Print Email Facebook Twitter The progression of replication forks at natural replication barriers in live bacteria Title The progression of replication forks at natural replication barriers in live bacteria Author Moolman, M.C. (TU Delft BN/Nynke Dekker Lab) Tiruvadi Krishnan, S. (TU Delft BN/Nynke Dekker Lab) Kerssemakers, J.W.J. (TU Delft BN/Technici en Analisten) de Leeuw, R. (TU Delft BN/Nynke Dekker Lab) Lorent, V.J.F. (Université Paris 13) Sherratt, David J. (University of Oxford) Dekker, N.H. (TU Delft BN/Nynke Dekker Lab) Date 2016 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. Subject Genome integrityrepair and replication To reference this document use: http://resolver.tudelft.nl/uuid:64c5ef9c-de66-4098-a5f8-39c3aed08a5b DOI https://doi.org/10.1093/nar/gkw397 ISSN 0305-1048 Source Nucleic acids research, 44 (13), 6262-6273 Part of collection Institutional Repository Document type journal article Rights © 2016 M.C. Moolman, S. Tiruvadi Krishnan, J.W.J. Kerssemakers, R. de Leeuw, V.J.F. Lorent, David J. Sherratt, N.H. Dekker Files PDF gkw397.pdf 3.46 MB Close viewer /islandora/object/uuid:64c5ef9c-de66-4098-a5f8-39c3aed08a5b/datastream/OBJ/view