fingers would affect function. the arginine finger mutants mcm4r701a, mcm6r708a and mcm7r593a were reconstituted with the corresponding wild-type proteins to yield mcm4ra/6/ mcm4/6ra/ and mcm4/6/7ra, respectively. next, the atpase and dna unwinding activities of the mutant complexes were compared to wild-type complex using the peak fractions from each of the columns. we first examined our results to determine whether the mutant complexes had formed. we noted that the elution of the mutant mcm4/6/ complexes from the gel filtration column was closely similar to that of wild-type complex and differed from the individual proteins. in addition, atpase activity was detected with the mutant complexes, albeit at a lower rate than wild-type complex. this lower activity did not appear to be due to contaminating activity from pair-wise combinations of mcm proteins. our conclusion is based on the observations that the pairwise combinations of mcm and and mcm and have little or no atpase activity compared to mcm4/6/7; and that the extent to which mutation of mcm and mcm affect atp hydrolysis differs between the pair-wise complexes and mcm4/6/ complexes. we conclude from these results that the mutant mcm4/6/ complexes had formed. with the assurance that the mutant complexes had formed, we then examined the results to determine whether mcm4/6/ functions as a trimer. dna unwinding above background was not detected with mcm4ra/6/ mcm4/6ra/ or mcm4/6/7ra, even at a concentration of protein at which wild-type complex unwinds approximately 70% of the substrate. atp hydrolysis was about 35-50% of that by non-mutant complex. these results support the idea that mcm4/6/ functions as a hexamer since all three arginine fingers and hence the formation of interfaces between each of the proteins is required for full function of mcm4/6/ mcm4/6/ binds dna as a hexamer if mcm4/6/ functions as a hexamer, then it should bind to dna as a hexamer. to test this idea, we crosslinked mcm4/6/ bound to single stranded dna using glutaraldehyde. to ensure single stranded dna, we used polydeoxythymidine as the substrate. mcm4/6/ was prepared in the absence of atp and then incubated with dna in the presence and absence of atp. after crosslinking, the products were analyzed on a native gel along with size markers. in the presence of mcm4/6/ and crosslinker, the dna migrated just below the thyroglobulin marker, consistent with the binding of hexamer to the dna. staining of the gel with colloidal coomassie blue detected a single band at the same position as the dna. furthermore, the migration of crosslinked mcm4/6/ through the gel is the same in the presence and absence of dna. the hexamer-sized band is protein dependent and is dependent on atp. these experiments suggest that mcm4/6/ binds to single stranded dna as a hexamer. thus, the accumulated evidence in this study suggests that mcm4/6/ functions as a hexamer and that formation of the hexamer is supported by atp. discussion we propose that mcm4/6/ exists in an equilibrium of oligomers that is shifted towards larger oligomers by atp binding. mcm4/6/ that elutes from the gel filtration column as small oligomers, probably trimers, appears to be active. this is in seeming contrast to the mutagenic analysis indicating that all three types of atp sites are required for mcm4/6/ function. it should be noted that the assays of mcm4/6/ function required the addition of atp and this likely accounts for the activity of the smaller oligomers; atp shifts the equilibrium towards larger oligomers, likely hexamers thus forming the active complex. these findings have implications for the use of mcm4/6/ as a probe for mcm2- mechanisms. mcm4/6/ oligomerization we suggest that mcm4/6/ oligomerizes into hexamers in the presence of atp. densitometry on the sds-page gels of the fractions from the gel filtration columns in the presence of atp and atpγs indicate that there are equal ratios of mcm mcm and mcm and thus are consistent with hexamers, but not other sized complexes that would be expected to contain extra copies of one or two of the subunits. furthermore, the hexameric form of mcm4/6/ is the form that is most frequently reported in other studies. here, the elution profiles from the gel filtration columns are relatively broad suggesting that there are different sized species. we suggest that mcm4/6/ exists in an equilibrium of different sized complexes that exchanges more rapidly than they are separated by gel filtration. we carried out additional biophysical measurements to assist in determining the size of the mcm4/6/ complex in the presence and absence of adenine nucleotides such as analytical ultracentrifugation and dynamic light scattering. while neither of these techniques was useful in determining a size, they both indicated that the size of mcm4/6/ increases when atp was added. given the opposing influences of shape on gel filtration and sedimentation equilibrium ultracentrifugation