HMGP - Robert Sclafani, Ph.D.

Robert A. Sclafani
Professor
Department of Biochemistry and Molecular Genetics
Program Director of Cell Biology-UCD Comprehensive Cancer Center
University of Colorado Denver at Fitzsimons
Department of Biochemistry and Molecular Genetics
Mail Stop 8101
P.O. Box 6511
Aurora, CO 80045

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 an eukaryotic microorganism is an excellent model system to study the cell cycle because facile molecular genetic techniques can be used in combination with classical biochemical and genetic methods. We use human cells in culture as a way to study the defects that are present in the cell cycle of cancer cells.

Yeast: Our current studies focus on the regulation of the yeast Cdc7/Dbf4 protein kinase, which regulates the initiation of DNA replication during the somatic cell cycle. Our current model is that Cdc7 kinase is activated by binding the Dbf4 protein during the G1 to S phase transition (Sclafani, 2000). Dbf4 protein levels are regulated by Rad53 protein kinase, which is also involved in DNA-checkpoint regulation, and by the APC (anaphase promotion complex). Active CDC7 kinase then phosphorylates the Mcm complex, a multi-subunit protein complex found at origins of DNA replication, to initiate the S phase. Both Cdc7 and Cdk1 proteins are needed for mtuliple steps during initiation (Sclafani et al., 2002). We have begun structure-function studies of the MCM complex with Dr. X. Chen by comparing Archaea MCM to Yeast MCM (Fletcher et al., 2003; Sclafani et al., 2004).

We have also defined a role of Cdc7/Dbf4 kinase in error-prone tyranslesion synthesis, which is important for mutagenesis (Pessoa-Brandão and Sclafani, 2004).

Humans: Our human studies are focused on the deregulation of the cell cycle which occurs in cancer cells. We have found that some lung, breast and ovarian cancers are defective for the tumor suppressor p16 (INK4a). Therefore, we have focused on determining the mechanism of action of p16 in order to test whether restoration of p16 gene function can be used as a therapy (Sclafani et al., 1998; Todd et al., 2000; Grimison et al, 2000; Mamay et al., 2001). We have also begun molecular studies of huCdc7, the human homologue of Cdc7, in cancer cells (Hess et al., 1998). We hypothesize that like yeast Cdc7, huCdc7 may regulate induced mutagenesis and therefore, be important in the multi-step progression to cancer.

Sclafani, R.A., Tecklenburg, M. and Pierce, A. The mcm5-bob1 bypass of Cdc7p/Dbf4p in DNA replication depends on both Cdk1-independent and Cdk1-dependent steps in S. cerevisiae Genetics, 161: 47-57 (2002).

Fletcher, R. J., B. E. Bishop, R. P. Leon, R. A. Sclafani, C. M. Ogata, and X. S. Chen. The structure and function of MCM from archaeal M. Thermoautotrophicum. Nat Struct Biol 10:160-167. (2003) (Selected by journal for cover figure and for "News and Views", see Kelman, Z., and J. Hurwitz. 2003. Structural lessons in DNA replication from the third domain of life. Nat Struct Biol 10:148-150.)

Agarwal, C., R. P. Singh, S. Dhanalakshmi, A. K. Tyagi, M. Tecklenburg, R. A. Sclafani, and R. Agarwal. 2003. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 22:8271-8282.

Pessoa-Brandão, L. and Sclafani, R.A. (2004) CDC7/DBF4 functions in the translesion synthesis branch of the RAD6 epistasis group in Saccharomyces cerevisiae. Genetics 167:1597-1610.

Sclafani, R. A., Fletcher, R. J., and Chen, X. S. (2004) Two heads are better than one: regulation of DNA replication by hexameric helicases. Genes Dev, 18: 2039-2045.

Tyagi, A.K, Singh, R.P., Agarwal, C., Sirirwardana, S., Sclafani R.A. and Agarwal, R. Resveratrol causes Cdc2-tyr15 phosphorylation via the ATM/ATR-Chk1/2-Cdc25C pathway as a central mechanism for S phase arrest in human ovarian carcinoma Ovar-3 cells. Carcinogenesis (2005) 26: 1978-87.

Dohrmann, P. and Sclafani, R.A. Novel Role for Checkpoint Rad53 Protein Kinase in the Initiation of Chromosomal DNA Replication in S. cerevisiae. Genetics, in press (2006).

Hoang, M. L., R. P. Leon, L. Pessoa-Brandao, S. Hunt, M. K. Raghuraman, W. L. Fangman, B. J. Brewer, and R. A. Sclafani (2007) Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in S. cerevisiae. Mol Cell Biol., 27:594-7602. PMCID: PMC2169039

Sclafani, R. A., and T. M. Holzen. (2007) Cell Cycle Regulation of DNA Replication. Annu Rev Genet., 41:237-280. PMCID: PMC2292467

S. Roy, M. Kaur, C. Agarwal, M. Tecklenburg, R. A. Sclafani and R. Agarwal. (2007) p21 and p27 induction by silibinin is essential for its cell cycle arrest effect in prostate carcinoma cells. Molecular Cancer Therapeutics 6: 2696-2707. PMID: 17938263

Roy, S., Singh, R.P., Siriwardana, S., Agarwal, C., Sclafani, R.A. and Agarwal, R. Down-regulation of both p21/Cip1 and p27/Kip1 produces a more aggressive prostate cancer phenotype. Cell Cycle, in press (2008).

Leon. R.P, Tecklenburg, M. and Sclafani, R.A. Functional conservation of b-hairpin DNA binding domains in the Mcm protein of M. thermoautotrophicum and Mcm5 protein of S. cerevisiae Genetics, in press (2008).