Analysis of DNA replication restart in bacteria at the single molecule level

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A very large and well conserved set of proteins is dedicated to ensure chromosomes integrity in all cells. Inability to repair DNA damage or fail to restart blocked replication forks are detrimental to cell survival, so understanding the mechanisms of how cells organize different repair pathways is important to understand life. Recently, it has become possible to study the interplay of proteins in real time, at a single molecule level. We will employ the new technology of single molecule tracking (SMT) to study the way replication forks deal with DNA damage in the model bacterium Bacillus subtilis. We will study the exchange of the two replicative DNA polymerases by the two alternative translesion polymerases, as well as their putative function outside of replication forks. We will also investigate the recruitment of known repair proteins to replication forks. An essential step forward will be taken by visualization of proteins at individual, stalled replication forks, using an inducible and reversible DNA roadblock. We will take advantage of our collection of major DNA repair proteins as functional fluorescent protein fusions, and a large mutant collection, and extend our study of replication restart enzymes and homologous recombination proteins at individual sites or replication forks at highest possible spatio-temporal resolution.