Functions Of Atr/mec1 In Meiosis And The Cell Cycle

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

Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control