| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 49, 48524-48528, December 5, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093
Received for publication, July 16, 2003 , and in revised form, September 8, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATP
S, not only increases both ORC and CDC6 t but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
, and RPA and the single-stranded binding protein (7) as well as suppresses the assembly of pre-RCs by inhibition of MCM loading (8). Thus, pre-RCs are only assembled during a defined window of the cell cycle.
In the yeast Saccharomyces cerevisiae, the high affinity of ORC for specific DNA sequences (ARS sequences) (9) directs pre-RCs to form at particular sites along the genome (10). The ORC hexameric complex consists of six related subunits, all of which are required for viability (1). ORC binding to the ARS protects approximately 50 bp of DNA, and ORC subunits 1, 2, and 4 make specific contacts within the 11-bp ARS consensus sequence (11). Binding to ARS inhibits the intrinsic ATPase activity of ORC1p. Although mutations in this subunit do not block the binding of ORC to DNA, they do abrogate the loading of Cdc6 (12). Although ORC and all of the other protein components involved in pre-RC formation are conserved between yeast and metazoans, metazoan equivalents of ARS sequences have yet to be found. Moreover, in metazoans, the number of pre-RCs that form appears to be under developmental control. For example, during early embryonic divisions in both Drosophila melanogaster and Xenopus laevis, pre-RCs form every 3 and 10 kb, respectfully, and pre-RC formation is DNA sequence-independent (13–15). As development proceeds, pre-RC formation gradually decreases as replicon size increases to 100–200 kb in somatic cells (14, 15).
At present, it is not known whether this change occurs as a result of post-translational modification of ORC, expression of new proteins that restrict where ORC can bind, or developmental modifications of the chromatin template that limit ORC binding (1). The composition of the embryonic and somatic ORC appears to be identical (16), suggesting that the decrease in pre-RC formation in somatic cells relative to early embryonic cells may largely be the result of developmental changes in the chromatin template.
The ability of Xenopus extracts to replicate DNA from any source provides an opportunity for carrying out detailed biochemical experiments designed to elucidate the kinetic parameters and mechanisms that limit and regulate the formation of pre-RCs in metazoans (17–20). Using this physiological system, we have measured the dissociation rate of ORC from chromatin at different stages of pre-RC assembly. Our results suggest that ORC by itself dissociates from nonspecific chromatin sequences with a t of 40 min and that ATP hydrolysis may play a critical role in timing how long ORC remains bound to DNA. We find that Cdc6 significantly stabilizes the association of ORC with chromatin increasing the t from 40 to 170 min. Following the loading of the MCM helicase into the pre-RC, the affinity of both ORC and Cdc6 for chromatin decreases significantly and ORC dissociates from chromatin with a t of 15 min. These findings suggest that ORC and Cdc6 may function in a catalytic manner, loading MCM onto DNA and then dissociating from the DNA to serve the same function at other locations. Furthermore, in the absence of ancillary factors or developmentally regulated chromatin modifications, the tight association of metazoan ORC with nonspecific sequences suggests that during a typical G1 period, it would be kinetically impossible for ORC to locate and bind to specific sites in the presence of the overwhelming number of tight binding nonspecific competitor sites present in the genome.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Immunodepletion—ORC, Cdc6, and MCM were depleted from 100 µl of egg cytosol by the addition of 15, 50, or 100 µl, respectively, of high titer affinity-purified -XORC2, -XCdc6, or -MCM7. Immunocomplexes were cleared three times with protein A-agarose (BD Biosciences) as previously described (7). For all of the proteins, monospecific sera were used for the depletion and preimmune sera were used for mock-depletion controls. -XORC2, -XCdc6, and -MCM3 sera were used as described previously (17, 23). Because -MCM3 only reacts with denatured MCM (data not shown), MCM7 antiserum was used for depletion. MCM7 antiserum was raised against a bacterially expressed C-terminal fragment of MCM7 (amino acids 545–720) that was purified using nickel-affinity chromatography. The antiserum specifically recognized a protein of a molecular mass of 
100 kDa and depleted the complete MCM complex (data not shown). In all of the cases, the extent of depletion was monitored by Western blotting and found to be at least 99%.
