Studies on the Checkpoint Regulation of DNA Synthesis

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DESCRIPTION (provided by applicant): Cell proliferation, the basis by which organisms survive, must lead to the accurate duplication of cells and then segregate them equally to two daughter cells. Loss of this tight control can lead to cell death or unregulated cell regulatory machinery, collectively triggered checkpoints. These surveillance systems are called into play when the normal course of replication is altered by DNA damage or by the collapse of replication forks. The activation of these complex surveillance systems regulates responses such as cell cycle arrest, programmed cell death and the activation of a large number of genes involved in DNA repair. Our goal is to examine the effects of the checkpoint regulation systems on a number of key proteins involved in initiation and synthesis of DNA. For this purpose, we plan to focus on the Rad3 and Cds1 kinases, two important signal transducers of checkpoint regulation in Schizosaccharomyces pombe. We plan to examine: 1) the role of the newly discovered Rad17p complexed to the small RFC subunit 2, 3, 4 and 5 and its interaction with the Rad family of gene Products HUS1, Rad1 and Rad9; 2) the interaction between Hus1, Rad1 and Rad9 and determine whether these components from a complex that con be loaded onto DNA by the Rad17-RFC complex; 3) the activation of the Rad3 and Cds1 kinases by various DNAs and evaluate their ability to phosphorylate and control the activities associated with putative targets of checkpoint regulation, RPA, the DNA polymerase alpha-primase complex and the Cdc7-Dbf4 kinase and 4) we plan to determine how Cid1 (for caffeine-induced death resistant), a fission yeast protein required for S-M checkpoint control, affects the activities associated with the replicative DNA polymerase delta. Cid1 is a homologue of the S. serevisiae Trf4 (Trf5) which was recently shown to possess beta-like DNA polymerase activity.