Project Description:
The Morrical Laboratory studies the structure and function of protein-DNA complexes required for the initiation and movement of DNA replication forks, for homologous genetic recombination, and for DNA repair processes including translesion DNA synthesis and Double-Strand Break Repair (DSBR). Our model system is the bacteriophage T4, which provides some of the best-characterized examples of multi-protein DNA replication, recombination, and repair systems. Our experimental approaches include structural biology and physical biochemistry applications. Currently, our efforts are focussed in the following areas: [1] X-ray crystallography and solution biochemistry of protein-DNA intermediates in homologous genetic recombination; [2] Thermodynamics of protein-ssDNA interactions; [3] Mechanisms of DNA helicase assembly at replication forks; and [4] Structure and function of DNA polymerases bound to lesion-containing templates. Two representative projects are summarized below: Structure & Function of T4 UvsY Protein. UvsY protein is the prototypical Recombination Mediator Protein or RMP (cf. Beernink & Morrical, 1999). UvsY facilitates homologous recombination by promoting the assembly of filaments of recombination proteins bound to ssDNA. Its filament assembly activity requires interactions both with ssDNA and with other protein members of the T4 recombination system (recombinase and SSB components). Our lab demonstrated that uvsY exists as a hexamer in solution, binds to ssDNA as a hexamer, and that the hexameric structure of uvsY is essential for high-affinity interactions with ssDNA and for recombination function. Recently we obtained high-quality crystals of uvsY-ssDNA complexes. Efforts are underway to solve this crystal structure and to determine biochemical and structural parameters of uvsY complexes with recombinase and SSB components. Facilitated Helicase Assembly by T4 Gp59 Protein. The acquisition of a DNA helicase by a nascent replication fork is essential for the reconstitution of processive, semi-conservative DNA synthesis. We are studying the mechanism by which a specific helicase assembly protein, gp59, loads the T4 replicative DNA helicase, gp41, onto the lagging strand of the replication fork during the early stages of T4 DNA replication. Elements of this project include determining biochemical parameters of gp59-helicase interactions and how they may influence the assembly of hexameric rings of the helicase, its active enzymatic form.

Ma, Y., Wang, T., Villemain, J.L., Giedroc, D.P., Morrical, S.W. Dual functions of single-stranded DNA-binding protein in helicase loading at the bacteriophage T4 DNA replication fork. J Biol Chem. 2004 Apr 30;279(18):19035-19045.

Bleuit, J.S., Ma, Y., Munro, J., Morrical, S.W. Mutations in a conserved motif inhibit single-stranded DNA binding and recombination mediator activities of bacteriophage T4 UvsY protein. J Biol Chem. 2004 Feb 13;279(7):6077-6086.

Bleuit, J.S., Xu, H., Ma, Y., Wang ,T., Liu, J., Morrical, S.W. Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8298-305.

Villemain, J.L., Ma, Y., Giedroc, D.P., Morrical, S.W. Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems.

Xu, H., Wang, Y., Bleuit, J.S., Morrical, S.W. Helicase assembly protein Gp59 of bacteriophage T4: fluorescence anisotropy and sedimentation studies of complexes formed with derivatives of Gp32, the phage ssDNA binding protein.

Beernink, H.T.H. & Morrical, S.W. (1999)RMPs: Recombination/replication mediator proteins. TrendsBiochem. Sci. 24: 385-389.

Ando, R.A. & Morrical, S.W. (1999) Relationship betweenhexamerization and ssDNA binding affinity in the uvsY recombinationprotein of bacteriophage T4. Biochemistry 38: 16589-16598.