Mechanisms and Regulation of Human and Viral DNA Replication  

The research interests of my laboratory are focused on the mechanism of human and viral DNA replication and how DNA replication is controlled. We use model viral DNA replication systems, such as simian virus 40 (SV40) and papillomaviruses (PV), both of which can replicate their DNA in human cells, as well as biochemical studies of human cellular proteins. Our studies on the mechanisms and regulation of DNA replication are intimately linked to our studies on novel PV and cellular DNA replication factors as well as our studies on how DNA damage inhibits DNA replication.

Our studies on PV DNA replication are focused on identifying cellular proteins that PVs require to replicate their DNA. We have previously shown that all the cellular factors required for in vitro SV40 DNA replication are also required for PV DNA replication in vitro. However, they are not sufficient; additional cellular factors are required to support PV DNA replication. We are currently using a reconstitution assay to purify those factors from human cells. In addition, a complementation assay, first developed in Dr. Louise Chow’s laboratory at the University of Alabama at Birmingham, is also being used to identify cellular factors in human 293 cell extracts that can complement human HeLa cell extracts for in vitro PV DNA replication.

One of the ways in which we are studying the mechanism of DNA replication is by looking at the protein-protein interactions that take place between DNA replication factors at the replication fork. I previously demonstrated that an interaction between SV40 T–antigen (a viral DNA replication protein) and the human Replication Protein A complex appears to be important for priming during DNA synthesis. We have now shown that the PV E1 protein also interacts with RPA, and this interaction modulates the activities of these proteins. The information we have generated has resulted in a new model for ssDNA binding protein recruitment at DNA replication forks. We are also investigating other interactions between the PV and cellular DNA replication proteins, and evaluating their biological significance.

We are also collaborating with Dr. Terry Beerman’s laboratory at Roswell Park Cancer Institute to study the molecular effects of DNA damage and anticancer drugs on DNA replication. Dr. Beerman’s laboratory has shown that DNA damaging anticancer drugs act to block cellular DNA replication at very low levels of drug. These responses seem to be similar to the very potent DNA damage dependent DNA replication arrest seen with ionizing radiation. We are currently investigating the mechanisms by which these drugs inhibit DNA replication. In addition to elucidating how these anticancer drugs act to arrest cancer cell growth, these studies will provide general information about how cells arrest DNA replication in response to DNA damage. 

Selected Recent Publications: 

Dimitrova, D.S., Todorov, I.T., Melendy, T., and Gilbert, D.M.  (1999).  Mcm-2, But Not RPA, is a Component of the Mammalian Early G1-Phase pre-Replication Complex.  Journal of Cell Biology.  146, 709-722.  (cover article) 

Han, Y., Loo, Y.-M., Militello, K.T., Melendy, T.  (1999).  Interactions of the papovavirus DNA replication initiator proteins simian virus 40 large T antigen and bovine papillomavirus type 1 E1 with human Replication Protein A.  Journal of Virology.  73, 4899-4907.   

Liu, J.-S., Kuo, S.-R., McHugh, M.M., Beerman, T.A., and Melendy, T.  (2000).  Adozelesin triggers DNA damage response pathways and arrests SV40 DNA replication through replication protein A inactivation.  Journal of Biological Chemistry.  275, 1391-1397.   

Loo, Y.-M., Melendy, T.  (2000).  The majority of human replication protein A remains complexed throughout the cell cycle.  Nucleic Acids Research.  28, 3354-3360.   

McHugh, M., Yin, X., Liu, J.-S., Kuo, S.-R., Melendy, T., and Beerman, T.A.  (2001).  The Cellular Responses to DNA Damage Induced by the Enediyenes, C-1027 and Neocarzinostatin Includes Hyperphosphorylation and Increased Nuclear Retention of Replication Protein A (RPA) and Trans Inhibition of DNA Replication.  Biochemistry.  40, 4792-4799.   

Liu, J.-S., Kuo, S.-R., Yin, X., Beerman, T.A., and Melendy, T.  (2001).  DNA Damage by the Enediyne, C-1027, Results in the Inhibition of DNA Replication by Loss of Replication Protein A Function and Activation of DNA-Dependent Protein Kinase.  Biochemistry.  40:14661-14667.    

