Analysis of the fission yeast cell cycle checkpoint gene rad3(+).

다국어 초록 (Multilingual Abstract)

Checkpoints maintain the proper order of cell cycle events. In <italic>Schizosaccharomyces pombe</italic>, the mitotic checkpoint pathway ensures that mitosis does not take place until DNA replication and DNA repair have been completed. The mitotic checkpoint pathway is a signal transduction pathway composed of many different proteins. The <italic>checkpoint rad</italic> proteins, which include Rad1p, Rad3p, Rad9p, Rad17p, Rad26p, and Hus1p, are believed to act upstream in the pathway, monitoring the DNA for the presence of incomplete DNA replication or DNA damage. In the presence of incomplete DNA replication or DNA damage, the Rad3p kinase is believed to phosphorylate downstream proteins, such as Cds1p and Chk1p. In turn, the Cds1p and Chk1p kinases phosphorylate the regulators of the cyclin-dependent kinase, Cdc2p, the activity of which controls entry into mitosis. Thus, the checkpoint pathway delays mitosis by downregulating the activity of Cdc2p.
The aim of this thesis was to further our understanding of the Rad3p kinase, which is believed to play a central role in this pathway. Rad3p is a member of a family of structurally and functionally related proteins from a number of diverse organisms, which includes human ATMp. Loss of <italic> ATM</italic> function in humans causes ataxia- telangiectasia, a disease in which patients suffer from a variety of symptoms, including a predisposition to cancer. Thus, we have studied fission yeast Rad3p as a model for understanding the function of mammalian homologues such as ATMp.
Two approaches were taken to investigate Rad3p function. First, to learn more about important domains in Rad3p, a structure/function analysis of the Rad3p protein was performed. The activity of a number of different <italic> rad3</italic> alleles was assessed using a variety of assays. We found that sequences outside the conserved kinase domain of Rad3p are required for checkpoint control. Using dominant negative analysis, we provide evidence that protein-protein interaction sites might be located in the N-terminus of the Rad3p protein. Second, we explored possible interactions between <italic>rad3</italic><super> +</super> and DNA replication genes. In this thesis, two novel <italic>rad3 </italic><super>+</super> overexpression phenotypes are described which strongly suggest that <italic>rad3</italic><super>+</super> can influence DNA replication in the cell. Different models which might account for these overexpression phenotypes are considered.