University of Colorado Department of Biochemistry and Molecular Genetics

The main area of focus of the laboratory is the regulation of the G1 to S phase transition of the cell cycle in yeast and human cells. Most cells coordinate growth and division in this phase of the cell cycle. Elucidation of the mechanisms of cell cycle control and cell commitment to DNA replication is important for determining the etiology of a number of diseases, especially cancer, in which the regulation is altered. Yeast (Saccharomyces cerevisiae), as a eukaryotic microorganism is an excellent model system to study the cell cycle because facile molecular and high-throughput genomic techniques can be used in combination with classical biochemical and genetic methods. We use molecular genetic high-throughput genomic analysis of cultured human cancer cells for our studies that are focused on exploiting the deregulation of the cell cycle that occurs in cancer cells as a target for chemoprevention and therapy.

Yeast: Our current studies focus on the regulation of the initiation of DNA replication in yeast. DDK (Dbf4-dependent kinase; also known as Cdc7/Dbf4) is a protein kinase that regulates the initiation of DNA replication during the somatic cell cycle. DDK is activated by binding the Dbf4 protein during the G1 to S phase transition (for a review, see Sclafani and Holzen, 2007). DDK phosphorylates the MCM complex, a multi-subunit DNA helicase found at origins of DNA replication, to initiate the S phase. Both DDK and CDK (cyclin-dependent kinase) protein kinases are needed for multiple steps that ultimately intersect to activate the MCM helicase and to load on DNA polymerases during initiation. Our structure-function studies of the MCM complex use the Archaeal MCM helicase as a structural model and yeast MCM as a functional, physiological model for replication (Fletcher et al., 2003; Leon et al., 2008). DDK also acts to regulate error-prone translesion synthesis by DNA polymerase , which is important for mutagenesis (Pessoa-Brandão and Sclafani, 2004). Rad53/Chk2 protein kinase also has a role in initiation of replication that is separate from its role in the replication checkpoint and involves changes in chromatin structure (Dohrmann and Sclafani, 2006; Holzen and Sclafani, 2010).

Humans: We are determining the mode of action of several compounds found in natural products that function in cancer chemoprevention (Grape Seed extract, silibinin, IP6, resveratrol). These compounds are known to prevent cancer in animal models and are not toxic to normal cells. We have shown that the cell cycle is frequently the target of these compounds resulting in G1 cell cycle arrest (Roy et al., 2007; 2009; Kaur et al., 2011). In contrast, the compound resveratrol that is found in red grape skins and wine activates the DNA damage checkpoint pathway and produces S phase arrest in cancer cells (Tyagi et al., 2005). These studies are in collaboration with Dr. Rajesh Agarwal of the University of Colorado School of Pharmacy.