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Interaction of Cdc2 with the Origin Recognition Complex at Origins of Replication in Schizosaccharomyces Pombe

Forschungsarbeit 2004 40 Seiten

Biologie - Genetik / Gentechnologie



1. Introduction
1.1. S. pombe as a Model System
1.2. Eukaryotic DNA Replication
1.3. The Origin Recognition Complex
1.4. Interactions between ORC and DNA Replication Proteins
1.5. Cdc2 Control of the Cell Cycle
1.6. Cdc2 Control of Replication
1.7. Project Goal

2. Materials
2.1. Sources of Used Chemicals, Enzymes, etc.
2.2. S. pombe Strains
2.3. Oligonucleotides
2.4. Solutions and Yeast Media
2.5. Equipment

3. Methods
3.1. Growth of S. pombe Strains
3.2. Chromatin Immunoprecipitation (ChIP)
3.3. Application of ChIP to S. pombe Strains
3.4. CsCl-Gradient Centrifugation
3.5. Phenol/Chloroform Extraction and Ethanol Precipitation .
3.6. PCR and Real-time PCR

4. Results
4.1. Cell Lysate Purification / CsCl Gradient
4.2. ORC Binds to Origins of Replication
4.3. Cdc2-GFP Co-immunoprecipitates with Origin DNA

5. Discussion
5.1. Development of a Working ChIP Assay for Routinely Lab Use
5.2. Verifying Previous Findings of ORC – Origin Interaction
5.3. Showing Cdc2 – ORC Interaction in vivo

6. Summary and Outlook





Chapter 1

We are unlikely to ever know everything about every organism. Therefore, we should agree on some convenient organism(s) to study in great depth, so that we can use the experience of the past to build on in the future” (Huxley, 1869).

1. Introduction

1.1 S. pombe as a Model System

Schizosaccharomyces pombe functions as a suitable model system since it is easy and inexpensive to rear, has a convenient size, a short life cycle, and is genetically manipulable. As a unicellular eukaryote, the fission yeast S. pombe can exist either in a haploid or diploid state and possesses two different mating types (h+ and h-). The wildtype however is h90, which means it can switch mating type. S. pombe shows a lot of similarity to Saccharomyces cerevisiae. However, the morphology of the two cells is different, with S. pombe being more rectangular than the circular-like S. cerevisiae cells (Hochstenbach, 1998).

S. pombe can undergo two different life cycles, either the vegetative (mitotic) cycle or the sporulation (meiotic) cycle, depending on the environment it is living in. These two cycles are shown in figure 2 with the change between the two occurring in cells at the G1 stage of the mitotic cycle. Under laboratory conditions, given all nutrients required, S. pombe prefers the haploid state. This makes it a favorable organism for genetic research since it ensures that introduced mutations are not masked by another wildtype allele.

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Fig. 1: Left, picture of S. pombe cells. At the top are two dividing cells in late Mitotic phase, showing the fission yeast typical septum at the point of cytoplasmic division. The lower cell is in early M phase, having its chromosomes already segregated.

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Fig. 2: Right, fission yeast cell cycle. Diagrammatic representation of the S. pombe cell cycles with the interchange between the two occurring in G1 phase (Figure obtained and used with permission from Trevor Pemberton, University of Sussex).

1.2 Eukaryotic DNA Replication - Once Per Cell Cycle Only

Transmission of genetic information from one cell generation to the next requires the accurate and complete duplication of each DNA strand exactly once before each cell division. Typically, this process begins with the binding of an “initiator” protein to a specific DNA sequence or “replicator”. In response to the appropriate cellular signals, the “initiator” directs a local unwinding of the DNA double helix and recruits additional factors to initiate the process of DNA replication. This paradigm describes most of the currently tractable replication systems and, although derived from prokaryotic and viral systems, there is no compelling reason to doubt that it will apply to all eukaryotic organisms. In fact, the proteins that regulate replication are highly conserved from yeast to humans, including the origin recognition complex (ORC), which binds directly to replication origin sequences in fission yeast (Leatherwood, 1996) as well as in all other eukaryotes tested (Diffley, 2001;Kelly, 2000).

1.3 The Origin Recognition Complex – a Closer Look

The origin recognition complex (ORC) plays a central role in initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. Competition binding experiments demonstrated that ORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. As shown in figure 3, from its six subunits, labeled accordingly to their size Orp1 – Orp6, only Orp4 binds through its N-terminal nine repeats long AT-Hooks directly to its cognate origins. Interestingly, although the remaining five subunits of ORC were able to interact with Orp4 bound to DNA, they did not appear to have any sequence-specific DNA binding activity on their own, nor did they alter the interaction of prebound Orp4 with the origin DNA (Dutta, 1997).