Melendy, T. and Li, R. (2001).  review:  Chromatin remodeling and initiation of DNA replication.  Frontiers in Biosciences.  6, d1048-1053.   

Liu, J.-S. and Melendy, T. (2002).  Papillomavirus DNA Replication.  in Human Papillomaviruses:  Perspectives in Medical Virology.  D. McCance (ed.) Elsevier.  Elsevier.  pp. 53-70.  

Liu, J.-S., Kuo, S.-R., Beerman, T.A., and Melendy, T. (2003).  Induction of DNA Damage Responses by Adozelesin Is S Phase-Specific and Dependent on Active Replication Forks.  Mol. Cancer Therapeutics.  2, 41-47.  

Cao, P.-R., McHugh, M.M., Melendy, T., and Beerman, T.A. (2003).  The DNA minor groove alkylating cyclopropylpyrroloindole  drugs bizelesin and adozelesin induce different DNA damage response pathways in human colon carcinoma HCT116 cells.  Mol. Cancer Therapeutics.  2, 651-659. 

Liu, J.-S., Kuo, S.-R., and Melendy, T.  (2003).  Comparison of Checkpoint Responses Triggered by DNA Polymerase Inhibition Versus DNA Damaging Agents.  Mutation Research.  532:215-226.  (cover article) 

Stracker, T.H., Cassell, G.D., Ward, P., Loo, Y.-M., van Breukelen, B., Carrington-Lawrence, S., Hamatake, R.K., van der Vliet, P.C., Weller, S.K., Melendy, T., Weitzman, M.D.  (2004).  The Rep protein of adeno-associated virus (AAV) interacts with single-stranded DNA-binding proteins that enhance viral replication.  Journal of Virology.  78, 441-453.  

Loo, Y.-M., Melendy, T. (2004).  Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA.  Journal of Virology.  78, 1605-1615.  

Tu, L.-C., Melendy, T., and Beerman, T.A. (2004).  DNA Damage Responses Triggered by a Highly Cytotoxic Monofunctional DNA Alkylator, Hedamycin, a Pluramycin Anti-tumor Antibiotic.  Mol. Cancer Therapeutics.   Mol. Cancer Therapeutics, 3, 577-585.

Clower, R.C., Fisk, J.C., and Melendy, T. (2006). Papillomavirus E1 protein binds to and stimulates human topoisomerase I. Journal of Virology . 80, 1584-1587.

Liu, J.-S., Kuo, S.-R., and Melendy, T. (2006). Phosphorylation of Replication Protein A by S Phase checkpoint kinases. DNA Repair. 5, 369-380 .

Hu, Y., Clower, R.C., and Melendy, T. (2006). Cellular topoisomerase I modulates origin binding by bovine papillomavirus type 1 E1. Journal of Virology 80, 4363-71.

Clower, R.C., Hu, Y., and Melendy, T. (2006). The PV E2 protein interacts with and stimulates DNA topoisomerase I. Virology 348, 13-18.

Narahari, J., Fisk, J.C., Melendy, T., and Roman, A. Interactions of the Cellular CCAAT Displacement Protein and Human Papillomavirus E2 Proteins with the Viral Origin of Replication Can Regulate DNA Replication. Virology 350, 302-311.

Clower, R.C., Hu, Y., and Melendy, T. A Helicase/Polymerase Interaction Reveals a Highly Processive Leading Strand DNA Polymerase Complex. Manuscript under review .

Fisk,J.C., and Melendy, T. Specific Evaluation of PIKK Activation in Human Cell Extracts. Manuscript submitted .

Clower RV. Hu Y. Melendy T. Papillomavirus E2 protein interacts with and stimulates human topoisomerase I. Virology. 348(1):13-8, 2006 Apr 25.

Liu JS. Kuo SR. Melendy T. DNA damage-induced RPA focalization is independent of gamma-H2AX and RPA hyper-phosphorylation. Journal of Cellular Biochemistry. 99(5):1452-63, 2006 Dec. 1.