BACKGROUND
dna replication in eukaryotes begins with the assembly of the pre-replicative complex comprising of replication initiators, the origin recognition complex, cdc <dig>  cdt <dig>  and the replicative helicase, mcm2- <dig> complex  <cit> .
increase in the activity of cyclin dependent kinases and loading of replication factor, mcm <dig> marks the transition from g <dig> to s phase.
these events promote the loading of cdc <dig>  rpa and dna polymerases at the replication origins to initiate dna synthesis.
the essential requirement of mcm <dig> in dna replication initiation and elongation has been exhibited across species  <cit> .
mutations in mcm <dig> cause a decrease in initiation of replication, slow progression of dna synthesis and stalling of replication forks during elongation  <cit> .
since mcm <dig> is essential for replication initiation and elongation, its activity is regulated in a cell-cycle manner to ensure a single round of replication.
s. cerevisiae mcm <dig> protein is present in all phases of the cell cycle though its association with chromatin is regulated to ensure replication licensing  <cit> .
human mcm <dig> protein is known to decrease in the early g <dig> phase  <cit> .
the levels of mcm <dig> mrna have been evaluated as the cells pass from m-phase into g <dig> phase.
though the mcm <dig> protein decreased, the levels of mcm <dig> mrna increased within the same time period.
these results demonstrate that the decrease in the mcm <dig> activity during the g <dig> phase is not due to decrease in transcription but because of protein turnover.
in this communication, we report that proteolysis of human mcm <dig> protein initiates before the onset of mitosis.
in an immunoblot with anti-mcm <dig> antibody, we observed that the m-phase blocked cells have downregulated the mcm <dig> protein.
on the basis of single cell immunofluorescence, we show that asynchronously growing cells in different phases of mitosis have reduced levels of mcm <dig>  also, mcm <dig> downregulation in m-phase is independent of the apc recognition motifs: the destruction and the ken box.
we demonstrate that mcm <dig> degradation is not dependent on anaphase promoting complex or on its recognition motifs but rather mediated by non-overlapping regions, apparently employing a redundant mechanism to ensure downregulation.
RESULTS
cell-cycle proteolysis of mcm <dig> initiates before the onset of mitosis
published reports indicate that the nocodazole blocked cells retain mcm <dig> protein which decreases in late m-phase and starts increasing  <dig> h after release  <cit> .
when mg <dig> was added to nocodazole released cells, there was an increase in mcm <dig> levels suggesting that the degradation that was occurring during the late m-phase was blocked by proteasome inhibitors.
in another report by the same group, a stable hela cell line which expressed green fluorescent protein-tagged mcm <dig> from a human cytomegalovirus immediate early promoter was established and authors observed that the gfp-mcm <dig> protein was detectable by immunofluorescence during mitosis  <cit> .
we looked at the levels of mcm <dig> and observed that they were significantly reduced in nocodazole blocked u2os cells .
mcm <dig> appeared after 4- <dig> hours of release from nocodazole block.
this is in contrast to the results obtained by hanaoka and coworkers, who observed mcm <dig> band by immunoblotting with anti-rabbit antibody, which was raised against 127- <dig> aa region of mcm <dig>  therefore, we wanted to rule out that the absence of mcm <dig> signal was due to limited immunoreactivity of the antibody used by us.
we have used a polyclonal rabbit antibody, ab , which is raised against the full-length protein, but showed weak immunoreactivity against the c-terminal protein  <cit> .
however, similar results were obtained with another polyclonal rabbit antibody, ab , which shows immunoreactivity against all regions of mcm <dig> .
as reported previously, the specificity of both these antibodies in immunoblotting assays have been established by rnai   <cit> .
in order to rule out that the decrease of mcm <dig> is an artifact generated by nocodazole toxicity, we blocked the cells with other reagents in m-phase and assayed the stability of mcm <dig>  vincristine binds to tubulin dimers and inhibits the assembly of microtubule structures, blocking cells in metaphase.
colchicine inhibits microtubule polymerization by binding to tubulin and thereby blocks cells in metaphase.
taxol hyper-stabilizes the beta subunit of tubulin, which is then unable to disassemble inducing a cell-cycle block at the metaphase/anaphase transition.
u2os cells blocked with any of these drugs displayed low levels of mcm <dig> .
on the basis of the above data, we conclude that human cells have low levels of mcm <dig> in mitosis.
as reported previously, we observed that the drop in mcm <dig> levels during mitosis is dependent on the proteasome .
to rule out that loss of recognition of mcm <dig> during mitosis could be due to epitope masking, we established that the mcm <dig> antibody recognizes the mitotic forms of mcm <dig>  since mcm <dig> is naturally proteolyzed during mitosis we expressed the ha-tagged ntd+id domain which is resistant to cell cycle-regulated degradation and tested whether the anti-mcm <dig> antibody  recognizes this forms of mcm <dig> during m-phase.
