How replication stress affects the developing organism

Accurate and complete DNA replication is important for healthy cell division. Slowing or stalling of DNA replication machinery can lead to formation of DNA breaks, in a process called replication stress. Replication stress can result from de-regulated cell cycle and proliferation after inhibiting the ATR checkpoint pathway or from activating oncogenes. Replication stress is nearly omnipresent in cancer, but it can also arrest growth of stem cells and cause growth and developmental defects in humans. ATR (zebrafish: atr) gene codes for a checkpoint kinase known to prevent replication stress in cells and developmental defects, such as microcephaly, in humans. However, link between replication stress and developmental defects is still not well understood. We plan to investigate this link in zebrafish, a powerful model organism with multiple advantages in studies of embryonic development.
Objectives:
1. Breed zebrafish strains heterozygous for mutations in atr, obtain and characterise homozygous mutant embryos;
2. Characterise the spatiotemporal features and molecular mechanisms of replication stress in WT and atr mutant embryos;
3. Investigate the spatiotemporal and genomic features of the DNA damage response to replication stress in wild-type and atr mutant embryos.
We will breed atr mutants and wild type zebrafish strains and optimise replication stress assays for use in fish embryos, including new protocols for DNA fibre microscopy, and use 53BP1 live imaging to measure replication dynamics in vivo. ChIP-seq will be used to identify hotspots of DNA damage response and to link genomic features and replication stress.
Characterisation of differences between mutant and wild-type embryos and replication stress responses exhibited in different growth stages and tissues would provide new insights into embryonic replication stress and functions of ATR kinase.