Chemical Inhibition of the Replication Stress Response Causes DNA Damage and Delayed Regeneration in S. mediterranea

Abstract

S. mediterranea is a planarian with extensive regenerative ability which requires substantial cell division. However, DNA replication stress occurs every cell division and must be resolved. From yeast to man, an evolutionarily conserved DNA replication stress response (RSR) is essential for life. The RSR is a signal transduction pathway regulated by Ataxia telangiectasia and Rad3‐related kinase (ATR) and Checkpoint 1 kinase (CHK1) to respond to replication stress. However, the RSR has not been established in planarian nor do we know if it functions in regeneration. We hypothesize that ATR may be essential for regeneration. Preliminary results show reduced regenerative ability in the presence of hydroxyurea (HU), a replication stress inducing agent. However, low concentrations (2mM) did not attenuate regeneration, indicating that ATR and the RSR may be present and able to overcome replication stress. To test this, we treated amputated tails with HU, ATR inhibitor (ATRi), and a combination of both. In 2mM HU, tails regenerated similarly to control. We observed only a one day delay in regeneration with ATRi alone. However, ATRi and 2mM HU in combination prevented planarian regeneration completely. In addition, replication stress was confirmed through comet assays, which quantified levels of DNA damage within each planarian experimental group. The ATRi and 2mM HU combination group exhibited the highest average comet tail moment. These synergistic effect suggests that ATR is significant in the response to replication stress during regeneration. Additionally, inhibition of CHK1, another kinase in the RSR pathway, exhibited similar tail moment values with ATRi. Future experiments will allow us to support the presence of CHK1 and its important role in resolving replication stress. Further, determining the importance of RSR genes in planarian will elucidate the conserved components and allow us to understand how ATR and the RSR contributes to the incredible regenerative abilities in planarian.Support or Funding InformationUST Committee for Student ResearchAlvarado Sanchez LabJim Jenkin for S. mediterraneaDepartment of Education HSI‐STEM grantUST Biology Smith ChairUST Biology Department