u2os cells expressing the ntd+id domain of mcm <dig> were blocked with nocodazole, released and harvested at regular intervals in order to collect cells in different phases of the cell cycle .
as observed previously, an anti-ha antibody immunoblot confirmed that the ntd+id domain of mcm <dig> was resistant to degradation in nocodazole blocked cells.
an anti-mcm <dig> antibody immunoblot displayed that the levels of endogenous mcm <dig> levels were low in m phase and increased around  <dig> h after nocodazole release but the ntd+id domain recognized through the same antibody did not show a decrease in levels, displaying that the mcm <dig> antibody recognizes the mitotic forms of mcm <dig>
single-cell imaging displays reduced levels of mcm <dig> during mitosis
we also looked at mcm <dig> levels in single cells as they passed through interphase and various phases of mitosis.
hela cells were fixed and visualized for endogenous mcm <dig> using rabbit polyclonal anti-mcm <dig> antibody, ab , in combination with an anti-rabbit tritc secondary antibody while microtubules were visualized with a mouse monoclonal antibody to alpha-tubulin conjugated to fitc .
the specificity of the antibody in immunofluorescence assays has been established by rnai .
from the asynchronously growing culture, we identified cells in prophase, metaphase, anaphase and telophase, and assayed for mcm <dig> and alpha-tubulin localization .
we observed that the cells in prophase or prometaphase, with condensed chromatin and bi-polar centrosomes, were negative for mcm <dig> signal .
in the same fields, we could observe interphase cells that were positive for mcm <dig> signal, ruling out errors in our visualization method .
as previously reported, not all interphase cells are positive for mcm <dig> signal and presumably these are the cells in early g <dig> phase.
similarly, cells in metaphase, identified by the presence of equatorial plate and polar microtubules, were also negative for mcm <dig> signal .
cells in anaphase and telophase identified by segregated sister chromatids and shortened kinetochore microtubules, were also negative for mcm <dig> signal .
to confirm that the loss of detection of mcm <dig> is not due to the protein becoming soluble during mitosis and that our immunofluorescence protocol can detect soluble proteins during mitosis, we have compared the levels of mcm <dig> and cyclin b in nocodazole blocked cells.
hela cells arrested in prometaphase  were negative for mcm <dig> signal but retained the cyclin b signal, establishing the accuracy of our immunofluorescence assay .
therefore, our results demonstrate that the levels of mcm <dig> protein are significantly reduced during mitosis.
it is known that nocodazole interferes with the polymerization of microtubules and cells treated with nocodazole enter mitosis but cannot form metaphase spindles thereby activating the spindle assembly checkpoint which causes an arrest in prometaphase.
as explained earlier in figure  <dig>  hela cells were treated with colchicine, nocodazole, taxol or vincristine for  <dig> h to obtain cells arrested in prometaphase.
incubation with any of these drugs resulted in disorganized microtubules and condensed chromosomes, confirming a m-phase block .
prometaphase cells have been marked by arrowheads and we observed that these cells were negative for mcm <dig> signal confirming that mcm <dig> degradation has been initiated at this stage of mitosis.
we determined the levels of endogenous mcm <dig> and cyclin a in an asynchronously growing culture of hela cells by immunofluorescence using a rabbit anti-mcm <dig> antibody, ab , and mouse anti-cyclin a antibody respectively .
utilizing the same antibodies, we have demonstrated that the cyclin a levels begin to increase in the s phase and it is degraded in the m-phase .
we observed that mcm <dig> was present till the g <dig> phase as almost all the cells  that retained cyclin a signal also stained positive for mcm <dig> .
combining the above results, we establish that mcm <dig> is present till the g <dig> phase and is degraded around the g2/m boundary.
live-cell imaging demonstrates that mcm <dig> is absent during the m-phase
an alternate approach to address the levels of mcm <dig> during mitosis would be to evaluate the cycling of mcm <dig> in live cells.
since it has been reported that mcm <dig> protein expression is not mainly regulated at the transcriptional level, we expressed egfp-tagged mcm <dig> from cytomegalovirus immediate early promoter  <cit> .
hela cells were transfected with pegfp c3-mcm <dig> and two days after transfection, we observed a  <dig> kda band with mcm <dig> immunoblot, confirming the expression of egfp-tagged mcm <dig> .
hela cells transfected with blank pegfp-c <dig> vector displayed cytoplasmic egfp signal while egfp expressed in fusion with mcm <dig> was guided to the nucleus .
we evaluated the egfp signal during mitosis, when the cells appear rounded in phase-contrast microscope.
we expressed the egfp-tagged full-length mcm <dig> in  <dig> cells and tracked the egfp signal and cell division of  <dig> cells .
the m-phase is known to last around 1- <dig>  h and as shown in figure  <dig>  we located a cell that is in mitosis and undergoes cytokinesis after  <dig> h  <dig> min .
