The Effect of Dia2 Protein Deficiency on the Cell Cycle, Cell Size, and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae.

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.

Parapatric genetic divergence among deep evolutionary lineages in the Mediterranean green crab, Carcinus aestuarii (Brachyura, Portunoidea, Carcinidae), accounts for a sharp phylogeographic break in the Eastern Mediterranean.

BACKGROUND: Recently, population genetic studies of Mediterranean marine species highlighted patterns of genetic divergence and phylogeographic breaks, due to the interplay between impacts of Pleistocene climate shifts and contemporary hydrographical barriers. These factors markedly shaped the distribution of marine organisms and their genetic makeup. The present study is part of an ongoing effort to understand the phylogeography and evolutionary history of the highly dispersive Mediterranean green crab, Carcinus aestuarii (Nardo, 1847), across the Mediterranean Sea. Recently, marked divergence between two highly separated haplogroups (genetic types I and II) of C. aestuarii was discerned across the Siculo-Tunisian Strait, suggesting an Early Pleistocene vicariant event. In order to better identify phylogeographic patterns in this species, a total of 263 individuals from 22 Mediterranean locations were analysed by comparing a 587 basepair region of the mitochondrial gene Cox1 (cytochrome oxidase subunit 1). The examined dataset is composed of both newly generated sequences (76) and previously investigated ones (187). RESULTS: Our results unveiled the occurrence of a highly divergent haplogroup (genetic type III) in the most north-eastern part of the Mediterranean Sea. Divergence between the most distinct type III and the common ancestor of both types I and II corresponds to the Early Pleistocene and coincides with the historical episode of separation between types I and II. Our results also revealed strong genetic divergence among adjacent regions (separating the Aegean and Marmara seas from the remaining distribution zone) and confirmed a sharp phylogeographic break across the Eastern Mediterranean. The recorded parapatric genetic divergence, with the potential existence of a contact zone between both groups in the Ionian Sea and notable differences in the demographic history, suggest the likely impact of paleoclimatic events, as well as past and contemporary oceanographic processes, in shaping genetic variability of this species. CONCLUSIONS: Our findings not only provide further evidence for the complex evolutionary history of the green crab in the Mediterranean Sea, but also stress the importance of investigating peripheral areas in the species' distribution zone in order to fully understand the distribution of genetic diversity and unravel hidden genetic units and local patterns of endemism.

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

BACKGROUND: The 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. FINDINGS: This 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. CONCLUSIONS: In 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.