University of Colorado Department of Biochemistry and Molecular Genetics

The main area of focus of the laboratory is the regulation and mechanisms of chromosomal DNA replication, mutagenesis and DNA repair in yeast and human cells. Elucidation of these mechanisms 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 because facile molecular and high-throughput genomic techniques can be used in combination with classical biochemical and genetic methods. We also 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 (for reviews, see Sclafani and Holzen, 2007; Rossbach and Sclafani, in press). 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 (Zhong et al., 2013). 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; Ramey and Sclafani, 2014). DDK also acts to regulate error-prone translesion synthesis by DNA polymerase , which is important for mutagenesis (Pessoa-Brandão and Sclafani, 2004; Brandão et al., 2014). Rad53/Chk2 protein kinase interacts with DDK function in initiation that is separate from its replication checkpoint function and involves changes in orgin 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 by producing replication stress resulting in S phase arrest and cell death in cancer cells (Tyagi et al., 2005 & 2011; Shrotriya et al., 2015). These studies are in collaboration with Dr. Rajesh Agarwal of the University of Colorado School of Pharmacy.