we observed that the cell was negative for mcm <dig> during mitosis but the daughter cells accumulated mcm <dig> after  <dig> h, during the g <dig> phase of the cell cycle.
the overexpression of mcm <dig> leads to a cell-cycle block during interphase and therefore the progression of cells from g <dig> to m could not be observed.
we have reported that the  <dig> aa zf motif  of mcm <dig> is sufficient for m phase proteolysis and therefore we expressed egfp-tagged zf motif of mcm <dig> and tracked the egfp signal and cell division of hela cells by fluorescence and phase-contrast microscopy respectively as the cells progressed through mitosis.
as shown in figure s4c , we located a mitotic cell that undergoes cytokines after  <dig> h  <dig> min .
we observed that the cell was negative for mcm <dig> during mitosis but the daughter cells accumulated mcm <dig> after around  <dig> h. therefore, utilizing a different cell-line, we establish that the full-length mcm <dig> is low during mitosis.
summing up, using this antibody-independent approach, we demonstrated that the mcm <dig> levels are low during mitosis.
apc is not required for cell cycle degradation of mcm10
apc/c or cyclosome is a complex of many proteins that functions as an e <dig> ubiquitin ligase during mitosis.
since mcm <dig> is low in the m-phase, there is a distinct possibility that apc is involved in degradation of mcm <dig>  notably, mcm <dig> contains destruction box sequence  and a ken box, motifs that are required for recognition of substrates by apc  <cit> .
to test whether apc mediates the m-phase degradation of mcm <dig>  we silenced apc <dig> subunit of apc, which would effectively debilitate its ubiquitination ability.
hela cells transfected with apc <dig> sirna for three consecutive days were blocked in the m-phase with nocodazole for  <dig> h and subsequently released into nocodazole free medium.
rnai against apc <dig> decreased the target protein and mrna but that did not increase the levels of mcm <dig> in nocodazole blocked cells .
rnai effectively inhibited the apc activity as evidenced by increase in the levels of cyclin b .
this strongly suggests that apc is not involved in the cell-cycle degradation of mcm <dig>  we have assayed the mcm <dig> levels in asynchronous populations of cells transfected with apc <dig>  cul <dig>  cdh <dig>  cdc <dig>  beta-trcp and fbxw <dig> or control gl <dig> sirna and observed that only minor variations were observed in mcm <dig> levels .
we have previously reported that uv-irradiation specifically proteolyses mcm <dig> and we have identified the e <dig> ubiquitin ligase mediates the uv-triggered and m-phase proteolysis of mcm <dig>  <cit> .
apc recognition motifs are not required for cell cycle degradation of mcm10
we have previously utilized stable u2os cells expressing ha-tagged mcm <dig>  to determine the segments of mcm <dig> that are essential for cell cycle-regulated degradation  <cit> .
full-length mcm <dig> can be broadly divided into n-terminal , inner , linker  and c-terminal  domains .
a coiled-coil motif is present within the n-terminus of mcm <dig>  which is required for homodimerization.
the inner and c-terminal domains, which contain zinc finger  and winged helix  motifs, bind to single- and double-stranded dna, and the p <dig> subunit of dna polymerase-alpha  <cit> .
as reported previously, we observed that the ha-tagged full length mcm <dig> protein behaves like the endogenous protein  <cit> .
u2os cells expressing ha-tagged full length mcm <dig> were blocked with nocodazole, released and harvested at regular intervals in order to collect cells in different phases of the cell cycle.
endogenous mcm <dig> levels were low in m phase and increased around  <dig> h after release from nocodazole block .
ha-tagged full-length mcm <dig> showed a degradation pattern similar to that of the endogenous protein, which demonstrates that our assay for evaluating the degradation of different regions of mcm <dig> is accurate.
we observed that the ntd+id domain of mcm <dig>  which contains the ken box, was resistant to cell cycle-regulated degradation.
in the present study, we tested whether the wh motif, that contains the destruction box, is proteolyzed during the m-phase.
stable u2os cells expressing wh motif of mcm <dig>  was blocked with nocodazole, which arrested the cells in m-phase.
the obtained mitotic cells were released from the block and harvested at regular intervals in order to collect cells in different phases of the cell cycle.
the flow cytometry profile of propidium iodide-stained dna and the levels of cyclin b demonstrate a block in m-phase and subsequent progression through the cell cycle .
as noted previously, mcm <dig> was absent in nocodazole-released cells, demonstrating the natural proteolysis of mcm <dig> in m phase which began to increase after  <dig> h, displaying the natural cycling of mcm <dig> levels .
full-length mcm <dig> expressed from the retroviral vector showed a degradation pattern similar to that of the endogenous protein, validating our assay for evaluating the cell cycle-regulated degradation of mcm <dig>  we observed that the wh motif fragment was resistant to cell cycle-regulated degradation.
