Homologous recombination requires nucleolytic degradation (resection) of DNA double-strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long-range resection is coordinated.
Microscopic bioluminescence imaging has been historically challenging due to a lack of detection methods and easily resolved probes. Here we combine bioluminescence with phasor analysis, an optical method commonly used to distinguish spectrally similar fluorophores. Bioluminescent phasor enabled rapid differentiation of multiple luciferase reporters and resonance energy transfer processes. The merger of bioluminescence and phasor analysis provides a platform for routine, time-lapse imaging of collections of cellular features.
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