Systems Biology of the Stress Response

1 PhD project proposal in the IPP summer call 2019

Scientific Background:

As DNA double-strand breaks (DSBs) pose a major threat to genomic integrity, cells evolved sophisticated pathways to repair these lesions. Which pathway is used to repair a given break depends on the nature of the lesion, the genomic context and the state of the cell cycle. However, cell cycle regulation is a dynamic process and cells often progress through the cell cycle despite the presence of DNA damage due to incomplete or compromised checkpoints. Therefore, the availability and suitability of repair pathways may change during the cellular response to DSBs and cells have to integrate DSB repair with other processes such as replication and chromosome segregation. For example, homology dependent repair (HDR) becomes available after the transition from G1 to S phase and it has been shown that error-prone recombination events take place at breaks induced during G1 phase. During the transition from G2 to M, cells need to handle breaks that are in the process of HDR and ensure that they are repaired in the following G1 phase. While we have a good understanding how repair pathway choice is regulated in each stage of the cell cycle at a given time, little is known about how DNA repair adjusts to dynamic process during cell cycle transitions. One of the main obstacles in gaining deeper insights into this process is the asynchrony and infrequence of such transitions, preventing application of most common approach from biochemistry and molecular biology.

PhD project proposal: Quantitative time-resolved analysis of DSB repair in individual living cells

In this project, we plan to investigate the repair of endogenous and induced DSBs during cell cycle progression using quantitative time-resolved analysis of individual living cells. To this end, we will combine live-cell imaging of fluorescent reporter cell lines with computational image processing and statistical analysis. We will initially follow DSBs using 53BP1 and MDC1 – based reporters and determine cell cycle progression using in-silico synchronization and cell cycle markers [1-2]. We will combine live-cell imaging with immunofluorescence staining of proteins involved in non-homologous end joining and HDR at different end-points to determine for each break the repair pathway usage after phase transitions. To follow DSB and pathway usage over time in the same living cell, we will establish reporters for indicative HDR components by using Cas9-mediated genome engineering [3]. To gain mechanistic insights, we will perform pharmacologically or genetically perturbations of repair and signaling pathways and monitor ensuing changes in DNA repair in living cells. Finally, we will use the resulting quantitative data to establish mathematical models of the interplay between DSB repair and cell cycle progression and validate these experimentally. [4]


Related publications:

[1] Loewer, A.#, Karanam, K., Mock, C., Lahav, G.#, (2013) “The p53 response in single cells is linearly correlated to the number of DNA breaks without a distinct threshold” BMC Biol 11:114
[2] Karanam, K., Kafri, R., Loewer, A., Lahav, G. (2012) „Quantitative Live Cell Imaging Reveals a Gradual Shift between DNA Repair Mechanisms and a Maximal Use of HR in Mid S Phase.“, Mol. Cell 47(2): 320-9
[3] Sheng, C., Mendler, I.-H., Rieke, S., Snyder, P., Jentsch, M., Friedrich, D., Drossel, B., Loewer, A. (2019) „PCNA-Mediated Degradation of p21 Coordinates the DNA Damage Response and Cell Cycle Regulation in Individual Cells”, Cell Reports, https://doi.org/10.1016/j.celrep.2019.03.031
[4] Toettcher, JE.*, Loewer, A.*, Ostheimer, GJ., Yaffe, MB., Tidor, B., Lahav, G. (2009) „Distinct mechanisms act in concert to mediate cell cycle arrest” PNAS 106(3): 788-90
* equal contribution # corresponding author 

Contact:

Prof. Alexander Löwer

Prof. Alexander Löwer
Technical University of Darmstadt
Biology Department
Schnittspahnstraße 13
64287 Darmstadt

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