Chromatin Binding Assay—15,000 demembranated sperm nuclei were incubated with 7.5 µl of membrane free egg cytosol supplemented with nocodazole and an ATP regeneration system (20 mM phosphocreatine, 5 µg/ml creatine kinase, and 0.2 mM ATP) for 30 min at room temperature. Samples were diluted in 45 µl of egg lysis buffer (ELB, 2.5 mM MgCl, 50 mM KCl, 10 mM Hepes, pH 7.7) + 2 mM ATP. Chromatin was isolated by spinning the diluted sample through 100 µl of ELB with 0.5 M sucrose for 15 s at 16,000 x g at 4 °C. All but 3 µl of supernatant were aspirated, and the remaining sample was boiled in 10 µl of SDS loading buffer.
DNA Replication Assays—DNA replication in NPE was measured as described previously (22). Sperm chromatin was incubated in 10 µl of extract for 30 min, and then 25 µl of NPE was added. [-32P]dATP was included in all of the replication assays at a concentration of 0.1 µCi/µl egg. Reaction products were visualized after running on 1% agarose gel that was then dried and exposed to a phosphorimaging screen (Amersham Biosciences).
Determination of ORC t—Standard chromatin binding assays were performed as described above. After the initial 30-min incubation of sperm chromatin in egg cytosol, 1 µg of supercoiled pBluescriptII SK(–) was added to the binding reaction with 45 µl of ELB + 2 mM ATP or 0.5 mM ATPS. During the initial 30-min binding reaction, ATPS was included in the ATP regeneration system where appropriate. t was determined by measuring the amount of ORC associated with chromatin as a function of time as assessed by Western blot analysis. The rate of dissociation was assumed to be the slope of the line generated by plotting the amount of ORC bound as a function of time using FigP software (Biosoft, Cambridge, United Kingdom). For each experiment, binding assays were quantified using a standard curve of bacterially expressed purified XORC2-His. Proteins were visualized by chemiluminescence (Pierce). All of the measurements were performed at least three times with consistent results, and the t represent results of one complete experiment. Western blot analysis was performed using primary antibodies described above and goat anti-rabbit IgG-horseradish proxidase (Jackson, West Grove, PA). All of the incubations were performed at 1:5000 dilutions. Western blot analysis was linear over 2 orders of magnitude as determined using purified XOR2-His.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
of ORC from pre-RCs, sperm chromatin was first incubated for 30 min in extract to allow pre-RCs to assemble and then a large excess of a 3-kb competitor plasmid DNA was added to the extract to trap both excess ORC present in the extract and ORC that dissociated from existing pre-RCs. At different times after the addition of the competitor plasmid DNA, brief centrifugation was used to separate the sperm chromatin from both the extract and the competitor DNA. Following this procedure, the amount of ORC2 remaining bound to the sperm was quantified by Western blot analysis. ORC2 is used as an indicator of the presence of the complete hexameric ORC complex. Using this approach, we found that excess plasmid efficiently competes for pre-assembled ORC in a dose- and time-dependent manner (Fig. 1, A and B). Plasmid also competes for preassembled Cdc6 (see Fig. 4C). Importantly, the plasmid does not compete for the pre-assembled MCM hexamer (Figs. 1A, and 4, A and C). This is consistent with data showing that after pre-RC assembly, both ORC and Cdc6 bind chromatin less tightly than MCM based on differential extraction of these proteins with high salt (18). Chromatin remains competent for replication after the removal of ORC and Cdc6 by plasmid competition (Fig. 1C). This is consistent with the finding that ORC and Cdc6 are not required for initiation once MCM is loaded onto DNA (18, 23).
|
|
from Chromatin— Using the competition assay described above, we measured the t of ORC by itself from sperm chromatin. To do this, Cdc6 was immunodepleted from the extract (Fig. 2A). Because ORC, Cdc6, and MCM are assembled into pre-RCs sequentially, depletion of Cdc6 from extracts blocks the binding of both Cdc6 and MCM (3). Therefore, under these conditions, the t of chromatin-bound ORC by itself can be determined from a competition assay. The percent of ORC remaining bound to chromatin at different times after the addition of excess competitor to the extract was measured by quantitative Western blot analysis using known quantities of purified XORC-His as a standard (Fig. 2B). The dissociation rate or t of ORC bound to sperm chromatin was determined from the slope of a plot of percent ORC bound versus time. Unexpectedly, the interaction of ORCs with chromatin under these conditions was extremely stable with a t of 40 min (Fig. 2C). This observation suggests that the binding of metazoan ORC to nonspecific DNA sequences, unlike S. cerevisiae ORC, is very tight and very stable (24, 25).
|
S) in the competition assay has an appreciable affect on the t of ORC bound to chromatin, increasing it from 40 to 169 min, a greater than 4-fold increase in the t (Fig. 2, B and C). Thus ATP hydrolysis, possibly via ORC itself, may regulate the interaction of ORCs with chromatin. The affect of ATPS is not simply due to the inhibition of activities that may be required for activation of the chromatin template. Chromatin decondensation takes place normally in these extracts. Moreover, the addition of ATPS to reactions in which chromatin is first assembled in cytosol depleted of ORC results in a similar t upon the addition of undepleted extract containing ORC (data not shown).
