Dr. Scott W. Morrical
DNA Repair- structure and function of protein-DNA complexes
My laboratory studies the structure and function of protein-DNA complexes required for the initiation and movement of DNA replication forks, for homologous recombination, and for DNA repair processes including double-strand break repair (DSBR). Our model systems include the bacteriophage T4, the extremely radiation-resistant bacterium Deinococcus radiodurans, the budding yeast Saccharomyces cerevisiae, and humans. Our experimental approaches include steady-state and pre-steady-state kinetics, fluorescence spectroscopy, site-directed mutagenesis, single-molecule enzmology, X-ray crystallography, and others. Current projects in the lab include:
Mechanisms of DNA helicase assembly and translocation at DNA replication forks. DNA helicase enzymes unwind double-stranded DNA at replication forks, allowing leading and lagging strand DNA synthesis to occur. Studies of two DNA helicases, Gp41 and Dda, in the bacteriophage T4 replication system have provided important insights on the roles of helicase in fork initiation and movement. Gp41 is a highly processive helicase, capable of driving a DNA replication fork over many kilobases of DNA without dissociating. The assembly of Gp41 helicase at replication forks is tightly regulated by its assembly factor, Gp59. Gp59 plays a critical role in targeting helicase assembly onto nascent DNA replication forks, and in coordinating helicase assembly with the initiation of both leading and lagging strand DNA synthesis. We are currently investigating the mechanism of this coordination by performing biochemical studies on both wild-type and mutated forms of Gp59. Dda is a non-processive helicase that drives DNA replication forks in the presence of the T4 ssDNA-binding protein, Gp32. Previous work in the lab established that Dda-Gp32 protein-protein interactions are essential for Dda-driven replication fork movement. We are currently investigating the effects of Gp32 on Dda self-association and kinetic properties.
Structure and function of presynaptic filaments. Recombinases of the highly conserved RecA/Rad51 family form presynaptic filaments on the single-stranded DNA that is generated during early stages of recombination and DNA repair processes. Filament formation activates the catalytic activities of a recombinase, including ATP hydrolysis and DNA strand exchange. The timing and location of presynaptic filament assembly and turnover are critical for the accurate repair of DNA double-strand breaks. To better understand presynaptic filament dynamics we are studying the UvsX recombinase of bacteriophage T4 and the RecA protein of Deinococcus radiodurans, using a variety of pre-steady-state, steady-state, and single-molecule methods to determine how these enzymes recognize and process their DNA and nucleotide substrates. These studies make heavy use of fluorescence spectroscopy directed towards native tryptophan fluorescence of the proteins or towards extrinsic fluorescence of labeled DNA, nucleotide, or protein components. Allosteric communication between adjacent recombinase subunits within the presynaptic filament is essential for recombination. To investigate recombinase allosterism, we generated a series of site-directed mutants in the yeast Rad51 recombinase that alter its allosteric, DNA-binding, and catalytic properties. Recently we, in collaboration with Dr. Mark Rould of UVM’s Center for X-ray Crystallography, solved the 2.5 Å X-ray crystallographic structure of a filamentous form of one such allosteric mutant of Rad51. Currently we are using structural and bioinformatic predictions to generate new allosteric mutants of Rad51, which we will characterize both biochemically and structurally.
Characterization of tumor-derived mutants of the human recombinase, hRAD51. Aberrant recombination, including defective presynaptic filament assembly, is a hallmark of many human cancers. We are interested to determine how mutations in the human hRAD51 recombinase, which is essential for the accurate repair of DNA double-strand breaks and for genome stability, are related to cancer. Recently, several tumor-derived mutations of hRAD51 have been identified. These mutant hRAD51 proteins have been expressed and purified in our lab, where we are conducting experiments to measure differences in DNA binding, enzymatic properties, and protein-protein interactions. Information from these studies will shed new light on how tumor cells subvert DNA repair processes to rapidly mutate and evolve. Ultimately we hope to put our knowledge of the basic biochemistry of homologous recombination to use in developing new treatment strategies for cancer.
Branagan, A., Maher, R., and Morrical, S. Assembly and Dynamics of Gp59-Gp32-Single-stranded DNA (ssDNA), a DNA Helicase Loading Complex Required for Recombination-dependent Replication in Bacteriophage T4. J Biol Chem. 2012 Jun 1;287(23):19070-81. Epub 2012 Apr 12.
Maher, R., Branagan, A., Morrical, S. Coordination of DNA replication and recombination activities in the maintenance of genome stability. J Cell Biochem. 2011 Jun 6. doi: 10.1002/jcb.23211. [Epub ahead of print]
Liu, J., Ehmsen, K., Heyer, W., Morrical, S. Presynaptic filament dynamics in homologous recombination and DNA repair. Crit Rev Biochem Mol Biol. 2011 Jun;46(3):240-70. doi: 10.3109/10409238.2011.576007
Chen, J., Villanueva, N., Rould, M., Morrical, S. Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant. Nucleic Acids Res. 2010 Aug;38(14):4889-906.
Xu, H., Beernink, H., Morrical, S. DNA-binding properties of T4 UvsY recombination mediator protein: polynucleotide wrapping promotes high-affinity binding to single-stranded DNA. Nucleic Acids Res. 2010 Aug;38(14):4821-33.