we confirmed that the nuclear localization signal expressed in fusion with the wh motif steered it to the nucleus and therefore change in cellular localization is not the reason for resistance to proteolysis .
we have previously reported that the lnk domain which does not have either the ken box or destruction box is proteolyzed as cells pass through m-phase.
therefore, we conclude that the ken and destruction box of mcm <dig> are neither essential nor sufficient for its m-phase proteolysis.
mcm <dig> proteolysis utilizes independent non-overlapping signals
to identify the domains required for mcm <dig> proteolysis during m-phase, we expressed different regions of mcm <dig> and assayed their stability.
as noted previously, the ntd+id domain was resistant to cell cycle-regulated degradation, but the lnk and ctd domains were proteolyzed in m phase .
though the zf motif was sufficient for m phase proteolysis of mcm <dig>  the ctd domain lacking the zf motif was also degraded in m phase, signifying that though zf motif is sufficient, it is not essential for m phase proteolysis of mcm <dig> .
we now wanted to identify the degron that is essential for degradation.
we further divided the ctd  into three fragments 607- <dig> aa, 707- <dig> aa  and 770- <dig> aa.
we observed that the 607- <dig> aa and 707- <dig> aa fragment were resistant to proteolysis .
however, the 770- <dig> aa fragment decreased in m-phase, demonstrating that the signal for degradation lies in this fragment.
in order to identify the minimum sequence required for mcm <dig> degradation, we further divided the 770- <dig> aa fragment into three parts: 770- <dig> aa, 783- <dig> aa and 843- <dig> aa.
770- <dig> aa fragment was partially stable indicating that there could be an incomplete degron in this fragment .
the 783- <dig> aa  and 843- <dig> aa decreased in m-phase demonstrating that there are non-overlapping regions that are sufficient for mcm <dig> proteolysis .
the zf motif was divided into  <dig> segments each of  <dig> amino acids: 783- <dig> aa, 803- <dig> aa and 823- <dig> aa.
we observed that 783- <dig> aa region was essential and sufficient for degradation while 803- <dig> aa and 823- <dig> aa were not proteolyzed .
therefore, we have narrowed to two regions that contain the degron: a  <dig> aa region  within the zf motif and c-terminal end of mcm <dig> .
another independent region that is sufficient for mcm <dig> downregulation is the linker domain .
we divided the linker domain into three parts: 440- <dig> aa, 471- <dig> aa and 525- <dig> aa.
we observed that though the linker domain is decreased during m-phase, none of the three constituent fragments were proteolyzed .
however, when we combined the 440- <dig> aa and 471- <dig> aa fragments, the degradation ability was restored .
combining 440- <dig> aa with 525- <dig> aa or combing 471- <dig> aa with 525- <dig> aa did not result in m-phase degradation .
therefore, the degron in the linker domain lies within the stretch of 440- <dig> aa.
hence, it seems that mcm <dig> degradation is mediated by non-overlapping regions, apparently to ensure downregulation of mcm <dig> even if proteolysis at any one region is somehow blocked.
there is no apparent sequence similarity between the three minimal regions that are competent for m-phase proteolysis: 440- <dig> aa, 783- <dig> aa and 843- <dig> aa.
this would suggest that the ubiquitination machinery is adaptable to recognize multiple sequences to ensure mcm <dig> proteolysis.
recognition of many biological targets is defined by similar 2-d structures rather than primary sequence and that possibility cannot be ruled for recognition of different domains of mcm <dig>
discussion and
CONCLUSIONS
we have observed that the mcm <dig> levels are significantly reduced in nocodazole blocked cells.
this is in contrast to the previous report where on the basis of estimation of mcm <dig> levels in asynchronous and nocodazole released cells by immunoblots, the authors inferred that mcm <dig> degradation begins after metaphase  <cit> .
it is possible that the mcm <dig> protein observed after nocodazole treatment was contributed by cells trapped at g2/m boundary.
the stabilization of mcm <dig> by mg <dig> in nocodazole released cells observed by them is likely due to non-degradation of mcm <dig> in these cells.
it has been suggested that a destruction box sequence  could be recognized by the anaphase-promoting complex for ubiquitination.
additionally, mcm <dig> also contains a ken box sequence  that is present in targets ubiquitinated by apc.
however, the ntd domain, which contains the ken box is not degraded during m-phase  <cit> .
similarly, the wh motif which contains the destruction box is stable during mitosis .
however, the linker region and zf motif, which do not have either destruction or ken box, are proteolyzed during m-phase.
in summation, the above data would suggest that the apc recognition motifs are not required for m-phase degradation of mcm <dig>  in this study, we have also demonstrated that inhibition of apc does not block cell-cycle degradation of mcm <dig>
this study has discovered at least three independent regions of mcm <dig> that are sufficient for proteolysis during m phase.
