Analysis of the Rad3 and Cds1 fission yeast checkpoint kinases.

摘要

All cells must duplicate and transmit their genetic material with great fidelity, as high error rates in this process lead to genomic instability and cell death. In the fission yeast Schizosaccharomyces pombe, the G2/M DNA damage and S/M DNA replication checkpoints help maintain genomic stability by enforcing a block in mitosis in the presence of unreplicated DNA or DNA damage, giving the cell time to complete DNA replication or repair its DNA. Another important response to a block in DNA replication is the recovery response. Identified through careful studies of checkpoint mutants, the recovery function permits successful exit from an S phase arrest. The mitotic arrest checkpoint function and the recovery response are mediated through similar pathways. Two of the central components in both pathways, the Rad3 kinase and the Cds1 kinase, have homologs in humans that when mutated result in the cancer-prone diseases, ataxia telangiectasia and Li-Fraumeni syndrome. This thesis will describe work on these two kinases.; The first aim of this thesis was to better understand the function of the Rad3 kinase. To do so, a structure/function analysis of the Rad3 protein was performed. Using a number of different assays, several rad3 alleles were characterized. This analysis revealed that regions outside the C-terminal kinase domain of Rad3 are required for its kinase activity. In addition, two sites not required for kinase activity but required for Rad3 function were identified in the N-terminus of the protein. Dominant negative analysis suggests that these sites may function as protein-protein interaction sites. Possible functions for these sites will be discussed.; Although the final targets of the mitotic arrest pathway are known to be components of the cell cycle machinery, the final receivers of the recovery pathway are not yet known. To understand the recovery pathway better, including its downstream targets, we studied an important regulator of this pathway, the Cds1 kinase. Several partial loss-of-function cds1 alleles were isolated and characterized. This analysis revealed a previously unknown role for the forkhead associated domain (FHA) domain of Cds1 in the phosphorylation of specific substrates. Suppressor analysis of the cds1 mutant alleles identified the Pap1 protein, an AP-1 transcription factor related to the mammalian c-Jun protein, as a potential regulator of a transcriptional response downstream of Cds1.