Real-Time Visualization of DNA-protein transactions during DNA...


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Title Real-Time Visualization of DNA-protein transactions during DNA Recombination: Analysis of Mechanistic Dynamics on Single DNA Molecules
Period 02 / 2007 - 02 / 2012
Status Completed
Research number OND1314669
Data Supplier Nederlandse Organisatie voor Wetenschappelijk Onderzoek


Summary of the proposed research The overall goal of our research is to understand the mechanisms and biological function of complex genome transactions such as homologous recombination. Homologous recombination, the exchange of sequences between homologous DNA molecules, is essential for accurate genome duplication, DNA damage repair and chromosome segregation. Recent advances in fluorescence detection and microscopy as well as our success in producing fluorescently labeled functional recombination proteins make it possible to analyze the mechanistic details of homologous recombination at the single molecule or single complex level. Single molecule analysis provides information on intermediate states, functional and structural variability and the distribution of variable states that cannot be recovered from bulk biochemical assays. Biologically, the importance of a reaction, such as homologous recombination, lies in the result of one such event occurring in the complex molecular environment of a cell. Thus the random variation in the details of molecular behavior, that we can now determine with single molecule mechanistic studies are of great importance for understanding how relatively simple biochemical activities are combined to create complex and adaptable living systems. Understanding the mechanism of homologous recombination as well as its control requires specific detailed descriptions of the conformational dynamics of the recombinase proteins and their DNA substrates, specifically the assembly and disassembly of the active recombinase-DNA nucleoprotein filament. We will use fluorescently labeled recombination proteins in single molecule fluorescence and fluorescent microscopy assays. By providing information on reaction intermediates as well as the stochastic behavior of molecules, analysis of single molecular entities provides a completely new picture of biologically important reaction mechanisms. The overall objectives of the research described in this proposal are: (1) Analyze the dynamic rearrangements between DNA and the core protein driving homologous recombination, Rad51, to gain insight into the key events that drive DNA strand exchange. (2) Analyze the effect of a number of accessory factors, regulators and mediators of Rad51 on its assembly onto and disassembly from DNA to gain insight into mechanisms that limit homologous recombination to appropriate locations. (3) Observe pairing of homologous DNA sequences by Rad51 to gain insight into the mechanism of DNA homology search/recognition within a genome. (4) Analyze the above reactions not only for the mitotically active Rad51 protein but also for the meiosis specific Dmc1 recombinase. The Dmc1 reaction also involves mediators whose mode of action will be analyzed to gain insight into interplay between the Dmc1 and Rad51 recombinases. We are uniquely suited to undertake this project and produce the highly relevant new information in a timely way. This project is embedded in a successful and internationally highly-respected research group devoted to understanding homologous recombination and other genome maintenance pathways through the application a wide variety of state of the art experimental tools. Our work spans the experimental range from animal models to single molecule imaging. Integrating information through this spectrum of techniques has a synergistic effect on our ability to understand fundamental biological processes. We have already developed the protein reagents needed to start the projects and immediately begin to apply this knowledge to the production of additional reagents. Expertise among our own group members and others at Erasmus MC as well as our collaborative work with nearby biophysics groups assure the success of the proposed sophisticated fluorescence detection, microscopy and analysis that we propose. Through these contacts we also have access to expertise that will help us apply and develop new methods in fluorescence detection and analysis.

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