Abstract
Single-particle (molecule) tracking (SPT/SMT) is a powerful method to study dynamic processes in living bacterial cells at high spatial and temporal resolution. We have performed single-molecule imaging of early DNA double-strand break (DSB) repair events during homologous recombination in the model bacterium Bacillus subtilis. Our findings reveal that DNA repair centres arise at all sites on the chromosome and that RecN, RecO and RecJ perform fast, enzyme-like functions during detection and procession of DNA double strand breaks, respectively. Interestingly, RecN changes its diffusion behavior upon induction of DNA damage, from a largely diffusive to a DNA-scanning mode, which increases efficiency of finding all sites of DNA breaks within a frame of few seconds. RecJ continues being bound to replication forks, but also assembles at many sites on the nucleoid upon DNA damage induction. RecO shows a similar change in its mobility as RecN, and also remains bound to sites of damage for few hundred milliseconds. Like RecN, it enters the nucleoid in damaged cells. Our data show that presynaptic preparation of DSBs including loading of RecA onto ssDNA is highly rapid and dynamic, and occurs throughout the chromosome, and not only at replication forks or only at distinct sites where many breaks are processes in analogy to eukaryotic DNA repair centres.
Highlights
SPT/SMT has revealed unprecedented insights into the mechanism of diverse cellular processes such as signal transduction[1], chromosome segregation[2,3], transcription[4], translation[5], replication[6] and DNA-repair[7,8,9]
The initial protein to visually assemble on the nucleoid in response to the induction of double-strand break (DSB) is the structural maintenance of chromosomes (SMC)-like protein RecN16,17, which binds to DNA ends in vitro[18] (Fig. 1)
To visualize RecJ, we generated a C-terminal yellow fluorescent protein (YFP) fusion, which was integrated into the original gene locus, and which is likewise expressed as sole source of the protein, and is under the control of the original promoter
Summary
SPT/SMT has revealed unprecedented insights into the mechanism of diverse cellular processes such as signal transduction[1], chromosome segregation[2,3], transcription[4], translation[5], replication[6] and DNA-repair[7,8,9]. We thereby obtained detailed and quantitative information on changes in diffusion patterns that are different from all proteins investigated, revealing that RecN and RecO change their diffusive behavior, massively entering the chromosome after induction of breaks, thereby maintaining a considerable pool of molecules that continuously scan the DNA for arising breaks throughout the entire genome. This mode of action ensures surveillance and safeguarding of all genetic information
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