DNArepair at 3Detail · Recombinational DNA repair analyzed by simultaneous scanning force and single molecule fluorescence microscopy: role of RAD54 in presynaptic and postsynaptic events
FP7 — People (Marie Curie Actions)
- Duration
- 2011-04-01 → 2014-03-31
- EU contribution
- €45,000
- Participants
- 1
- Scheme
- MC-ERG
Lines connect the coordinator with its partners.
Results in brief
Recombinational DNA repair analyzed by simultaneous scanning force and single molecule fluorescence microscopy: role of RAD54 in presynaptic and postsynaptic events
Efficient treatment of genetic diseases like cancer requires detailed analysis of DNA repair processes. Here, we describe how combining two types of microscopes make it possible to recognize different components of DNA repair machinery at the nanometer level. Essential components of cellular repair systems are recombinases proteins like RAD51. These proteins bind to damaged DNA forming filaments that do the work of DNA recombination and repair. These filaments can find the complementary DNA sequence that is used as a backup for recovering missing information. Mediator proteins, like RAD54 and BRCA2, regulate these filament functions for effective repair. However, the molecular mechanisms of this regulation remain largely unknown. We were able to describe the localization of different mediators on RAD51-DNA filaments representing different stages in the repair reaction. Scanning Force Microscopy (SFM) can visualize proteins and their complex assemblies working on DNA with nm resolution. Nevertheless, this technique fails in recognizing two different proteins when they have similar shape, a common situation in DNA metabolism. So, we combined SFM with fluorescence microscopy. We have solved a long-standing problem in structural biology allowing us to produce photographs of DNA associated with recombinases and identify the relative position of other components important for genetic stability. This methodology and the analysis tools that we have developed, will open the door to novel and exciting possibilities for understanding the molecular mechanism of cancer.
Data: CORDIS, © European Union
Project objective
The goal of the research is to understand the mechanistic of human genetic recombination at the single molecular level.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.Understanding the mechanism of DNA repair by homologous recombination requires detailed structural descriptions of recombination intermediates.We are uniquely poised to unravel key steps in homologous recombination at the molecular mechanistic level using state of the art imaging tools. We have a substantial track record in applying SFM topographic imaging to the understanding of DNA break repair mechanisms and other genome transactions. The recently developed method that combined SFM with single molecule sensitivity fluorescence will expand the information we can obtain from molecular imaging. In particular, our aims will be:1 Simultaneous localization of multiple human DNA repair factors acting on recombination intermediates.2 Analysis of DNA replication after repair.Homologous recombination proteins are the target for important treatment modalities against cancer. By analysing the mechanism through which these proteins cooperate in DSB repair, we expect to provide insights into their molecular assembly.Recombination proteins and DNA substrates labelled with flourophores will be used in single molecule microscopy assays. We have developed methods to combine SFM nm resolution topography and single molecule sensitivity fluorescence (Sanchez, et al., 2010). This SFM-fluorescence microscopy will be exploited here for specifically recognizing and localizing DNA repair factors.
Original text from CORDIS.
Participants
- ERASMUS UNIVERSITAIR MEDISCH CENTRUM ROTTERDAM · RotterdamCoordinatorNetherlands
Links
Data: CORDIS, © European Union