as well as the dramatic increase in size in the cross-linking experiments, we concluded that the changes in elution in the gel filtration experiments dependent on atp are not solely due to conformational changes rather than changes in oligomeric forms. the observation of smaller oligomers in the absence of nucleotide is consistent with studies on mammalian mcm4/6/ expressed in insect cells where trimers have been observed under certain conditions, but contrasts with the observation of only hexamers with budding and fission yeast mcm4/6/. the strongest contrast is the atp-dependence for oligomerization found in this study and the isolation of budding yeast mcm4/6/ hexamers by the scwacha group. there are many differences in the preparation of these complexes and a single factor that contributes to the differing oligomeric states cannot be identified. however, it is interesting to note that there are also differences in activity between mcm4/6/ isolated as a hexamer versus mcm4/6/ isolated as a trimer. specifically, mcm4/6/ isolated as a trimer binds to circular single stranded dna. in contrast, with mcm4/6/ isolated as a hexamer, single stranded circular dna was unable to compete mcm4/6/ away from linear oligonucleotides. consistent with this contrast, we have found that binding of mcm4/6/ to circular single stranded dna is inhibited by first incubating mcm4/6/ in atp or atpγs, which promotes hexamerization. one explanation of these findings is that mcm4/6/ hexamers are unable to bind to circular single stranded dna because dna is unable to access its binding sites in the interior of the ring. a similar effect has been observed with other helicases. note that crosslinking experiments suggest that mcm4/6/ bound to dna is a hexamer. thus, it is the formation of hexamer before binding to dna and not formation of the hexamer per se that is inhibitory. consistent with the lower dna binding when incubated in atp before dna, mcm4/6/ reconstituted in the presence of atp also had lower dna unwinding activity. in addition to hexamers, there are also instances of mcm complexes as double hexamers. based on its dna unwinding behaviour at "y"-shaped forks, s. pombe mcm4/6/ is proposed to be more processive as a double hexamer. more recently, it was shown that s. cerevisiae mcm2- assembles on origins as a double hexamer. these observations are similar to double hexamers proposed for viral and bacterial replicative helicases. formation of double hexamers of mcm complexes likely requires the presence of dna and/or origin assembly factors required for dna replication initiation. atp and the oligomerization of helicases nucleotides affect the oligomerization of other helicases such as bacteriophage t gene 4a, e. coli dnab, bacillus subtilus dnac and sv tag. similar to mcm proteins, the atp sites of the aforementioned proteins are located at the interface of subunits and thus are ideally located to affect oligomerization. however, unlike mcms these helicases are comprised of several copies of the same subunit. thus, subunit interfaces differ only by the nucleotide binding state of the subunits involved. conversely, mcm4/6/ not only has nucleotide-defined differences at its subunit interfaces, but also differences in interfaces defined by subunit composition. our model predicts that mcm4/6/ trimers would oligomerize into hexamers through interaction between mcm and mcm the predicted interaction between mcm and mcm may be the weak link in formation of mcm4/6/ hexamers. in our previous studies, we did not detect a pair-wise physical interaction between mcm and mcm under conditions where interactions of mcm with mcm or mcm were detected. in another study, co-expression of s. cerevisiae mcm and mcm in insect cells did not yield a stable complex. furthermore, we were unable to detect interaction between mcm and mcm in the presence of atp and glutaraldehyde, although a weak atpase activity was noted when mcm and mcm were combined. in contrast, a synthetic lethal interaction between temperature sensitive alleles of genes encoding the s. pombe homologues of mcm and mcm as well as a weak physical interaction between the human proteins have been reported. it may be that a robust interaction between mcm and mcm requires mcm in budding yeast. alternatively, the relative weakness of a mcm6-mcm interaction may be compensated for by the presence of two mcm6- interfaces in the mcm4/6/ hexamer. mcm4/6/ atp site utilization mutation of the arginine fingers in mcm mcm or mcm resulted in complexes that were unable to unwind dna and had reduced atpase activity. these observations suggested to us that mcm4/6/ does not function as a trimer. in addition, the effects of the arginine finger mutation on atp hydrolysis by mcm4/6/ are not what one would expect if each of the atp sites functioned independently of the others and contributed equally to atp hydrolysis by the complex. rather, larger decreases in hydrolysis were