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Fig 3: The Origin Recognition Complex

Binding of S. pombe ORC (SpORC) to its cognate origins involves a clearly distinct mechanism from the one in S. cerevisiae involving nine repeats of an AT-hook motif found uniquely at the N-terminus of the S. pombe Orp4 subunit. A discrete binding site has not been identified, however recent studies indicate that SpORC recognizes stretches of A-rich DNA.

In most eukaryotic systems, binding of ORC to DNA is intimately linked to ATP binding and hydrolysis by ORC (Dutta, 1997). But in S. pombe, however, it is not. Although ATP binding is not substantial for a stable DNA binding by S. pombe ORC it might be needed for the recruiting of other proteins necessary for the initiation of replication. The function as an ATPase of ORC remains to be determined for all eukaryotes. Possible events that could be coupled to ATP hydrolysis include the assembly of protein complexes at the origin, changes in the origin DNA or the associated protein complexes during the initiation of DNA replication, or disassembly of origin-protein complexes after initiation has occurred. The function of ORC in eukaryotic cells is to select genomic sites where pre-replicative complexes (pre-RCs) can be assembled. Subsequent activation of these pre-RCs results in bi-directional DNA replication that originates at or close to the ORC DNA binding sites. Since ORC binding to DNA is the first step in pre-RC assembly, ORC determines where DNA replication can begin by binding specific DNA sites in the genome.

Initiation sites for DNA replication are distributed throughout all eukaryotic genomes. This insures that genomes can be duplicated in a few minutes (e.g. rapidly cleaving embryos of flies, frogs, and fish) to a few hours (e.g. mammalian cells) without having to change the basic replication fork mechanism by which DNA is replicated. In yeast, flies and mammals, cis-acting sequences (replication origins) determine where DNA replication will begin (DePamphilis 1999, Altman and Fanning 2001, Lu 2001, Bell and Dutta 2002), but with the exception of the small (app. 0.1 Kb) replication origins in S. cerevisiae, no consensus sequence has been identified that is required for replication. Replication origins in fission yeast, flies and mammals are five to 20 times larger than those in budding yeast. They frequently contain large AT-rich regions and genetically identifiable sequences that are required for origin function. The activity of these sequences is often orientation or distance dependent.

1.4 Interactions between ORC and DNA Replication Proteins – the Pre-RC

ORC binding to the replicator is the first step in the establishment of a multifactor assembly called the pre-replicative complex (pre-RC, for review see Bell, 2002). But how does ORC find its way to the tiny motifs within the genome, given an average spacing between strong origin sequences of app. 20-30 Kb? It seems unlikely that the sequence specificity of ORC is sufficient for localizing these origin sequences all by itself. Studies of the interaction of ORC with origin DNA in the presence of other pre-RC components suggest that these factors may contribute to the specificity of ORC localization. The recruitment of these factors occurs in an ordered process: Cdc18 (S. pombe name; also known as Cdc6 in S. cerevisiae) and Cdt1 load first followed by Mcm2-7, which may function as the replicative helicase (Tye, 1999). As cells pass through the G1 to S phase transition additional replication factors are recruited to the origin. These factors include Mcm10 and Cdc45, the three eukaryotic DNA polymerases, and the eukaryotic ssDNA binding protein, RPA (for review, see Bell, 2002). As with the components of the pre-RC, either cells or extracts lacking ORC fail to assemble these additional factors at the origin. Although it is clear that ORC is required to initiate the assembly of all of these factors at the origin, ORC has only been shown to interact directly with a small subset of these factors. The preinitiation complex is apparently fully assembled in G1 phase, and yet initiation does not occur. Initiation is not triggered until two protein kinases become active. These kinases are Hsk1 (S. pombe name; also known as Cdc7 in S. cerevisiae) and Cdc2 (Vas, 2001).

