DYNASING

Dynamic single-molecule approach to DNA homologous recombination

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 Obiettivo del progetto (Objective)

'The goal of the 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. 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. 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. Recombination proteins labelled with a flourophore will be use in single molecule fluorescence microscopy assays. The main objectives are: 1 Analyze the dynamic rearrangements between DNA and Rad51 to gain insight into the key events that drive DNA strand exchange. 2 Analyze the effect of accessory factors of Rad51 on its assembly/disassembly from DNA to gain insight into mechanisms that limit homologous recombination to appropriate locations. DNA damaging agents, such as ionizing radiation and interstrand DNA crosslinking compounds, provide important treatment modalities against cancer. Among the proteins implicated in repair of DNA damage induced by these agents are the homologous recombination. By analyzing the mechanism through which these proteins cooperate in DSB repair, we expect to provide insights into the molecular assembly pathways of the ‘guardians of the genome’.'

Introduzione (Teaser)

High-resolution techniques offer much in the way of uncovering molecular behaviour. This is necessary for the study of how healthy as well as diseased living systems develop.

Descrizione progetto (Article)

The 'Dynamic single-molecule approach to DNA homologous recombination' (Dynasing) project studied the mechanisms and biological function of homologous recombination. This is the intricate exchange of sequences between homologous deoxyribonucleic acid (DNA) molecules, and is essential for genome duplication, DNA damage repair and chromosome segregation.

The project's main objectives were to analyse the dynamics behind DNA strand exchange and protein assembly/disassembly, so as to better understand how homologous recombination in the form of double-strand break (DSB) repair is restricted to appropriate locations.

Single molecule analysis can provide information on what is happening at this level, as well as on differences in the function and structure of these molecules. This is important in learning more about the proteins involved in this recombination process and how their DNA substrates form or become deformed.

Dynasing used a combination of methods including scanning force, fluorescent microscopy and imaging techniques to conduct their investigations of DNA-bound proteins and molecular dynamics at never-before-seen resolutions. This multi-approach will pave the way for development of new tools to further examine complex protein assemblies.

On the strength of such breakthroughs, researchers have been able to show that the RAD50/MRE11/NBS1 (RMN) protein complex plays a vital role in the repair of DSBs through homologous recombination. Other project successes include analysing the movement of human RAD54 protein with nanometre and millisecond resolution.