The ability to maintain genomic stability is vital to the survival of all organisms. One crucial mechanism to detect and respond to DNA damage involves a signaling pathway mediated by the ATR and Chkl kinases. Defects in this pathway, known as the ATR checkpoint, are associated with a variety of human cancers. Activation of Chkl by ATR is dependent upon a key mediator protein, Claspin. My preliminary results show that Claspin is targeted for ubiquitylation and proteasome-mediated degradation in response to DNA damage. These findings suggest a surprising involvement of protein ubiquitylation in the regulation of ATR checkpoint. My proposal aims to determine the molecular mechanism responsible for, and the biological consequences of damage-induced Claspin degradation. Furthermore, I propose to use proteomic approaches to identify novel proteins that are specifically ubiquitinylated after DNA damage, and to systematically investigate the functions of ubiquitylation in ATR checkpoint response.
Specific Aims : 1) Determine the molecular mechanisms responsible for UV-induced Claspin degradation, 2) Investigate the functions of Claspin degradation on ATR checkpoint response and regulation of stressed replication forks, 3) Conduct a proteomic screen to identify novel ubiquitinylated proteins that regulate the ATR checkpoint. Using a combination of molecular biology, mammalian tissue culture, and biochemical techniques, I plan to first test specific mutants of Claspin that are predicted to fail to be degraded by the proteasome. I will also determine how Claspin ubiquitylation is regulated by ATR checkpoint. Once a stabilized Claspin mutant has been identified, I will use it to determine the biological consequences of UV-induced Claspin degradation. I will study the effects of Claspin stabilization on checkpoint recovery, Chkl translocation from chromatin, and DNA repair using biochemical, cell biological, and immunofluorescence techniques. Finally, I propose to conduct a proteomic screen to determine all proteins that are ubiquitinylated in a DNA damage-inducible manner that regulate ATR checkpoint response. Once new proteins have been identified with these techniques, I will use the approaches outlined in Specific Aims 1-2 to determine the mechanism by which these post-translational modifications occur as well as their effects on the cellular DNA damage response. Public Health Relevance: Proteasome inhibitors have emerged as promising drugs for the treatment of cancers. This proposed study may reveal the mechanisms by which proteasome inhibitors eliminate cancer cells, and may facilitate the development of new targeted therapies for cancer patients.
|Havens, Courtney G; Shobnam, Nadia; Guarino, Estrella et al. (2012) Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2. J Biol Chem 287:11410-21|
|Flynn, Rachel Litman; Centore, Richard C; O'Sullivan, Roderick J et al. (2011) TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA. Nature 471:532-6|
|Centore, Richard C; Havens, Courtney G; Manning, Amity L et al. (2010) CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase. Mol Cell 40:22-33|