since the non-overlapping regions of mcm <dig> sufficient for m-phase proteolysis did not display sequence similarity, it suggests of a limited degeneracy of the e <dig> ligase to allow for recognition of multiple substrates.
crl <dig> is known to mediate the ubiquitination of almost two dozen substrates, including cdt <dig>  pcna and histones that do not share sequence similarity  <cit> .
it is widely believed that the proteolysis of mcm <dig> during mitosis is a mechanism to prevent aberrant initiation of replication and therefore it is possible that utilization of independent regions is a means to ensure downregulation of mcm <dig> even if proteolysis at any one region is somehow blocked.
the cloning of stable mutants of full length mcm <dig> would help us evaluate the effect of stable mcm <dig> on cell-cycle progression and genomic stability.
it is believed that the proteolysis of mcm <dig> during mitosis is a vital mechanism to prevent aberrant initiation of replication and the present study describes the regulation of mcm <dig> during this phase of the cell-cycle.

 dna replication in eukaryotes begins with the assembly of the pre-replicative complex comprising of replication initiators, the origin recognition complex, cdc cdt and the replicative helicase, mcm2- complex. increase in the activity of cyclin dependent kinases and loading of replication factor, mcm marks the transition from g to s phase. these events promote the loading of cdc rpa and dna polymerases at the replication origins to initiate dna synthesis. the essential requirement of mcm in dna replication initiation and elongation has been exhibited across species. mutations in mcm cause a decrease in initiation of replication, slow progression of dna synthesis and stalling of replication forks during elongation. since mcm is essential for replication initiation and elongation, its activity is regulated in a cell-cycle manner to ensure a single round of replication. s. cerevisiae mcm protein is present in all phases of the cell cycle though its association with chromatin is regulated to ensure replication licensing. human mcm protein is known to decrease in the early g phase. the levels of mcm mrna have been evaluated as the cells pass from m-phase into g phase. though the mcm protein decreased, the levels of mcm mrna increased within the same time period. these results demonstrate that the decrease in the mcm activity during the g phase is not due to decrease in transcription but because of protein turnover. in this communication, we report that proteolysis of human mcm protein initiates before the onset of mitosis. in an immunoblot with anti-mcm antibody, we observed that the m-phase blocked cells have downregulated the mcm protein. on the basis of single cell immunofluorescence, we show that asynchronously growing cells in different phases of mitosis have reduced levels of mcm also, mcm downregulation in m-phase is independent of the apc recognition motifs: the destruction and the ken box. we demonstrate that mcm degradation is not dependent on anaphase promoting complex or on its recognition motifs but rather mediated by non-overlapping regions, apparently employing a redundant mechanism to ensure downregulation.  cell-cycle proteolysis of mcm initiates before the onset of mitosis published reports indicate that the nocodazole blocked cells retain mcm protein which decreases in late m-phase and starts increasing h after release. when mg was added to nocodazole released cells, there was an increase in mcm levels suggesting that the degradation that was occurring during the late m-phase was blocked by proteasome inhibitors. in another report by the same group, a stable hela cell line which expressed green fluorescent protein-tagged mcm from a human cytomegalovirus immediate early promoter was established and authors observed that the gfp-mcm protein was detectable by immunofluorescence during mitosis. we looked at the levels of mcm and observed that they were significantly reduced in nocodazole blocked u2os cells. mcm appeared after 4- hours of release from nocodazole block. this is in contrast to the results obtained by hanaoka and coworkers, who observed mcm band by immunoblotting with anti-rabbit antibody, which was raised against 127- aa region of mcm therefore, we wanted to rule out that the absence of mcm signal was due to limited immunoreactivity of the antibody used by us. we have used a polyclonal rabbit antibody, ab, which is raised against the full-length protein, but showed weak immunoreactivity against the c-terminal protein. however, similar results were obtained with another polyclonal rabbit antibody, ab, which shows immunoreactivity against all regions of mcm. as reported previously, the specificity of both these antibodies in immunoblotting assays have been established by rnai. in order to rule out that the decrease of mcm is an artifact generated by nocodazole toxicity, we blocked the cells with other reagents in m-phase and assayed the stability of mcm vincristine binds to tubulin dimers and inhibits the assembly of microtubule structures, blocking cells in metaphase. colchicine inhibits microtubule polymerization by binding to tubulin and thereby blocks cells in metaphase. taxol hyper-stabilizes the beta subunit of tubulin, which is then unable to disassemble inducing a cell-cycle block at the metaphase/anaphase transition. u2os cells blocked with any of these drugs displayed low levels of mcm. on the basis of the above data, we conclude that human cells have low levels of mcm in mitosis. as reported previously, we observed that the drop in mcm levels during mitosis is dependent on the proteasome. to rule out that loss of recognition of mcm during mitosis could be