S. mediterranea Ataxia telangiectasia and Rad3‐related kinase (SmedATR) May be Required to Prevent DNA Replication Stress

Abstract

DNA replication is an important mechanism involved in cellular processes like reproduction, repair, and regeneration. S. mediterranea, a planarian with an amazing regenerative ability, goes through continuous rounds of cell division. Consequently, DNA replication stress occurs with each cell division and must be resolved. From yeast to man, The DNA replication stress response (RSR) is evolutionarily conserved and necessary for life among organisms. 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, including DNA damage. However, the RSR has not yet been established in the planarian and its involvement in regeneration is currently unknown. We hypothesize that ATR may be essential in replication by preventing and resolving DNA damage. Regeneration experiments were conducted and results indicated that the regenerative ability of planaria were reduced in the presence of hydroxyurea (HU), a replication stress inducing agent. Low HU concentrations (2mM) had similar phenotypic results to the control which suggests that ATR and the RSR may be present in this low dosage to overcome the replication stress. However, when treated with a combination of 2mM HU and ATR inhibitor (ATRi), planarian regeneration was severely hindered. To confirm replication stress shown phenotypically in the regeneration experiments, comet assays were performed to quantify the levels of DNA damage in each experimental group. The ATRi and 2mM HU combination group exhibited the highest average comet tail moment, a parameter used to measure the amounts of fragmented DNA within each planarian cell. The synergistic effects of ATR in combination with HU suggest 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 as ATRi. Future experiments will allow us to support the presence of CHK1 and its important role in resolving replication stress. 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 Research Alvarado Sanchez Lab, Stowers Institute for Medical Research Jim Jenkin for S. mediterranea Department of Education HSI‐STEM grant UST Biology Smith Chair UST Biology Department