Final Report Summary - DNAREPAIR AT 3DETAIL (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.
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.