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.
Inhaltsverzeichnis (Table of Contents)
- Introduction
- S. pombe as a Model System
- Eukaryotic DNA Replication
- The Origin Recognition Complex
- Interactions between ORC and DNA Replication Proteins
- Cdc2 Control of the Cell Cycle
- Cdc2 Control of Replication
- Project Goal
- Materials
- Sources of Used Chemicals, Enzymes, etc.
- S. pombe Strains
- Oligonucleotides
- Solutions and Yeast Media
- Equipment
- Methods
- Growth of S. pombe Strains
- Chromatin Immunoprecipitation (ChIP)
- Application of ChIP to S. pombe Strains
- CsCl-Gradient Centrifugation
- Phenol/Chloroform Extraction and Ethanol Precipitation
- PCR and Real-time PCR
- Results
- Cell Lysate Purification / CsCl Gradient
- ORC Binds to Origins of Replication
- Cdc2-GFP Co-immunoprecipitates with Origin DNA
- Discussion
- Development of a Working ChIP Assay for Routinely Lab Use
- Verifying Previous Findings of ORC – Origin Interaction
- Showing Cdc2 - ORC Interaction in vivo
- Summary and Outlook
- Acknowledgements
- Figures
- Tables
- References
Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)
This work focuses on understanding the role of Cdc2 in the regulation of DNA replication in the fission yeast Schizosaccharomyces pombe. The study aims to investigate the interaction of Cdc2 with the origin recognition complex (ORC) at origins of replication in S. pombe and its implications for cell cycle control.
- The role of Cdc2 in regulating DNA replication in S. pombe.
- The interaction of Cdc2 with the ORC at origins of replication.
- The significance of this interaction for cell cycle control.
- The development and application of a chromatin immunoprecipitation (ChIP) assay.
- The validation of previous findings regarding ORC-origin interactions.
Zusammenfassung der Kapitel (Chapter Summaries)
The introduction chapter sets the stage by discussing the significance of S. pombe as a model system for studying eukaryotic DNA replication. It provides a concise overview of eukaryotic DNA replication, the role of the origin recognition complex (ORC), and the importance of Cdc2 in regulating the cell cycle. The chapter concludes with outlining the project's specific objectives.
The materials and methods chapter details the procedures used throughout the study, including strains, chemicals, equipment, and specific techniques such as chromatin immunoprecipitation (ChIP) and real-time PCR.
The results chapter presents the key findings of the study, including evidence for ORC binding to origins of replication and the co-immunoprecipitation of Cdc2-GFP with origin DNA.
The discussion chapter interprets the results, highlighting the significance of the findings for understanding the role of Cdc2 in DNA replication and providing a framework for future research directions.
Schlüsselwörter (Keywords)
The primary keywords and focus topics of this work include Schizosaccharomyces pombe, DNA replication, origin recognition complex (ORC), Cdc2, cell cycle control, chromatin immunoprecipitation (ChIP), and real-time PCR.
- Arbeit zitieren
- Michael Sassen (Autor:in), 2004, Interaction of Cdc2 with the Origin Recognition Complex at Origins of Replication in Schizosaccharomyces Pombe, München, GRIN Verlag, https://www.hausarbeiten.de/document/41889
