The impact of the integrated stress response on DNA replication
by Josephine Ann Mun Yee Choo
Date of Examination:2019-12-12
Date of issue:2020-05-27
Advisor:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Matthias Dobbelstein
Referee:PD Dr. Halyna Shcherbata
Persistent Address:
http://dx.doi.org/10.53846/goediss-7917
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Abstract
English
The integrated stress response (ISR) is activated following various stress stimuli which leads to the phosphorylation of the translation initiation factor eIF2alpha. Phosphorylation of eIF2alpha blocks cap−dependent translation as it prevents the recruitment of ribosomes and initiator tRNAs to the 5’ cap of the mRNA. On the other hand, translation of certain mRNAs coding for stress-responsive proteins is upregulated through cap-independent translation. One major downstream target of the ISR is ATF4, whose translation is enhanced when cap-dependent translation is impaired. Therefore, stimulation of the ISR leads to a block in global protein synthesis and also enrichment of the ATF4 transcription factor. Both inhibiting protein synthesis and upregulating ATF4 play important roles in ensuring survival during cellular stress. ATF4 can activate genes involved in maintaining survival of these cells. In addition, inhibiting protein synthesis helps the cell preserve energy and nutrients when conditions are unfavourable. As DNA replication is a highly regulated and energy-consuming process, we hypothesised that ISR activation should also hinder DNA replication for the same reason. Indeed, we found that activation of the ISR dramatically impairs DNA synthesis. This was observed within an hour of ISR stimulation and occurred independently of ATF4. Furthermore, this inhibition of DNA replication was not accompanied by an induction of the DNA damage response signalling. ISR led to the depletion of newly synthesised histones, likely through its role in blocking protein synthesis. Lack of histones upon ISR favoured a more open chromatin and accumulation of DNA:RNA hybrids (R−loops) which are responsible for inhibiting DNA replication. Conversely, the addition of histones or removal of R-loops following ISR induction significantly restored DNA replication progression. More importantly, the stalling of DNA replication in the context of ISR aids in cellular survival as removal of R−loops negatively impacted the long-term proliferation of these cells. Taken together, our study further expands the role of the ISR from blocking protein synthesis to directly hindering DNA replication. Due to its pro-survival role, some tumours have been shown to rely on the ISR to grow in nutrient−limiting conditions. In addition, the ISR has also been implicated in chemoresistance, although most of these studies involve the transcriptional programme changes following ATF4 induction. Our study suggests that the ISR could also mediate chemoresistance in tumours through slowing down DNA replication. Moreover, we found that this impairment in DNA replication protects cellular viability during stress. Although this warrants further investigation, inhibiting the ISR would be an attractive therapeutic option for cancer. This is especially important for solid tumours growing in areas with limited access to nutrients and oxygen, and are therefore dependent on the ISR for survival
Keywords: Integrated stress response; PKR; PERK; GCN2; eIF2alpha; R-loops; DNA replication; DNA fiber assays