due to epitope masking, we established that the mcm antibody recognizes the mitotic forms of mcm since mcm is naturally proteolyzed during mitosis we expressed the ha-tagged ntd+id domain which is resistant to cell cycle-regulated degradation and tested whether the anti-mcm antibody recognizes this forms of mcm during m-phase. u2os cells expressing the ntd+id domain of mcm were blocked with nocodazole, released and harvested at regular intervals in order to collect cells in different phases of the cell cycle. as observed previously, an anti-ha antibody immunoblot confirmed that the ntd+id domain of mcm was resistant to degradation in nocodazole blocked cells. an anti-mcm antibody immunoblot displayed that the levels of endogenous mcm levels were low in m phase and increased around h after nocodazole release but the ntd+id domain recognized through the same antibody did not show a decrease in levels, displaying that the mcm antibody recognizes the mitotic forms of mcm single-cell imaging displays reduced levels of mcm during mitosis we also looked at mcm levels in single cells as they passed through interphase and various phases of mitosis. hela cells were fixed and visualized for endogenous mcm using rabbit polyclonal anti-mcm antibody, ab, in combination with an anti-rabbit tritc secondary antibody while microtubules were visualized with a mouse monoclonal antibody to alpha-tubulin conjugated to fitc. the specificity of the antibody in immunofluorescence assays has been established by rnai. from the asynchronously growing culture, we identified cells in prophase, metaphase, anaphase and telophase, and assayed for mcm and alpha-tubulin localization. we observed that the cells in prophase or prometaphase, with condensed chromatin and bi-polar centrosomes, were negative for mcm signal. in the same fields, we could observe interphase cells that were positive for mcm signal, ruling out errors in our visualization method. as previously reported, not all interphase cells are positive for mcm signal and presumably these are the cells in early g phase. similarly, cells in metaphase, identified by the presence of equatorial plate and polar microtubules, were also negative for mcm signal. cells in anaphase and telophase identified by segregated sister chromatids and shortened kinetochore microtubules, were also negative for mcm signal. to confirm that the loss of detection of mcm is not due to the protein becoming soluble during mitosis and that our immunofluorescence protocol can detect soluble proteins during mitosis, we have compared the levels of mcm and cyclin b in nocodazole blocked cells. hela cells arrested in prometaphase were negative for mcm signal but retained the cyclin b signal, establishing the accuracy of our immunofluorescence assay. therefore, our results demonstrate that the levels of mcm protein are significantly reduced during mitosis. it is known that nocodazole interferes with the polymerization of microtubules and cells treated with nocodazole enter mitosis but cannot form metaphase spindles thereby activating the spindle assembly checkpoint which causes an arrest in prometaphase. as explained earlier in figure hela cells were treated with colchicine, nocodazole, taxol or vincristine for h to obtain cells arrested in prometaphase. incubation with any of these drugs resulted in disorganized microtubules and condensed chromosomes, confirming a m-phase block. prometaphase cells have been marked by arrowheads and we observed that these cells were negative for mcm signal confirming that mcm degradation has been initiated at this stage of mitosis. we determined the levels of endogenous mcm and cyclin a in an asynchronously growing culture of hela cells by immunofluorescence using a rabbit anti-mcm antibody, ab, and mouse anti-cyclin a antibody respectively. utilizing the same antibodies, we have demonstrated that the cyclin a levels begin to increase in the s phase and it is degraded in the m-phase. we observed that mcm was present till the g phase as almost all the cells that retained cyclin a signal also stained positive for mcm. combining the above results, we establish that mcm is present till the g phase and is degraded around the g2/m boundary. live-cell imaging demonstrates that mcm is absent during the m-phase an alternate approach to address the levels of mcm during mitosis would be to evaluate the cycling of mcm in live cells. since it has been reported that mcm protein expression is not mainly regulated at the transcriptional level, we expressed egfp-tagged mcm from cytomegalovirus immediate early promoter. hela cells were transfected with pegfp c3-mcm and two days after transfection, we observed a kda band with mcm immunoblot, confirming the expression of egfp-tagged mcm. hela cells transfected with blank pegfp-c vector displayed cytoplasmic egfp signal while egfp expressed in fusion with mcm was guided to the nucleus. we evaluated the egfp signal during mitosis, when the cells appear rounded in phase-contrast microscope. we expressed the egfp-tagged full-length mcm in cells and tracked the egfp signal and cell division of cells. the m-phase is known to last around 1- h and as shown in figure we located a cell that is in mitosis and undergoes cytokinesis after h min. we observed that the cell was negative for mcm during mitosis but the daughter cells accumulated mcm after h, during the g phase of the cell cycle. the overexpression of mcm leads to a cell-cycle block during interphase and therefore the progression of cells from g to m could not be observed. we have reported that the aa zf motif of mcm is sufficient for m phase proteolysis and therefore we expressed egfp-tagged zf motif of mcm and tracked the egfp signal and cell division of