Figure S1C shows that this more sensitive method detects higher numbers of foci even in undamaged cells, but with a similar timing. Thus, in order to follow RPA dynamics in real time, we employed live-cell imaging. Results The Dynamics of MMS-Induced RPA Foci Largely Reflects Postreplicative Events but Provides No Evidence for a Congregation of Lesions into Repair Centers Thus, we provide evidence that in budding yeast non-DSB lesions are processed predominantly in a manner that is both temporally and spatially well segregated from ongoing replication. Subsequent Exo1-dependent expansion over time contributes to the gradual emergence of discernable RPA foci in regions of replicated DNA that are resolved locally by the action of the RAD6 pathway and by salvage HR. Instead, they suggest a model where these foci originate in the wake of replication forks as local clusters of ssDNA due to multiple replisome re-priming events in spatial proximity. Moreover, our observations challenge the prevalent view that spontaneous or replication stress-induced repair foci generally represent stalled replication forks. In contrast to HR foci arising from the processing of DSBs or damaged replication forks, we find no evidence for a congregation of individual RPA foci or a significant overlap with other known repair centers such as nuclear pores, the nuclear periphery or intra-nuclear quality control (INQ) centers. They are largely resolved via the RAD6 pathway, involving both TS and TLS activities but also complemented by salvage recombination. We report here that under conditions of replication stress induced by polymerase-blocking lesions in the template, RPA foci can indeed serve as faithful indicators of DNA damage bypass tracts. We propose that most RPA and recombination foci induced by polymerase-blocking lesions in the replication template are clusters of repair tracts arising from replication centers by polymerase re-priming and subsequent expansion of daughter-strand gaps over the course of S phase. Instead, they represent sites of postreplicative DNA damage bypass involving translesion synthesis and homologous recombination. We now report that in budding yeast they predominantly form far away from sites of ongoing replication, and they do not overlap with any of the repair centers associated with collapsed replication forks or double-strand breaks. RPA foci emerging during S phase in response to tolerable loads of polymerase-blocking lesions are generally thought to indicate stalled replication intermediates. Most repair pathways involve single-stranded DNA (ssDNA), making replication protein A (RPA) a hallmark of DNA damage and replication stress. Tracing DNA repair factors by fluorescence microscopy provides valuable information about how DNA damage processing is orchestrated within cells.
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