1.5 Cdc2 Control of the Cell Cycle in S. pombe

Cdc2 is a cyclin-dependent kinase (CDK), which is activated at different stages throughout the cell cycle mainly by the cyclins Cig2 and Cdc13. In S. pombe only one CDK, namely Cdc2 regulates the cell cycle (Moreno, 1989). Starting with no activity during G1 phase it allows the pre-RC to be assembled in preparation for DNA replication. The progression into S phase is allowed by the raising activity of Cdc2. This is accomplished in part because Rum1, a Cdc2 inhibitor, is degraded and the cyclins Cic1 and Cig2, lacking in G1, are synthesized and bound to Cdc2. At the same time the increased Cdc2 activity does not allow new formation of pre-RC and therefore prevents re-replication (Correa-Bordes, 1995). More cyclins accumulate during S phase and G2 but their effect on Cdc2 is held in check by inhibitory phosphorylation by the Wee1 and Mik1 kinases. In order to trigger mitoses, the activity of Cdc2 becomes higher at the end of G2 by its dephosphorylation via Cdc25 phosphatase and its binding to the mitotic cyclin Cdc13. At the very end of M phase Cdc2 is inactivated by expression of Rum1, destruction of Cdc13 and several other mechanisms (Lee, 1999; Booher, 1989).

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Fig 4: Cdc2 activity throughout the cell cycle. Cdc2 as the major cyclin-dependent kinase in S. pombe controls most of the cell’s cycle related activity. Cdc2 in return is activated at certain checkpoints by different cyclins. The most important one is Cdc13 because, although it is not present at all stages throughout the cell cycle, it can substitute the other cyclins and therefore activate Cdc2 all by itself. Cdc25 also has an activator function as it removes the inhibitory Tyr15 phosphorylation at Cdc2. Inhibitors including Wee1, Mik1, and Rum1 play the counterpart of activating cyclins and Cdc25. The whole cell cycle is a very precisely balanced system of inhibition and activation of Cdc2.

The function of Cdc2 is not limited to the control of DNA replication. It also takes part in control mechanisms of growth polarity, spindle pole body duplication, chromosome condensation, mitotic spindle functions, mitosis, and cytokinesis (Nurse, 1990).

1.6 Cdc2 Control of Replication

As shown in figure 5, the main CDK concentration in S. pombe, Cdc2, remains at a constant level throughout the cell cycle. Its regulatory function is established as its activity changes (Hendrickson, 1996). This change is triggered by Cdc2 binding to its cyclin partners Cdc13 and Cig2. Both cyclins act at different stages of the fission yeast cell cycle. Cdc13 formation peaks at the onset of M-Phase, causing a rapid rise of Cdc2 activity once it binds to it. Cig2 is responsible for raising the Cdc2 activity level at the beginning of S-Phase, allowing DNA replication to start. But whereas Cdc13 is essential, Cig2 is not. The whole cell cycle can be driven by Cdc13 alone (Nguyen, 2001). Therefore it is the cyclic destruction, reforming and binding to Cdc2 of the cyclins that state the internal clock of the cell cycle.

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Fig 5: Cyclic activation of Cdc2. Cdc13 and Cig2, the main cell cycle driving cyclins, act at different stages of the fission yeast cell cycle. Both are B-type cyclins and activate Cdc2 by binding either during S- or M-phase. It is known that Cdc2 binds also to Cig1 and Puc1 cyclins, which have an overlapping role with Cig2 in the initiation of S phase and are therefore included under Cig2 activity.

Initiation of replication depends on Cdc2 kinase activity, and a number of origin binding proteins including Cdc18, Mcm4, Orc2, and Orc6 are targets for Cdc2-dependent phosphorylation in vitro and in vivo (Vas, 2001; Lopez-Girona, 1998). How can changes in Cdc2 activity control DNA replication? Proteins belonging to the MCM family have been identified as being associated with G1 chromatin, which is in a permissive state for replication. Those MCM family proteins are absent from G2 chromatin, which cannot normally replicate. Interestingly, a feature of these proteins is that they are hypophosphorylated in G1 and become phosphorylated during S phase. This phosphorylation is performed by Cdc2. The changes in phosphorylation are correlated with changes in MCM affinity for chromatin providing a strong link between the Cdc2 activity cycle and DNA replication licensing. Mcm4 on the other hand requires Orc1 and Cdc18 to function in a proper way, whereas Cdc18 itself is dependent on ORC (Kearsey, 2000; Lopez-Girona, 1998). These findings suggest ORC working as a “landing pad” to assemble the various replication proteins of the pre-RC at the origin of replication (Prasanth, 2002).

Although ORC binds constitutively to chromatin throughout the fission yeast cell cycle, the prereplicative complex only forms on origins during G1. In recent publications (Wuarin 2002 and citations therein) it is reasoned that a factor may stably bind ORC during G2 and early M phase to prevent reinitiation of S phase. Since the Cdc13 protein resides in the chromatin domain of fission yeast nuclei and is required to prevent re-replication, this factor might be the Cdc13/Cdc2 complex itself. In cells lacking Cdc13, association of Cdc2 to origins was abolished and conversely, the association of Cdc13 to origins was greatly diminished in cdc2 ts-mutants. This mechanism is another example of the regulatory role of the Cdc13/Cdc2 complex. As with the phosphorylation of the Mcm proteins, prevention of re-replication by Cdc2 binding to ORC is mediated via association with the main cyclin Cdc13.