observed, in keeping with the idea that atp binding and/or hydrolysis at one site affects the activity of other subunits in the helicase ring. a similar effect has been observed in other helicases, including mcm2-. indeed, mcm complexes provide an excellent system to study this phenomenon since mutation of one or two subunits with defined positions in the helicase ring is more easily achieved in a heterohexamer than a homohexamer. the contribution that each atp site makes to the activity of the complex appears to be roughly equal. this observation contrasts to the studies of you and colleagues on mouse mcm4/6/ which indicated that each atp site made different contributions to atp hydrolysis, dna binding and dna unwinding. the mouse study used mutations in the walker a and b motifs, which in some proteins affects atp binding as well as hydrolysis and this may account for the differences. mcm4/6/ within mcm2-7 one of the motivations for studying mcm4/6/ is to discern differences between mcm4/6/ and mcm2- that may yield clues to the mechanism of mcm2- all of the subunits are essential for dna unwinding in vivo. in contrast, mcm4/6/ is sufficient for dna unwinding in vitro and mcm or mcm3/ inhibit dna unwinding. these observations have led to the suggestion that mcm and may play essential regulatory roles within mcm2- recent mutational studies are consistent with that idea. however, the proposal that only a subset of mcm2- subunits participates directly in dna unwinding must be reconciled with studies on homohexameric helicases in which all subunits participate directly in the molecular mechanisms required for dna translocation and dna unwinding. our conclusion that the active form of mcm4/6/ is the hexamer and not a trimer is a first step towards addressing this issue. still to be addressed are the questions of whether the requirement of a hexameric mcm4/6/ is due to all six subunits participating directly in dna unwinding or to formation of the ring, which tethers the helicase to dna and/or forms a steric block for dna unwinding.  we conclude that s. cerevisiae mcm4/6/ exists in an equilibrium of different oligomers in solution. when atp, atpγs or adp is added to mcm4/6/ the equilibrium shifts towards formation of larger oligomers. finally, mutation of a residue important for the function of intersubunit atp sites indicates that the three types of atp site interfaces are required for full activity, consistent with mcm4/6/ functioning as a hexamer. 
in addition, we found that atp, which is required for mcm4/6/ activity, shifts the equilibrium towards larger oligomers, likely hexamers of mcm4/6/ atpγs and to a lesser extent adp also shift the equilibrium towards hexamers. the functional significance of the different mcm4/6/ oligomeric states has not been assessed. study of mcm4/6/ complexes containing mutations that interfere with the formation of inter-subunit atp sites indicates that full activity of mcm4/6/ requires all of its atp sites, which are formed in a hexamer and not a trimer. the results of such an assessment would have implications for studies of both mcm4/6/ and mcm2- here, we show that saccharomyces cerevisiae mcm4/6/ reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of atp. minichromosome maintenance proteins and are related by sequence and form a variety of complexes that unwind dna, including mcm4/6/ a mcm4/6/ trimer forms one half of the mcm2- hexameric ring and can be thought of as the catalytic core of mcm2- the replicative helicase in eukaryotic cells.
 minichromosome maintenance proteins and are related by sequence and form a variety of complexes that unwind dna, including mcm4/6/ a mcm4/6/ trimer forms one half of the mcm2- hexameric ring and can be thought of as the catalytic core of mcm2- the replicative helicase in eukaryotic cells. oligomeric analysis of mcm4/6/ suggests that it forms a hexamer containing two mcm4/6/ trimers, however, under certain conditions trimeric mcm4/6/ has also been observed. the functional significance of the different mcm4/6/ oligomeric states has not been assessed. the results of such an assessment would have implications for studies of both mcm4/6/ and mcm2-  here, we show that saccharomyces cerevisiae mcm4/6/ reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of atp. in addition, we found that atp, which is required for mcm4/6/ activity, shifts the equilibrium towards larger oligomers, likely hexamers of mcm4/6/ atpγs and to a lesser extent adp also shift the equilibrium towards hexamers. study of mcm4/6/ complexes containing mutations that interfere with the formation of inter-subunit atp sites indicates that full activity of mcm4/6/ requires all of its atp sites, which are formed in a hexamer and not a trimer. in keeping with this observation, mcm4/6/ binds dna as a hexamer.  the minimal functional unit of mcm4/6/ is a hexamer. one of the roles of atp binding by mcm4/6/ may be to stabilize formation of hexamers. 