BackgroundThe S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established.FindingsThis work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation.ConclusionsIn S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCFDia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4—an essential DNA replication protein and substrate of Dia2—prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.