The parABS system is a widely employed mechanism for plasmid partitioning and chromosome segregation in bacteria. ParB binds to parS sites on plasmids and chromosomes and associates with broad regions of adjacent DNA, a phenomenon known as spreading. Although essential for ParB function, the mechanism of spreading remains poorly understood. Using single-molecule approaches, we discovered that Bacillus subtilis ParB (Spo0J) is able to trap DNA loops. Point mutants in Spo0J that disrupt DNA bridging are defective in spreading and recruitment of structural maintenance of chromosomes (SMC) condensin complexes in vivo. DNA bridging helps to explain how a limited number of Spo0J molecules per parS site (~20) can spread over many kilobases and suggests a mechanism by which ParB proteins could facilitate the loading of SMC complexes. We show that DNA bridging is a property of diverse ParB homologs, suggesting broad evolutionary conservation.

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SUMMARY Repair of DNA double-strand breaks (DSBs) is essential for genomic stability. The most common DSB repair mechanism in human cells, non-homologous end joining (NHEJ), rejoins broken DNA ends by direct ligation. It remains unclear how components of the NHEJ machinery assemble a synaptic complex that bridges DNA ends. Here, we use single-molecule imaging in a vertebrate cell-free extract to show that synapsis of DNA ends occurs in at least two stages that are controlled by different NHEJ factors. DNA ends are initially tethered in a long-range complex whose formation requires the Ku70/80 heterodimer and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The ends are then closely aligned, which requires XLF, a non-catalytic function of XRCC4-LIG4, and DNA-PK activity. These results reveal a structural transition in the synaptic complex that governs alignment of DNA ends. Our approach provides a means of studying physiological DNA double-strand break repair at single-molecule resolution.