This proposal is the first renewal of our studies into the control of genome integrity by the G2 DNA damage checkpoint effector kinase, Chk1. DNA damage checkpoints halt cell cycle progression when genomic lesions are detected and thus allow time for DNA repair. They are crucial to maintain genome stability, and when they fail, the ensuing instability allows for a heightened rate of molecular evolution that is a hallmark of cancer initiation and progression. Checkpoints are also central to the biology of aging, response to environmental mutagens, and in developmental signals essential for tissue homeostasis. Whilst loss of p53-dependent checkpoints are common in tumors, Chk1 loss is not seen and suggests that Chk1 is essential for tumor cell viability, and thus targeting Chk1 with small molecule inhibitors may be efficacious in caner therapy. Chk1 prevents entry into mitosis in response to DNA damage through modulating Cdc2 kinase activity. This pathway and the G2/M cell cycle machinery are ancient in origin and highly conserved. Our studies therefore utilize the fission yeast Schizosaccharomyces pombe as model for gene and pathway discovery, and with the high conservation, these data inform studies in mammalian cell systems. Our long-term goal is to understand mechanisms that control the activation of Chk1, that maintain Chk1 activity during DNA repair, and that then inactivate Chk1 to allow resumption of the cell cycle. Moreover, we aim to understand this biology in the context of other DNA damage response pathways, particularly those controlled by p53, which are crucial to tumor biology in mammals but are not present in yeast. Our ongoing analysis of Chk1 function and regulation is structured with three specific aims. Firstly, we aim to determine mechanisms of Chk1 activation in S. pombe. Chk1 is activated by Rad3-mediated phosphorylation mediated by the BRCT-domain protein Crb2. We have developed tools to dissect inter- and intramolecular interactions within active and inactive Chk1, and based on preliminary data propose that Chk1 activity is regulated through altered intramolecular interactions. Secondly, we investigate negative regulation of checkpoint signaling, both through its direct termination and analysis of Chk1 antagonists. Finally, experiments are described to dissect regulation of human Chk1, the effects of Chk1 dysfunction and to understand this biology in the context of crosstalk to and from p53-derived signals to control cell fate decisions. These experiments are particularly important in the rational development of Chk1 inhibitors for use as anti-cancer agents, where it is essential to know the combined effect of tumor genotype and Chk1 inhibition.
Cells proliferate with remarkable fidelity, and defects in these quality control processes, known as checkpoints, are at the root cause of the genetic basis of cancer. This project studies an enzyme known as Chk1, which is essential to prevent cell division in the presence of damaged. Our experiments regarding the regulation and function of Chk1 are thus at the coalface of the biology of cancer initiation, progression and therapy.
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| Calonge, Teresa M; Eshaghi, Majid; Liu, Jianhua et al. (2010) Transformation/transcription domain-associated protein (TRRAP)-mediated regulation of Wee1. Genetics 185:81-93 |
| Kuntz, Karen; O'Connell, Matthew J (2009) The G(2) DNA damage checkpoint: could this ancient regulator be the Achilles heel of cancer? Cancer Biol Ther 8:1433-9 |
| Irmisch, Anja; Ampatzidou, Eleni; Mizuno, Ken'ichi et al. (2009) Smc5/6 maintains stalled replication forks in a recombination-competent conformation. EMBO J 28:144-55 |