Cdc6 Stabilizes ORC Binding of Origins—To determine the influence of Cdc6 on the t of ORC, we allowed pre-RCs to progress to an intermediate step in assembly by immunodepletion of MCM complexes from the cytosolic extract. Depletion of the complete hexameric MCM complex was achieved using an antibody to XMCM7 (Fig. 3A) (27). These extracts remained competent for loading both ORC and Cdc6. In the presence of Cdc6, the t of ORC increased from 40 to 128 min (Fig. 3, B and C), a 3-fold increase. This finding suggests that recruitment of Cdc6 confers stability to the ORC/chromatin complex, possibly through the inhibition of the intrinsic ATP hydrolysis activity of ORC. In support of this possibility, non-hydrolyzable ATP does not affect the t of the ORC·Cdc6 complex (<2-fold increase) as much it does ORC alone (greater than 4-fold).
|
of ORC from a mature pre-RC is 15 min. This is a 3-fold decrease relative to ORC alone (t = 40 min) and an 8.5-fold decrease relative to ORC-Cdc6 (t = 128 min) (Fig. 4, A and B). This dramatic change in the t suggests that ORC and Cdc6 are destabilized upon MCM loading possibly because of the recruitment of other replication factors or via changes in DNA topology such as the generation of localized single-stranded DNA (26). The switch from a tightly bound complex to a relatively looser protein/DNA complex upon loading of the putative replication helicase suggests that ORC and Cdc6 may function catalytically. That is, once ORC and Cdc6 have loaded MCM onto DNA, they can dissociate from the DNA to load MCM onto other sites during the G1 window of pre-RC assembly. When non-hydrolyzable ATP is added to mock-depleted cytosol, ORC and Cdc6 associate with chromatin but MCM binding is blocked (Fig. 4C). Moreover, under these conditions both ORC and Cdc6 are resistant to competition by plasmid DNA (Fig. 4C). Similar to the ORC1p subunit, Cdc6 contains nucleotide-binding Walker A and B motifs. Mutations in these sites inactivate hydrolytic activity in vitro and abrogate the loading of MCMs onto chromatin (28–30). Thus, dissociation of ORC and Cdc6 may be linked to an ATP hydrolysis step required for the loading of the MCM hexamer (Fig. 5).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
40 min. The long association time between ORC and nonspecific DNA sequences has important ramifications for models that assume that ORC normally binds and assembles pre-RCs only at specific DNA sequences. That is, for ORC to bind specific sites requires that it sample and bind many nonspecific sites during the course of a search for a specific binding site. Presumably, if the affinity of ORC for nonspecific sites is very weak then it would remain bound for a short time before dissociating. This does not appear to be the case for the metazoan ORC based on the data presented in this report. Rather, a 40-min resident time suggests that ORC could only sample 18 sites in a somatic replicon of 100–200 kb during a typical 12-h G1 phase. Clearly, this possibility suggests that if metazoan pre-RCs form at unique sequences then either ancillary factors must exist, which reduce the affinity of somatic ORC for nonspecific DNA sites, or large regions of the cells chromatin must be modified so that these nonspecific sites are no longer accessible to ORC. With respect to ancillary proteins that could function to reduce the affinity of ORC for nonspecific DNA sites, Cdc6 might be an excellent candidate. Indeed, in a recent report (25) using purified ORC and Cdc6 from yeast, it was concluded that Cdc6 reduced the affinity of ORC for nonspecific sites. However, in our studies under more physiological conditions, we find that when Cdc6 is incorporated into the pre-RC, the stable association of ORC with nonspecific sites increases greater than 4-fold, from 40 to 128 min. This disparity may be because of the different assay conditions employed. We have used chromatinized DNA template and crude extracts mimicking in vivo conditions, whereas the yeast study employed purified ORC, Cdc6, and naked 290-bp DNA fragments. It is possible, however, that the results may also be species-dependent. In yeast, ORC clearly binds to well defined sequences, whereas in metazoans, comparable sequences may not exist. As such, Cdc6 in yeast may have adapted an additional function (decreasing the association of ORC with nonspecific sequences) that is absent in the Cdc6 from metazoan organisms.
