Dokument

Summary:
DNA double strand breaks (DSBs) are a severe threat to genome integrity and thus a variety of proteins are dedicated to repair such threats. The major repair route in bacteria is that of homologous recombination (HR), with the ATPase RecA as a key player. In HR, a broken DNA strand is repaired using a second intact DNA copy present on a homologous chromosome. This process involves the exchange of DNA strands, mediated by RecA, which forms filamentous polymers on ssDNA and initiates strand exchange. Prior to RecA, a plethora of Rec-proteins (Rec NJORFX) act to initiate HR. In Bacillus subtilis this process starts with RecN forming foci 15 minutes after DSB induction and is finished when RecA filaments disassemble and cell growth resumes after three hours. I wished to obtain a more detailed view on the dynamics of these proteins, and therefore employed single molecule fluorescence microscopy in live cells. Using 40 ms stream acquisition, I detected the movement of single Rec proteins and analyzed these trajectories mathematically. In exponentially growing cells I observed that RecN, RecO and, partially, RecJ continuously scan the nucleoid, supporting a distributive search model of individual molecules. In contrast to RecN and RecO, a fraction of the exonuclease RecJ is retained at the replication machinery. Upon induction of DSBs, RecNJO arrest at several sites on the nucleoid. RecN does not form static repair centers as proposed for eukaryotes, but short-lived (~2.5 s) clusters that act as repair enzyme recruitment platforms. Thus the local concentration of Rec-proteins increases to trap interaction partners out of a pool of diffusive enzymes. A majority of the RecNJO molecules keep on scanning for lesions or interaction partners, even in the presence of DSBs. In toto, my work indicates that the initial detection of a DSB, processing of free DNA ends, and loading of RecA on the generated ssDNA site takes place in a very short time frame, performed by a minority of the protein population.

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