Investigating the Roles of Mec1 and Rad53 in the S-Phase Checkpoint in Saccharomyces cerevisiae

The S-phase checkpoint, developed by the cell in response to DNA damage, is critical to maintaining cellular function. The S-phase checkpoint operates through two separate pathways, the DNA damage checkpoint and the DNA replication checkpoint. Both pathways of the S-phase checkpoint respond to DNA damage in Saccharomyces cerevisiae, or budding yeast, through separate mechanisms both relying on two critical kinases, Mec1 and Rad53. My goal in my master’s thesis research was to work toward elucidating the details of these two kinases’ roles in both pathways of the S-phase checkpoint. To do so, I subjected highly conserved, hydrophobic residues on the surfaces of Mec1 and Rad53 to extensive mutagenesis using a novel technique designed to identify rare separation-of-function mutations, as well as traditional loss-of-function screens. By conducting a thorough genetic screen of the two kinases, I sought to aid efforts in uncovering additional protein factors involved in the S-phase checkpoint and in clarifying how the two different pathways work in conjunction to respond to DNA damage throughout the cell. Determining specifically how Mec1 and Rad53 work within the two pathways can lead to similar discoveries about the DNA damage response pathways in mammalian cells, facilitating the development of future treatments of cancer and other debilitating genetic diseases