It is also clear from our studies that the very stable association of the metazoan ORC·Cdc6 complex with random DNA sites would make a search for "specific" sites very slow. ORC·Cdc6 would only be able to sample six sites within a 100–200-kb somatic replicon during a typical 12-h G1 phase. Again, this finding suggests that if specific sites exist in metazoans, then yet to be identified ancillary factors must function to inhibit the association of somatic ORC with nonspecific sites. These factors could associate with either ORC or chromatin.
With respect to the role of ATP hydrolysis in pre-RC assembly, we observe that the presence of non-hydrolyzable ATP significantly stabilizes the association of ORC with chromatin, increasing its t 4-fold to 169 min. Interestingly, previous studies have demonstrated that the ATP hydrolysis rate of purified yeast ORC is reduced 7–8-fold when bound to DNA and that in this bound state ORC hydrolyzes approximately 1 ATP/every 30 min (24).2 This is very similar to the time we measured for the t of metazoan ORC from chromatin (40 min) and is consistent with the hypothesis that ATP hydrolysis might serve as a timing mechanism regulating the association of ORC with DNA. For example, if bound ATP stabilizes a conformation of ORC that has high affinity for DNA, the duration of this high affinity state (association of ORC with DNA) would be linked to the rate of ATP hydrolysis. Moreover, if Cdc6 served to inhibit the intrinsic ORC ATP hydrolysis rate further, this could account for the 4-fold increase in t of an ORC·Cdc6 complex relative to ORC alone.
Prior to the loading of MCM, the ORC·Cdc6 complex is bound to DNA very tightly with a t of 128 min. However, following the loading of MCM, the t of ORC decreases 8.5-fold to 15 min. This MCM-dependent decrease is consistent with observations showing that after MCM is loaded, ORC is extracted from DNA in 250 mM salt, whereas prior to the loading of MCM, ORC is resistant to salt washes (18). The fast t of ORC that we observe suggests that once MCM is stably loaded onto chromatin, ORC and Cdc6 can dissociate and function to load MCM at other locations. In this sense, ORC and Cdc6 act catalytically (23). By contrast, MCM remains permanently at the original pre-RC to function in the initiation of replication during S phase of the cell cycle. In support of this observation, we find that following MCM loading, displacement of ORC by the addition of competitor DNA does not inhibit subsequent initiation of DNA replication.
This conclusion might seem at odds with the observation that ORC remains associated with DNA throughout the cell cycle until mitosis (31). However, even after MCM is loaded onto DNA, ORC and Cdc6 still associate with DNA for a relatively long period (15 min). Therefore, steady-state techniques such as immunofluorescence would not reveal either a decrease in the amount of ORC bound following pre-RC formation or the dynamic nature of the association between ORC and DNA following pre-RC formation.
|
FOOTNOTES |
|---|
To whom correspondence should be addressed: Division of Biological Sciences, University of California San Diego, Bonner Hall 4202 (M/C 0349), 9500 Gilmann Dr., La Jolla, CA 92093. Tel.: 858-534-3423; Fax: 858-822-3531; E-mail: jnewport{at}ucsd.edu.
1 The abbreviations used are: pre-RC, pre-replication complex; NPE, nucleoplasmic extract; ELB, egg lysis buffer; ORC, origin recognition complex; ATPS, adenosine 5'-3-O-(thio)triphosphate; ARS, autonomous replication sequence.
2 S. Bell, personal communication.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Zellner, T. Herrmann, C. Schulz, and F. Grummt Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation Nucleic Acids Res., November 29, 2007; 35(20): 6701 - 6713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Siddiqui and B. Stillman ATP-dependent Assembly of the Human Origin Recognition Complex J. Biol. Chem., November 2, 2007; 282(44): 32370 - 32383. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. E. Arias and J. C. Walter Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells Genes & Dev., March 1, 2007; 21(5): 497 - 518. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Waga and A. Zembutsu Dynamics of DNA Binding of Replication Initiation Proteins during de Novo Formation of Pre-replicative Complexes in Xenopus Egg Extracts J. Biol. Chem., April 21, 2006; 281(16): 10926 - 10934. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Y. Takeda, Y. Shibata, J. D. Parvin, and A. Dutta Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells Genes & Dev., December 1, 2005; 19(23): 2827 - 2836. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
