The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

Abstract

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.

Mrc1-Dependent Chromatin Compaction Represses DNA Double-Stranded Break Repair by Homologous Recombination Upon Replication Stress

“…In the initial 2–4 h, resection is severely suppressed, but the suppression becomes alleviated afterwards even in the presence of constant HU treatment. This is consistent with the observation that cells can bypass the S phase checkpoint and proceed through the cell cycle after prolonged HU incubation (Uzunova et al, 2014 ).…”

Rad53- and Chk1-Dependent DNA Damage Response Pathways Cooperatively Promote Fungal Pathogenesis and Modulate Antifungal Drug Susceptibility

“…In S. cerevisiae and humans, Rad53 and the homologous protein kinase Chk2 are localized in the nucleus (28, 29). To examine the cellular localization of Rad53 in C.…”

Cdc48 targets INQ-localized Mrc1 to facilitate recovery from replication stress

“…The transient dissociation of replisome components Mcm2, Ctf4 and Cdc45 from Mrc1 was confirmed by Western blotting of immunoprecipitated C-terminally FLAG-tagged Mrc1 (Figure 1D). Moreover, in an MMS-stressed S phase, Mrc1-FLAG interaction with the fork components was reduced to the same degree as in an untreated tof1∆ mutant, in which Mrc1 localization to the fork has been shown to be compromised (Bando et al, 2009;Uzunova et al, 2014), and completely abolished in the MMS-treated tof1∆ 6 mutant. Concurrently, formation of Mrc1 foci rose to a four-fold higher level in MMStreated tof1∆ mutant cells compared to that observed in wild-type cells (Figure 1E).…”