|
|
           |
|
Peter Bullock, Ph.D. Professor, Tufts Department of Biochemistry Ph.D. Molecular Biology |
||||
Links:ResearchLab MembersRecent Publications |
Research Summary:Studies in our laboratory are designed to address, in molecular terms, basic issues related to the initiation of DNA replication in tumor viruses. Topics under investigation include, 1) the recognition of origins of replication by virally encoded "initiator" proteins, 2) the mechanism of origin unwinding, 3) the mechanism by which DNA is pumped through eukaryotic helicases and 4) the regulation of these processes. We are also very interested in how a viral protein, termed T-antigen, interacts with the cellular repair and checkpoint machinery in order to drive infected cells into S-phase. A combination of biochemical, molecular biology and structural techniques are employed in these studies (the latter conducted in collaboration with members of the Bachovchin and Bohm laboratories). We are also working with members of the Wright laboratory to identify cyclic peptide inhibitors of viral replication using genetic techniques. |
||||
I. Initiation of DNA Replication and the regulation of this process:Initiation of DNA replication is a complicated process that is poorly understood in molecular terms. Therefore, we are studying this process using a viral model system, SV40, and assorted biophysical, biochemical and genetic techniques. Summarized below are some of our recent advances in understanding how the initiation of DNA replication takes place. Structural studies: SV40 encodes a multi-domain protein termed T-antigen (T-ag) that performs a number of tasks during initiation of replication. Owing to the work of many investigators, critical insights into the structure of T-ag have recently been obtained. For example, in Meinke et al. (2006) we reported that the origin-binding domain (T-ag-obd) forms a left-handed “spiral hexamer”. An intriguing feature of the spiral hexamer is the presence of a gap between the first and last subunits. This gap may play an important role in the subsequent extrusion of ssDNA from the double hexamer. |
||
|
||
In related studies, conducted in collaboration with Andrew Bohm and Gretchen Meinke, we determined the co-structure of the T-ag-obd bound to a GAGGC containing oligonucleotide. We are conducting a number of additional structural studies designed to further our understanding of how viral "initiators", such as T-ag, catalyze the early stages of DNA replication. Origin recognition and DNA melting: We have also investigated the protein/DNA interactions that T-ag makes during recognition of the viral origin. Studies described in Reese et al (2004) led to the identification of the “beta-hairpin” motif in T-ag (red residues in figure below) and established the critical roles it plays in origin recognition and DNA melting. Based on extensive sequence homology, Reese et al. proposed that this motif is present in many other DNA helicases. In addition to origin recognition, it is now clear from a number of studies conducted by other groups that “beta-hairpins” play critical roles in the ATP dependent movement of ssDNA through the helicases encoded by DNA tumor viruses.
|
||
|
||
In recent studies, we have analyzed the role played by the "beta-hairpin" during the unwinding process (Kumar et al., (2007)). These studies demonstrated that residues at the tip of the beta-hairpin are needed to melt the origin. They also indicate that subsequent oligomerization events take place on only one of the strands generated by the melting process. How DNA unwinding is coupled to "initiator assembly" is a topic that is being actively pursued in our laboratory. Transit of DNA through a eukaryotic helicase: DNA helicases are essential for DNA metabolism; however, little is known about how DNA is pumped through these complexes. Therefore, in Reese et al (2006) we used NMR methods to establish the path taken by ssDNA past the T-ag-obd. This information has helped us to refine our model for how ssDNA transits through the double hexamer helicase that is depicted in Fig 3. We have initiated a number of projects designed to confirm this model. |
||
|
||
II. A chemical-genetic approach to the isolation of inhibitors of viral replication:We are also interested in the identification of inhibitors of viral replication. Therefore, it is of interest that vast peptide libraries, can be prepared in E. coli using intein based vectors which give rise to cyclic peptides (reviewed in Horswill and Benkovic (2005) Cell Cycle 4: 552-555). In addition, powerful genetic methods have been described that enable the selection of cyclic peptides that interact with specific protein targets. To extend these studies, members of the Bullock and Wright laboratories are using genetic methods to identify cyclic peptides that disrupt the assembly of viral "initiators" on origins of replication. |
Graduate Students
Anu Kumar
Technician
Stephanie Moine
Fradet-Turcotte A, Vincent C, Joubert S, Bullock PA, Archambault J (2007). Quantitative analysis of the binding of simian virus 40 large T antigen to DNA. J. of Virology. 81(17), 9162-74.
Kumar A, Meinke G, Reese DK, Moine S, Phelan PJ, Fradet-Turcotte A, Archambault J, Bohm A, Bullock PA (2007). Model for T-antigen-dependent melting of the simian virus 40 core origin based on studies of the interaction of the beta-hairpin with DNA. J. of Virology. 81 (9), 4808-18.
Meinke, G., Phelan, P., Moine, S., Bochkareva, E., Bochkarev, A., Bullock, P.A. and Bohm, A. (2007). The Crystal Structure of the SV40 T-antigen Origin Binding Domain in Complex with DNA. PLOS Biology. 5, 144-156.
Reese, D.K., Meinke, G., Kumar, A., Moine, S., Chen, K., Sudmeier, J.L., Bachovchin, W., Bohm, A. and Bullock, P.A. (2006). Analyses of the Interaction between the Origin Binding Domain from Simian Virus 40 T-antigen and Single-Stranded DNA Provides Insights into DNA unwinding and Initiation of DNA Replication. J. of Virology. 80, 12248-12259.
Meinke, G., Bullock, P.A. and Bohm, A. (2006). The crystal structure of the SV40 large tumor antigen origin-binding domain. J. of Virology 80, 4304-4312.
Bradshaw, E. M., D. G. Sanford, et al. (2004). "T antigen origin-binding domain of simian virus 40: determinants of specific DNA binding." Biochemistry 43(22): 6928-36.
Reese, D. K., K. R. Sreekumar, et al. (2004). "Interactions required for binding of simian virus 40 T antigen to the viral origin and molecular modeling of initial assembly events." J Virol 78(6): 2921-34.
Sudmeier, J. L., E. M. Bradshaw, et al. (2003). "Identification of histidine tautomers in proteins by 2D 1H/13C(delta2) one-bond correlated NMR." J Am Chem Soc 125(28): 8430-1.
Kim, R. J., S. Moine, et al. (2002). "Peptides containing cyclin/Cdk-nuclear localization signal motifs derived from viral initiator proteins bind to DNA when unphosphorylated." J Virol 76(23): 11785-92.
Purviance, J. D., A. E. Prack, et al. (2001). "In the simian virus 40 in vitro replication system, start site selection by the polymerase alpha-primase complex is not significantly altered by changes in the concentration of ribonucleotides." J Virol 75(14): 6392-401.
Sreekumar, K. R., B. A. Barbaro, et al. (2001). "Methods for studying interactions between Simian virus 40 T-antigen and the viral origin of replication." Methods Mol Biol 165: 49-67.
Barbaro, B. A., K. R. Sreekumar, et al. (2000). "Phosphorylation of simian virus 40 T antigen on Thr 124 selectively promotes double-hexamer formation on subfragments of the viral core origin." J Virol 74(18): 8601-13.
Sreekumar, K. R., A. E. Prack, et al. (2000). "The simian virus 40 core
origin contains two separate sequence modules that support T-antigen double-hexamer
assembly." J Virol 74(18): 8589-600.
