Technische Universität Bergakademie Freiberg
Fakultät für Chemie und Physik

Interaction of Cdc2 with the origin recognition
complex at origins of replication in
Schizosaccharomyces pombe

by

Michael Sassen

2004

TABLE OF CONTENT

1. Introduction ...  1
1.1. S. pombe as a Model System  ...   1
1.2. Eukaryotic DNA Replication  ...   2
1.3. The Origin Recognition Complex ...   3
1.4. Interactions between ORC and DNA Replication Proteins  ...  4
1.5. Cdc2 Control of the Cell Cycle  ...  5
1.6. Cdc2 Control of Replication  ...   6
1.7. Project Goal  ...  8

2. Materials ...   9
2.1. Sources of Used Chemicals, Enzymes, etc.  ...   9
2.2. S. pombe Strains  ...  10
2.3. Oligonucleotides  ...  10
2.4. Solutions and Yeast Media  ...  10
2.5. Equipment ...  12

3. Methods ...   13
3.1. Growth of S. pombe Strains  ...   13
3.2. Chromatin Immunoprecipitation (ChIP) ...   13
3.3. Application of ChIP to S. pombe Strains  ...   16
3.4. CsCl-Gradient Centrifugation  ...   18
3.5. Phenol/Chloroform Extraction and Ethanol Precipitation  ...   19
3.6. PCR and Real-time PCR  ...  20

4. Results  ...  23
4.1. Cell Lysate Purification / CsCl Gradient  ...  23
4.2. ORC Binds to Origins of Replication  ...   24
4.3. Cdc2-GFP Co-immunoprecipitates with Origin DNA ...  26

5. Discussion  ...   28
5.1. Development of a Working ChIP Assay for Routinely Lab Use ...  28
5.2. Verifying Previous Findings of ORC – Origin Interaction  ...  29
5.3. Showing Cdc2 – ORC Interaction in vivo  ...  30

6. Summary and Outlook  ...   31

Acknowledgements  ...   33
Figures  ...   33
Tables  ...  34
References  ...   35

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).

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. 

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.

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).

[...]

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).

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).

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 occurredThe function of ORC in eukaryotic cells is to select genomic sites where pre-replicative complexes (pre-RCs) can be assembled.

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