Genetic and biochemical analysis of DNA damage checkpoint signalling in budding yeast
Final Report Abstract
The stability of the genetic information is constantly threatened by damages to DNA. In order to signal the presence of damaged DNA eukaryotic cells have evolved a pathway termed DNA damage checkpoint. The checkpoints constituted of a phosphorylation cascade, which acts on damaged chromatin. Interestingly, this signalling involves not only the DNA directly at the site of the lesion, but also the surrounding chromatin. In this project we investigate the contribution of these two chromosomal domains using the DNA damage response of budding yeast as a model. Previous results indicated that the two signalling domains may have redundant functions, for example in the targeting of specific checkpoint proteins, such as the mediator protein Rad9. However, our results challenge this view, as we find that targeting mechanisms directly at the site of the DNA lesion and in the surrounding chromatin collaborate, for example in the tethering of checkpoint proteins to damaged chromatin or in the establishment of activating phosphorylation marks on checkpoint proteins. While our results provide mechanistic insights into the collaboration of checkpoint signalling complexes at different chromosomal locations, we also find evidence for the existence of different checkpoint signalling circuits that lead to the phosphorylation of soluble or chromatin-associated proteins. Surprisingly, the circuit that leads to the phosphorylation of a soluble target (Rad53) is much more dependent on the initial checkpoint signal, compared to a chromatin-associated target (histone H2A). Our data thus supports a view where the DNA damage checkpoint is not one single cellular mechanism, but rather consists of several signalling circuits that collectively drive the cellular response to DNA damage.
Publications
- (2014). A cell cycle-regulated Slx4-Dpb11 signalling complex controls the resolution of DNA repair intermediates linked to stalled replication. Genes Dev. 28:1604-1619
Gritenaite, D., Princz, L. N., Szakal, B., Bantele, S. C. S, Wendeler, L., Schilbach, S., Habermann, B. H., Lisby, M., Branzei, D. and Pfänder, B.
(See online at https://doi.org/10.1101/gad.240515.114) - (2015). Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage. eLife. e12256
Lee, S. H., Princz, L. N., Klügel, M. F., Habermann, B., Pfänder, B., and Biertümpfel, C.
(See online at https://doi.org/10.7554/eLife.12256) - (2015). The Slx4-Dpb11 scaffold complex: coordinating the response to replication fork stalling in S-phase and the subsequent mitosis. Cell Cyle. Cell Cycle, 14, 488-94
Princz, L. N., Gritenaite, D., and Pfänder, B.
(See online at https://doi.org/10.4161/15384101.2014.989126) - (2016) Regulation of the Initiation of DNA Replication upon DNA Damage in Eukaryotes. in: The initiation of DNA replication in eukaryotes. Springer
Koehler, K., Ferreira, P., Pfänder, B., and Boos, D.
(See online at https://doi.org/10.1007/978-3-319-24696-3_22) - (2016). Roles of Sld2, Sld3, and Dpb11 in Replication Initiation. in: The initiation of DNA replication in eukaryotes. Springer
Reußwig, K.U., Boos, D., and Pfänder, B.
(See online at https://doi.org/10.1007/978-3-319-24696-3_15)
