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Genetic landscape of the homology search

Periodic Reporting for period 1 - RecPAIR (Genetic landscape of the homology search)

Reporting period: 2019-06-08 to 2021-06-07

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.
The work within the RecPAIR project can be divided into four different sections:

(1) We have created a highly controlled, inducible genetic system to create site-specific DSB on demand. To achieve this goal we used cutting-edge molecular biology and microfluidic tools. The Cas9 under inducible promoter and DNA damage reporter was cloned into a plasmid. In combination with another plasmid containing a specific guide RNA, we were able to create a large number of repairable DSBs. We optimised the induction of the DSB system using mother-machine microfluidic chips to maximise the number of cells undergoing the DNA repair which allowed us to generate sufficient amounts of data to perform a detailed, quantitative description of the DSB repair and homology search.

(2) New image processing code was written to analyse and plot the data. We developed a neural network (U-Net) based segmentation tool which shortened the analysis time and increased the accuracy of cell detection.

(3) A RecA fusion to the ALFA epitope tag was cloned and imaged with STED microscopy to visualise the RecA filaments beyond the resolution limit of conventional microscopy. This fusion allowed us to discover the architecture of the RecA-ssDNA filaments during the search phase within a cell.

(4) A Quantitative model of the homology search was developed to explain the experimental results generated in the RecPAIR project. The model takes the observations of the dynamics of the search and the architecture of the RecA filament into account. The predictions of the model were validated with the results of the experiments.

Main results of the RecPAIR project:
In the project, we have imaged the DSB repair process in live cells with unmatched precision. We discovered that the repair of a single DSB is finished in under 15 minutes with little fitness cost. Next, using fluorescent fusions of RecA, we showed that the repair is facilitated by thin and highly dynamic filaments of RecA. We described a new quantitative model in which the architecture of the RecA filament effectively reduces search dimensionality to two dimensions. The RecPAIR project led to a manuscript that is currently available on the bioRxiv preprint server titled “Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search”.
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 linked to it.

Our results show that the long and stretched RecA-ssDNA filament can sample many different sites along the cell length, and reduces the dimensionality of the search, thus, allowing for repair to be finished in only 15 minutes while scanning a vast amount of DNA. We can speculate that the defects in the formation of the filament, or changes in the time which segments of RecA filament scan the potential double-stranded DNA will greatly affect the efficiency of HR. Our results and conclusions are consistent with the biochemical description of the search from in vitro studies of the RecA protein. We show that in bacteria, the search is governed by simple biophysical principles. An interesting question is whether complex organisms with larger genomes compacted in a nucleus also take advantage of the ‘reduced dimensionality’ mechanism and if the Rad51 (RecA homolog) filament architecture resembles that of the E. coli.
Example of image data and our interpretation of these