Eukaryotic DNA replication depends on the origin recognition complex (ORC) which is conserved from yeast to humans. Activity of replication factors including ORC is coordinated with the cell cycle progression to ensure that the entire genome is copied exactly once per cell cycle. This coordination depends on Cdc2 (cell division cycle 2), also highly conserved across all eukaryotes, which binds to the origin recognition complex.
This book presents research results of the chromatin cross-linking immuno-precipitation (ChIP) procedure to determine (1) if Cdc2 kinase is localized at DNA replication origin as predicted by current models, (2) when this happens and (3) what the genetic requirements for that localization are. Furthermore the binding ability of different ORC and cdc2 mutants was tested, as well as the influence of Cdc18 on the ORC-Cdc2 interaction. This is particularly interesting, since those regulations ensure genome stability. Studying these pathways in yeast gives us insights into the mechanisms that are critical in mammalian cells to prevent cancer.
Table of Contents
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
Research Objectives and Key Topics
The primary research objective of this work is to demonstrate the in vivo binding of the Cdc2 kinase to the origin recognition complex (ORC) in the fission yeast Schizosaccharomyces pombe. By further developing and optimizing a chromatin immunoprecipitation (ChIP) assay, the author seeks to provide in vivo evidence for the interaction of replication control proteins at DNA replication origins, thereby elucidating the mechanisms that prevent re-replication and ensure genomic stability.
- Optimization of Chromatin Immunoprecipitation (ChIP) techniques for fission yeast
- Characterization of ORC binding to replication origins in vivo
- Investigation of the interaction between Cdc2 kinase and the origin recognition complex
- Evaluation of cell cycle control mechanisms governing DNA replication
- Implementation of sensitive detection methods such as real-time PCR for protein-DNA interactions
Excerpt from the Book
4.3 Cdc2-GFP co-immunoprecipitates with origin DNA
Although ORC binds constitutively to chromatin throughout the fission yeast cell cycle, the prereplicative complex only forms on origins in G1. So presumably there is a factor, which inhibits reformation of the pre-RC once origins are fired. This factor might stably bind ORC during G2 and early M phase to prevent reinitiation of S phase. Postulating that this factor is the Cdc13/Cdc2 complex itself a GFP-tagged cdc2 mutant and chromatin immunoprecipitation (ChIP) analysis were used to identify a possible binding of Cdc2 to ORC. The GFP is attached at the C-terminus Cdc2 and since the cells do not show any mutant phenotype the assumption can be made, that the GFP tag does not affect Cdc2 function in vivo.
Summary of Chapters
1. Introduction: Outlines the use of S. pombe as a model system for DNA replication and introduces the role of the ORC and Cdc2 kinase in cell cycle control.
2. Materials: Lists all strains, chemicals, buffers, and equipment used throughout the experimental procedures.
3. Methods: Details the cultivation of yeast strains and the optimization of the chromatin immunoprecipitation (ChIP) assay, including sonication, CsCl-gradient centrifugation, and real-time PCR.
4. Results: Presents the findings regarding lysate purification and provides evidence for the in vivo binding of ORC and Cdc2 to origins of replication.
5. Discussion: Evaluates the success of the developed ChIP assay and interprets the data in the context of cell cycle regulation and genome stability.
6. Summary and Outlook: Synthesizes the experimental findings and proposes future studies, such as genome-wide "ChIP on a chip" experiments, to further understand replication initiation.
Keywords
Schizosaccharomyces pombe, DNA Replication, Origin Recognition Complex, ORC, Cdc2, Chromatin Immunoprecipitation, ChIP, Cell Cycle, Pre-replicative Complex, ARS3001, Gene Regulation, Genomic Stability, Kinase, Fission Yeast, Protein-DNA Interaction
Frequently Asked Questions
What is the core focus of this research?
This work focuses on the interaction between the cyclin-dependent kinase Cdc2 and the origin recognition complex (ORC) in the fission yeast Schizosaccharomyces pombe to better understand how DNA replication is controlled.
Which organisms are investigated in this study?
The research is conducted exclusively on the fission yeast Schizosaccharomyces pombe, which serves as a model system for eukaryotic DNA replication.
What is the main goal of the project?
The goal is to prove the in vivo binding of Cdc2 to the origin recognition complex at replication origins, validating models previously based only on in vitro data.
What scientific methodology was utilized?
The author utilized and optimized the chromatin immunoprecipitation (ChIP) method, combined with CsCl-gradient centrifugation and real-time PCR, to detect protein-DNA interactions.
What does the main body of the work cover?
It covers the systematic optimization of the ChIP protocol, the demonstration of ORC binding to the ARS3001 origin, and the identification of the interaction between Cdc2 and ORC.
Which keywords define this research?
Key terms include Schizosaccharomyces pombe, ORC, Cdc2, ChIP, DNA replication, and cell cycle regulation.
Why was the CsCl-gradient centrifugation implemented?
It was implemented as a cleanup step to increase the stringency of the ChIP assay, effectively reducing contamination by free protein and uncrosslinked DNA.
What is the significance of the ARS3001 origin?
ARS3001 is a well-characterized replication origin in S. pombe located within ribosomal repeats, providing high copy numbers for robust detection of protein-DNA complexes.
How does the author verify that the ChIP signal is specific?
Specificity is verified through the use of negative controls, including wildtype strains and non-origin specific primers, as well as melting curve analysis in real-time PCR.
- Quote paper
- Michael Sassen (Author), 2004, Interaction of Cdc2 with the Origin Recognition Complex at Origins of Replication in Schizosaccharomyces Pombe, Munich, GRIN Verlag, https://www.grin.com/document/41889
