When exposed to replication stress, the factors involved in DNA replication display differing responses that in many cases remain unexplained. In this study, we generated a series of genomically engineered embryonic stem cell lines with fluorescently tagged replication factors to track their spatiotemporal dynamics in live-cell microscopy during and after treatment with the replication inhibitors aphidicolin and hydroxyurea. All 3 replicative polymerases were found to accumulate upon stress, with the primase subunits of polymerase alpha retaining catalytic activity. This accumulation was dependent on new origin firing and accompanied by 9-1-1 clamp loading in the vicinity of stalled forks. Pulse-stress-pulse experiments and spatial correlation analyses of replicons indicated that, after stress is removed, replication preferentially restarts at new sites surrounding previously stalled forks. These findings shed light on the replisome's immediate reaction to stalled replication forks and provide comprehensive kinetic maps of the replisome under stress.
Schulte, L., Pabba, M. K., Kamkaew, M., Prorok, P., Arroyo, M., Cardoso, M. C.
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