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

The archaeal "7kDa DNA-binding" proteins: extended characterization of an old gifted family

International audienceThe " 7 kDa DNA-binding " family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 μM to 22 μM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative " 7 kDa DNA-binding " family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents. In living organisms, the long genomic DNA has to be packed in order to fit into cells, while the genetic information must stay accessible for replication and transcription events. To this aim, organisms have developed different compaction systems, such as the wrapping of DNA around histones to form the chromatin in Eukarya, and the supercoiling of DNA with the help of non-histone proteins to form the nucleoid in Bacteria. Archaea often live in extreme environments and have the additional challenge to protect their genomic DNA from extreme conditions, such as high temperatures. Many Archaea contain homologs of eukaryotic histones, but Desulfurococcales, Thermoplasmatales and Sulfolobales use a different kind of packaging proteins 1,2. Hyperthermophile and acidophile archaea of the Sulfolobales order from the Crenarchaeota kingdom express small basic DNA-binding proteins, which represent about 5% of the total soluble cellular proteins, sufficient to coat the entire genome of a Sulfolobus cell 3. These proteins constitute the family called " 7 kDa DNA-binding " or Sul7d 4. They were first isolated from Sulfolobus acido-caldarius which produces five of them, named Sac7a, b, c, d, and e. Sac7d and Sac7e are encoded by distinct genes, while Sac7a and b are truncated versions of Sac7d 5–7. Highly similar homologs have been found in all Sulfolobus species, such as Sso7d from Sulfolobus solfataricus 8 , and Ssh7a and Ssh7b from Sulfolobus shibatae-two proteins encoded by two distinct genes 3,9. Sac7d and Sso7d are the two most studied proteins of this family. They have been characterized for their structure, function, chemical stability and biophysical properties 7. Sac7d and Sso7d are hyperthermostable (T m = 90.4 °C and 100.2 °C, respectively) 10,11 and are resistant from pH 0 up to at least pH 12 12,13. Although Sac7d and Sso7d sequences show only few differences, Sso7d is more stable than Sac7d. Their three-dimensional structures show that they both fold as an SH3-like domain capped by a C-terminal α-helix 14,15 and that they sharply kink the double DNA helix upon binding into the minor groove 16,17. It has been shown that Sac7d and Sso7d are general dsDNA binders with K D values varying in a salt dependent manner from 20 nM (low salt) to 3.8 μ M (high salt) for Sac7d, and from 116 nM to 12.8 μ M for Sso7d, and with a preference for G/C rich sequences 18,19. Sac7d has the property to increase the thermal stability of DNA duplexes by as much as 43.5 °C 6,15. Furthermore, Ssh7a and Ssh7b have been partially characterized and an affinity for dsDNA of about 100 n