Accumulation of single-stranded DNA in tumour cells as a result of replicative stress
by Meike Kunze
Date of Examination:2018-01-30
Date of issue:2018-01-15
Advisor:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Heidi Hahn
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Abstract
English
Cells are constantly subjected to DNA damaging agents like reactive oxygen species, UV light or, in the case of cancer cells, chemotherapeutic drugs. All these agents can lead to replicative stress, meaning that the most sensitive part of the cell cycle, namely S phase where cells replicate their DNA, is compromised. Cells developed several mechanisms to respond to replicative stress. One of the most prominent is the stalling of replication forks. Stalled forks can either be rapidly stabilised and eventually restarted or they collapse, leaving behind unfinished sites of replication that later on need to be dealt with. The stalling of forks leads to the exposure of stretches of single-stranded DNA (ssDNA). In this study we use a new DNA staining method to detect these stretches of ssDNA and refine it for use in a high-content, semi-automated setup. With this method we then conduct a mini-screen including proteins known to be involved in the DNA damage response and identify two candidates that are investigated further. Knockdown of G2E3, an E3 ubiquitin ligase, together with gemcitabine treatment leads to much higher levels of ssDNA as a read-out for stalled replication forks and γ-H2AX, a well established marker of DNA damage. We further provide evidence that G2E3 is a novel regulator of ATR/Chk1 signalling as it inhibits the phosphorylation and hence activation of Chk1, one of the key kinases of the DNA damage response. Based on these results, G2E3 is a potential new drug target for sensitising cancer cells for chemotherapy. The second protein investigated is MK2, a kinase known to be involved in signalling following DNA damage. We show that inhibition of MK2 rescues the normally observed induction of stalled forks and γ-H2AX accumulation upon gemcitabine treatment defining it as a regulator of replication fork stalling during replicative stress.
Keywords: Molecular Oncology; Cancer; Replicative stress; Chemosensitisation; G2E3; MK2; Gemcitabine; Chemotherapy