UCLA

Claspin is a Chk1 regulatory protein that acts at replication forks in both the DDR and replication pathways to ensure the fidelity of DNA prior to entry into mitosis and to initiate DDR in the case of damaged DNA. Though many of Claspin’s functional domains have been identified it is unclear what activates and localizes it to DNA. ATM is a kinase that regulates the replication and DDR pathways at SQ and TQ sites. Here we propose using caffeine, an inhibitor of ATM, to determine the importance of ATM in Claspin activation and localization, and following that with a combination of rescue experiments with Claspin deficient mice that utilizes phospomimetic and unphosphorylatable versions of Claspin to determine the importance of phosphorylation at ATM targets sites for Claspin activation. We will then use immunocytochemistry to determine if Claspin mutants are successfully recruited to DNA.