Analysis of Saccharomyces cerevisiae Mec1 and Rad9 functions in the DNA Damage Response

Abstract

The maintenance of genome integrity is critical for cell proliferation and survival of all organisms. Central to the DNA Damage Response are signal-transduction pathways, termed DNA damage checkpoints, conserved from yeast to human cells. Failure to respond properly to DNA damage, through the activation of these pathways, results in increased mutagenesis and genetic instability. In addition, in higher organisms, defects in the DNA damage checkpoints are frequently associated with cancer, ageing and several other pathologies. Clearly, to understand oncogenesis, and in particular its early stages, it will be important to fully understand the DNA damage response. The phosphatidylinositol-3-kinase-like kinase Mec1, the ATR orthologue, is the master of the DNA damage checkpoints in Saccharomyces cerevisiae and is essetntial for cell viability. In chapter 2 of this thesis, we investigate the structurefunction relationship of Mec1. In particular, we examine the role of tandem helical motifs, called HEAT repeats, that comprise the non-kinase portion of Mec1 and other PIKKs. In this study, we found that mec1 mutants, expressing a Mec1 protein version where single/multiple HEATs were removed or replaced by equivalent HEATs from related PIKKs, behaved similarly to the null strain. This suggests that none of these mutants were proficient for Mec1¿s essential function. Our data indicates that the essential function of Mec1 is dependent on the integrity of its HEAT repeats. Rad9, the prototypal checkpoint mediator, also plays a pivotal role in the DNA damage response in S. cerevisiae. In chapter 4 of this thesis we investigate the role of the cell cycle-dependent phosphorylation of Rad9 and we show that this process is dependent on B-type cyclin (Clbs) forms of Cdc28 (the single cyclin-dependent kinase in budding yeast). Based on this study, we propose that Cdc28 fine tunes Rad9 DNA damage response functions and we particularly focus on Rad9 regulatory functions in Chk1 activation. We found that the integrity of nine putative Cdc28 phosphorylation sites located in the N-terminal region of Rad9, especially T143, is required for Chk1 activation and Rad9-Chk1 interaction in the G2/M phase of the cell cycle. Our data suggests a novel PIKK- and Rad9-dependent model for Chk1 activation in response to DNA damage.