Analysis of DNA repair in bacteria in real time and at the single molecule level

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The integrity of chromosomes is ensured by a very large yet well conserved set of proteins in all cells. As inability to repair DNA damage or an overshooting response are detrimental to cell survival, knowledge on the detailed mechanisms of DNA 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 extend and deepen our knowledge on DNA repair in the model bacterium Bacillus subtilis. We will study the exchange of the two replicative DNA polymerases by translesion polymerases, the recruitment of repair proteins to replication forks, and the dynamics of repair proteins at distinct double strand breaks, using an inducible endonuclease system. An essential step forward will be taken by visualization of proteins at individual, stalled replication forks, using an inducible DNA roadblock. We will take advantage on our collection of functional fluorescent protein fusions of major DNA repair proteins involved in repair by homologous recombination, and a large mutant collection, and extend our study of replication restart enzymes and homologous recombination proteins at individual sites or replication forks or DNA breaks sites at highest possible spatio-temporal resolution.