In the RecPAIR project, we focused on the search for the repair template during double-stranded break (DSB) repair via the homologous recombination (HR) pathway using E. coli as a model organism. We used microfluidics and quantitative fluorescence microscopy to unravel the molecular mechanism of the search for the template in live cells.
The DSB repair by homologous recombination (HR) is a conserved and abundant mechanism. It deals with a particularly toxic type of DNA damage and serves an important protective role in cell metabolism. Defects in the recombination machinery are linked to genetic diseases and to a high risk of cancer. A detailed description of the molecular mechanism of the HR will help to better understand, and potentially prevent, the pathologies associated with the defects in recombination. DSB repair relies on that the damaged DNA strand can locate the right repair template fast and accurately, but how this is achieved in a living cell has, until now, been misapprehended. The core components and basic mechanistic principles of the DSB repair are shared between even distantly related organisms. The fundamental principles of the recombination mechanism discovered in bacteria likely also apply to eukaryotic cells.
The search relies on a prototypic strand exchange protein, RecA in E. coli, which forms a filament of single-stranded DNA (ssDNA) that can locate a homologous target within a double-stranded DNA. In this project, we show that it usually takes a bacterium 10-20 minutes to repair a DSB and that the homology search is completed in less than 9 ± 3 minutes by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose a model in which the architecture of the RecA filament effectively speeds up the search by reducing it from three to two dimensions.
Our efforts also resulted in a flexible and universal system to study the process of DSB repair, or other types of DNA damage repair, that can be easily implemented by us, or other researchers in the future. The mentioned system consists of fluorescent fusions of RecA protein, inducible Cas9 nuclease to create DNA damage, microfluidics, and image analysis software.