hela cells by fluorescence and phase-contrast microscopy respectively as the cells progressed through mitosis. as shown in figure s4c, we located a mitotic cell that undergoes cytokines after h min. we observed that the cell was negative for mcm during mitosis but the daughter cells accumulated mcm after around h. therefore, utilizing a different cell-line, we establish that the full-length mcm is low during mitosis. summing up, using this antibody-independent approach, we demonstrated that the mcm levels are low during mitosis. apc is not required for cell cycle degradation of mcm10 apc/c or cyclosome is a complex of many proteins that functions as an e ubiquitin ligase during mitosis. since mcm is low in the m-phase, there is a distinct possibility that apc is involved in degradation of mcm notably, mcm contains destruction box sequence and a ken box, motifs that are required for recognition of substrates by apc. to test whether apc mediates the m-phase degradation of mcm we silenced apc subunit of apc, which would effectively debilitate its ubiquitination ability. hela cells transfected with apc sirna for three consecutive days were blocked in the m-phase with nocodazole for h and subsequently released into nocodazole free medium. rnai against apc decreased the target protein and mrna but that did not increase the levels of mcm in nocodazole blocked cells. rnai effectively inhibited the apc activity as evidenced by increase in the levels of cyclin b. this strongly suggests that apc is not involved in the cell-cycle degradation of mcm we have assayed the mcm levels in asynchronous populations of cells transfected with apc cul cdh cdc beta-trcp and fbxw or control gl sirna and observed that only minor variations were observed in mcm levels. we have previously reported that uv-irradiation specifically proteolyses mcm and we have identified the e ubiquitin ligase mediates the uv-triggered and m-phase proteolysis of mcm. apc recognition motifs are not required for cell cycle degradation of mcm10 we have previously utilized stable u2os cells expressing ha-tagged mcm to determine the segments of mcm that are essential for cell cycle-regulated degradation. full-length mcm can be broadly divided into n-terminal, inner, linker and c-terminal domains. a coiled-coil motif is present within the n-terminus of mcm which is required for homodimerization. the inner and c-terminal domains, which contain zinc finger and winged helix motifs, bind to single- and double-stranded dna, and the p subunit of dna polymerase-alpha. as reported previously, we observed that the ha-tagged full length mcm protein behaves like the endogenous protein. u2os cells expressing ha-tagged full length mcm were blocked with nocodazole, released and harvested at regular intervals in order to collect cells in different phases of the cell cycle. endogenous mcm levels were low in m phase and increased around h after release from nocodazole block. ha-tagged full-length mcm showed a degradation pattern similar to that of the endogenous protein, which demonstrates that our assay for evaluating the degradation of different regions of mcm is accurate. we observed that the ntd+id domain of mcm which contains the ken box, was resistant to cell cycle-regulated degradation. in the present study, we tested whether the wh motif, that contains the destruction box, is proteolyzed during the m-phase. stable u2os cells expressing wh motif of mcm was blocked with nocodazole, which arrested the cells in m-phase. the obtained mitotic cells were released from the block and harvested at regular intervals in order to collect cells in different phases of the cell cycle. the flow cytometry profile of propidium iodide-stained dna and the levels of cyclin b demonstrate a block in m-phase and subsequent progression through the cell cycle. as noted previously, mcm was absent in nocodazole-released cells, demonstrating the natural proteolysis of mcm in m phase which began to increase after h, displaying the natural cycling of mcm levels. full-length mcm expressed from the retroviral vector showed a degradation pattern similar to that of the endogenous protein, validating our assay for evaluating the cell cycle-regulated degradation of mcm we observed that the wh motif fragment was resistant to cell cycle-regulated degradation. we confirmed that the nuclear localization signal expressed in fusion with the wh motif steered it to the nucleus and therefore change in cellular localization is not the reason for resistance to proteolysis. we have previously reported that the lnk domain which does not have either the ken box or destruction box is proteolyzed as cells pass through m-phase. therefore, we conclude that the ken and destruction box of mcm are neither essential nor sufficient for its m-phase proteolysis. mcm proteolysis utilizes independent non-overlapping signals to identify the domains required for mcm proteolysis during m-phase, we expressed different regions of mcm and assayed their stability. as noted previously, the ntd+id domain was resistant to cell cycle-regulated degradation, but the lnk and ctd domains were proteolyzed in m phase. though the zf motif was sufficient for m phase proteolysis of mcm the ctd domain lacking the zf motif was also degraded in m phase, signifying that though zf motif is sufficient, it is not essential for m phase proteolysis of mcm. we now wanted to identify the degron that is essential for degradation. we further divided the ctd into three fragments 607- aa, 707- aa and 770- aa. we observed that the 607- aa and 707- aa fragment were resistant to proteolysis. however, the 770- aa fragment decreased in m-phase, demonstrating that the signal for degradation lies in this fragment. in order