Open Access Dissertation
Date of Award
George S. Brush
Mec1 is a protein kinase in S. cerevisiae that is critical for the DNA damage checkpoint response, and is the yeast orthologue of the human ATR protein. Cancer cells rely on ATR to arrest the cell cycle and allow sufficient time to repair DNA damage before proceeding through the cell cycle, and ATR inhibitors have been developed as possible anti-cancer agents. DBF4 is the regulatory subunit of DBF4-dependent kinase (DDK) that regulates initiation of DNA replication and is overexpressed in a number of different cancer types. To better understand ATR and DBF4 function, we took advantage of yeast genetics to examine the role of Mec1 and Dbf4 in prevention of DNA rereplication during meiosis. We found that Dbf4 phosphorylation sites and Mec1 were essential for prevention of DNA rereplication when the meiotic recombination checkpoint was activated by deletion of DMC1. We further found evidence that Dbf4 was phosphorylated by a kinase other than the canonical Rad53 or Mek1 protein kinases.
We also examined genes that synthetically interact with MEC1 in order to better understand the function of Mec1 during the normal cell cycle. Synthetic genetic interactions occur when fitness is not affected by a single mutation of one of two genes, but when both of the two genes are mutated a significant loss in fitness occurs. Among the many genes identified to synthetically interact with MEC1, many are involved in mRNA transcription, including several subunits of the Mediator complex. We also found genes involved in DNA replication to synthetically interact with MEC1, such as RAD27 and DBF4. We assessed whether these synthetic interactions exist in human non-small cell lung cancer (NSCLC) cell lines. We discovered that inhibitors of ATR and DDK act synergistically in p53 null cell lines but not in p53 wild type cell lines. This data suggests a synthetic interaction between ATR and genes involved in transcriptional regulation or DNA replication in NSCLC cells that is dependent on the absence of p53. These studies have provided insight into novel targets to inhibit in combination with ATR inhibitors as a treatment regimen for NSCLC.
Weatherford, Layne, "Functions Of Atr/mec1 In Meiosis And The Cell Cycle" (2017). Wayne State University Dissertations. 1892.