A third mechanism of DNA replication control by Cdc2 is the phosphorylation of the M phase checkpoint control factor Cdc18 (Ayté et al., 2001). Cdc2 may regulate replication licensing by catalyzing the destruction of nonchromatin bound Cdc18 protein. cdc18 and cig2 mRNAs and their corresponding proteins are both periodically expressed in synchronous endoredublicating cells lacking Cdc13 (cdc13::ura4 nmt41-cdc13). Cdc18 protein is degraded coincidently with the peak of Cig2 protein expression during each endoredublication cycle (Wuarin 2002). This suggests that Cig2/Cdc2 kinase prevents relicensing of origins both by catalyzing the periodic destruction of Cdc18 and by regulating its own synthesis. Once more this regulatory pathway shows the dependence of Cdc2 on its cyclin partner, here Cig2.

As previously found in vitro (Vas et al. 2001), Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. The cell cycle coupled regulation of replication is crucial for genome stability in every organism. I am particularly interested in the regulations that ensure genome stability because studying these pathways in yeast gives us insights into the mechanisms that are critical in mammalian cells to prevent cancer.

1.7 Project Goal

The goal of my project is to show an in vivo binding of Cdc2 to the origin recognition complex in S. pombe. The existing model of initiation of DNA replication is based on in vitro findings of a Cdc2 – ORC interaction in fission yeast (Leatherwood 1996). To show this interaction in vivo, a working chromatin immunoprecipitation (ChIP) assay had to be further developed to be employed routinely in the lab. The ChIP assay is a valuable method to show DNA – protein as well as protein – protein interactions in vivo. Existing protocols had to be modified and extended as well as new parts were to be added. Especially measures to enhance the stringency of the assay, such as the CsCl gradient centrifugation had to be implemented. Additionally more sensitive detection methods, such as optimized PCR programs and real time PCR detection should be employed.

To ensure the reliability of the ChIP assay, previously published ChIP experiments (reviewed in Gavin KA et al. 1995) were redone and the results were compared as control. It was to show that proteins of the origin recognition complex bind to origins of replication in S. pombe.

Chapter 2


2.1 Sources of Used Chemicals, Enzymes, and Kits.

American National Can, USA

BioRad Laboratories, Hercules, CA, USA

Fisher Scientific, Pittsburgh, PA, USA

J.T. Baker, Phillipsburg, NJ, USA

Mettler Toledo, Switzerland

Molecular Probe, Eugene, OR, USA

Nortech Laboratories Inc., Farmingdale, NY, USA

Pharmacia Biotech, Uppsala, Sweden

Q-Bio gene, USA

Qiagen Inc., Valencia, CA, USA

Roche Diagnostics GmbH, Mannheim, Germany

Sigma Chemical Co., St. Louis, MO, USA

VWR Scientific, USA

2.2 S. pombe Strains

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Table 1: S. pombe strains

2.3 Oligonucleotides

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Table 2: Oligonucleotides

The important 3’ region for specific priming is free of secondary structures, repetitive sequences, palindromes, and highly degenerated sequences.

2.4 Solutions and Yeast Media

All used chemicals were of analytical quality. The content of kit solutions can be obtained from the kit’s user manual.

2.5 M Glycine

37 % Formaldehyde

70 % Ethanol

Anti-GFP antibody (Molecular Probe)

Bradford reagent (BioRad Laboratories)

Glass beads (Sigma 212-300 microns)

Protein A coupled to agarose beads (Pharmacia Biotech)

Qiaquick PCR Purification Kit #28104

LaRoche FastStart High Fidelity PCR System Kit #03553426001

Yes Medium 5 g Yeast Extract, 30 g Glucose, 0.19 g Adenine, 0.19 g Histadine, 0.19 g Leucine, 0.19 g Uracil. Distilled water ad 1000 ml, for plates add 20 g agar/L

TE 10mM Tris HCl (pH 8.0), 1mM EDTA (pH 8.0)



ISBN (eBook)
ISBN (Buch)
993 KB
Institution / Hochschule
Technische Universität Bergakademie Freiberg
Interaction Cdc2 Origin Recognition Complex Origins Replication Schizosaccharomyces Pombe




Titel: Interaction of Cdc2 with the Origin Recognition Complex at Origins of Replication in Schizosaccharomyces Pombe