The progression of replication forks at natural replication barriers in live bacteria
M. Charl Moolman (TU Delft - BN/Nynke Dekker Lab)
Sriram Tiruvadi Krishnan (TU Delft - BN/Nynke Dekker Lab)
J.W.J. Kerssemakers (BN/Technici en Analisten)
R. de Leeuw (TU Delft - BN/Nynke Dekker Lab)
V.J.F. Lorent (Université Paris 13)
David J. Sherratt (University of Oxford)
Nynke Dekker (TU Delft - BN/Nynke Dekker Lab)
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Abstract
Protein-DNA complexes are one of the principal barriers the replisome encounters during replication. One such barrier is the Tus-ter complex, which is a direction dependent barrier for replication fork progression. The details concerning the dynamics of the replisome when encountering these Tus-ter barriers in the cell are poorly understood. By performing quantitative fluorescence microscopy with microfuidics, we investigate the effect on the replisome when encountering these barriers in live Escherichia coli cells. We make use of an E. coli variant that includes only an ectopic origin of replication that is positioned such that one of the two replisomes encounters a Tus-ter barrier before the other replisome. This enables us to single out the effect of encountering a Tus-ter roadblock on an individual replisome. We demonstrate that the replisome remains stably bound after encountering a Tus-ter complex from the non-permissive direction. Furthermore, the replisome is only transiently blocked, and continues replication beyond the barrier. Additionally, we demonstrate that these barriers affect sister chromosome segregation by visualizing specific chromosomal loci in the presence and absence of the Tus protein. These observations demonstrate the resilience of the replication fork to natural barriers and the sensitivity of chromosome alignment to fork progression.
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