(C) Stability of Pol2-FLAG and Mcm4 shown in B was quantified as described in Figure 1. in the absence of CHX. However CHX affected cell cycle progression after the release from G2/M. (B) Cellular amounts of Pol2-FLAG and Mcm4 were determined at the indicated times after the release from G2/M. (C) Stability of Pol2-FLAG and Mcm4 shown in B was quantified as described in Physique 1. Pol2-FLAG was unstable in were quantified using EZQuant-Gel 2.1. Relative intensity of protein bands at 0 h FHF4 was set to 1 1 in each experiment. Relative Pol2-FLAG Xylometazoline HCl levels in cells at 25 and 35C shown in (Physique 3D) were also quantified.(EPS) pgen.1003213.s003.eps (6.0M) GUID:?EB0FB0C3-98EF-4B6D-9F6D-202041047D8F Physique S4: cells with or without genotoxic brokers were investigated. Exponentially growing cells were shifted to 30C for 3 h and fixed in ethanol and stained with DAPI. cells undergo mitotic catastrophes. Quantification of cells with defective chromosome segregation was performed. More than 300 cells were counted at each time point. (B) Representative images of observed nuclear phenotypes in are shown. The scale bar represents 10 m. (C) DNA damage sensitivity of mutation. Five-fold serial dilutions of cells were incubated on YES agar medium supplemented with the indicated amounts of HU and CPT for 4 to 5 days at 25C. (D) The checkpoint-dependent cell elongation phenotype of mutation. Cells of the indicated genotypes were incubated on YES agar medium made up of 5 mM HU or 5 M CPT for 2 days at 25C and photographed. The scale bar represents 10 m. (E) mutation exacerbates replication recovery defects of strains due to recombination at rDNA repeats. (F) Quantification of DNA replication recovery shown in strains used in this study.(DOCX) pgen.1003213.s007.docx (104K) GUID:?16FC18EA-B6B6-4B4F-9E4A-67F3F71424E7 Table S2: Primers used in this study.(DOCX) pgen.1003213.s008.docx (114K) GUID:?98AFE9EB-3B41-45B6-8979-978CEB8E08B1 Abstract The stabilization of the replisome complex is essential in order to achieve highly processive DNA replication and preserve genomic integrity. Conversely, it would also be advantageous for the cell to abrogate replisome functions to prevent inappropriate replication when fork progression is usually adversely perturbed. However, such mechanisms remain elusive. Here we report that replicative DNA polymerases and helicases, the major components of the replisome, are degraded in concert in the absence of Swi1, a subunit of the replication fork protection complex. In sharp contrast, ORC and PCNA, which are also required for DNA replication, were stably maintained. We demonstrate that this degradation of DNA polymerases and helicases is dependent around the ubiquitin-proteasome system, in which the SCFPof3 ubiquitin ligase is usually involved. Consistently, we show that Pof3 interacts with Xylometazoline HCl DNA polymerase . Remarkably, forced accumulation of replisome components leads to abnormal DNA replication and mitotic catastrophes in the absence of Swi1. Swi1 is known to prevent fork collapse at natural replication block sites throughout the genome. Therefore, our results suggest that the cell elicits a program to degrade replisomes upon replication stress in the absence of Swi1. We also suggest that this program prevents inappropriate duplication of the genome, which in turn contributes to the preservation of genomic integrity. Author Summary Replication stress interferes with the normal progression of the replication fork. Under these conditions, cells activate the replication checkpoint to coordinate DNA repair with cell cycle arrest. The current understanding is usually that, in response to replication Xylometazoline HCl block, this checkpoint stabilizes replication forks and replisome structures to achieve accurate DNA replication. However, it would also be Xylometazoline HCl advantageous for the cell to stop DNA replication and reorganize the replisome structures when conditions are not ideal, but such mechanisms have not been explored. In this study, we describe a mechanism that regulates replisome stability in response to replication stress. We found that replisome components become highly unstable and degraded when replication forks are perturbed in the absence of Swi1, a subunit of replication fork protection complex. We demonstrate that replisome degradation is dependent around the SCFPof3 ubiquitin ligase complex. Strikingly, when we forced cells to stabilize replisome components, cells underwent abnormal DNA replication, leading to mitotic catastrophes. Thus, our study provides novel mechanistic insights into understanding how the replication machinery is usually regulated to achieve faithful duplication of the genome upon replication stress. Introduction Initiation of DNA replication is usually directed by the formation of the pre-replication complex (pre-RC) at the origin of replication [1]. The pre-RC includes a number of essential replication proteins such.