to identify the minimum sequence required for mcm degradation, we further divided the 770- aa fragment into three parts: 770- aa, 783- aa and 843- aa. 770- aa fragment was partially stable indicating that there could be an incomplete degron in this fragment. the 783- aa and 843- aa decreased in m-phase demonstrating that there are non-overlapping regions that are sufficient for mcm proteolysis. the zf motif was divided into segments each of amino acids: 783- aa, 803- aa and 823- aa. we observed that 783- aa region was essential and sufficient for degradation while 803- aa and 823- aa were not proteolyzed. therefore, we have narrowed to two regions that contain the degron: a aa region within the zf motif and c-terminal end of mcm. another independent region that is sufficient for mcm downregulation is the linker domain. we divided the linker domain into three parts: 440- aa, 471- aa and 525- aa. we observed that though the linker domain is decreased during m-phase, none of the three constituent fragments were proteolyzed. however, when we combined the 440- aa and 471- aa fragments, the degradation ability was restored. combining 440- aa with 525- aa or combing 471- aa with 525- aa did not result in m-phase degradation. therefore, the degron in the linker domain lies within the stretch of 440- aa. hence, it seems that mcm degradation is mediated by non-overlapping regions, apparently to ensure downregulation of mcm even if proteolysis at any one region is somehow blocked. there is no apparent sequence similarity between the three minimal regions that are competent for m-phase proteolysis: 440- aa, 783- aa and 843- aa. this would suggest that the ubiquitination machinery is adaptable to recognize multiple sequences to ensure mcm proteolysis. recognition of many biological targets is defined by similar 2-d structures rather than primary sequence and that possibility cannot be ruled for recognition of different domains of mcm discussion and  we have observed that the mcm levels are significantly reduced in nocodazole blocked cells. this is in contrast to the previous report where on the basis of estimation of mcm levels in asynchronous and nocodazole released cells by immunoblots, the authors inferred that mcm degradation begins after metaphase. it is possible that the mcm protein observed after nocodazole treatment was contributed by cells trapped at g2/m boundary. the stabilization of mcm by mg in nocodazole released cells observed by them is likely due to non-degradation of mcm in these cells. it has been suggested that a destruction box sequence could be recognized by the anaphase-promoting complex for ubiquitination. additionally, mcm also contains a ken box sequence that is present in targets ubiquitinated by apc. however, the ntd domain, which contains the ken box is not degraded during m-phase. similarly, the wh motif which contains the destruction box is stable during mitosis. however, the linker region and zf motif, which do not have either destruction or ken box, are proteolyzed during m-phase. in summation, the above data would suggest that the apc recognition motifs are not required for m-phase degradation of mcm in this study, we have also demonstrated that inhibition of apc does not block cell-cycle degradation of mcm this study has discovered at least three independent regions of mcm that are sufficient for proteolysis during m phase. since the non-overlapping regions of mcm sufficient for m-phase proteolysis did not display sequence similarity, it suggests of a limited degeneracy of the e ligase to allow for recognition of multiple substrates. crl is known to mediate the ubiquitination of almost two dozen substrates, including cdt pcna and histones that do not share sequence similarity. it is widely believed that the proteolysis of mcm during mitosis is a mechanism to prevent aberrant initiation of replication and therefore it is possible that utilization of independent regions is a means to ensure downregulation of mcm even if proteolysis at any one region is somehow blocked. the cloning of stable mutants of full length mcm would help us evaluate the effect of stable mcm on cell-cycle progression and genomic stability. it is believed that the proteolysis of mcm during mitosis is a vital mechanism to prevent aberrant initiation of replication and the present study describes the regulation of mcm during this phase of the cell-cycle. 
it is believed that the proteolysis of mcm during mitosis is a vital mechanism to prevent aberrant initiation of replication and the present study describes the regulation of mcm during this phase of the cell-cycle. further, the proteolysis in m-phase can be independently mediated by non-overlapping regions of mcm apparently employing a redundant mechanism to ensure downregulation.
 mcm protein is essential for initiation and elongation phases of replication. human cells proteolyze mcm during mitosis, presumably to ensure a single round of replication. it has been proposed that anaphase promoting complex ubiquitinates mcm in late m and early g phases.  in contrast to the previous work, we report that the degradation of mcm is initiated at the onset of mitosis. immunoblotting and immunofluorescence assays display that mcm levels are low in all phases of mitosis. we report that mcm degradation is not dependent on anaphase promoting complex. further, the proteolysis in m-phase can be independently mediated by non-overlapping regions of mcm apparently employing a redundant mechanism to ensure downregulation.  it is believed that the proteolysis of mcm during mitosis is a vital mechanism to prevent aberrant initiation of replication and the present study describes the regulation of mcm during this phase of the cell-cycle. 