The objective of this research program is to determine the mechanisms by which recombinase and helicase enzymes are assembled onto single-stranded DNA (ssDNA) in the bacteriophage T4 DNA replication/recombination system. We will study the assembly of the T4 UvsX recombinase into presynaptic filaments, and we will study the assembly of two different DNA helicases, Gp41 and Dda, onto ssDNA at replication forks and in recombination intermediates. All three enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound Gp32, the T4 ssDNA-binding protein. UvsX and Gp41 both require the activity of a specific mediator protein, UvsY or Gp59, respectively, for proper assembly onto Gp32-ssDNA complexes, whereas Dda achieves the same effect through direct protein-protein interactions with Gp32. We will explore all three enzyme loading mechanisms using classical biochemical methods (kinetics, thermodynamics, fluorescence, sedimentation, crosslinking), single- molecule approaches (fluorescence imaging, force spectroscopy), and mutagenesis.
Our SPECIFIC AIMS are: (1) Determine the kinetic mechanism of UvsX-ssDNA presynaptic filament assembly and collapse. We will test a model in which UvsY protein selectively enhances filament nucleation, UvsX actively displaces gp32 from ssDNA, and filaments exhibit dynamic instability linked to ATP hydrolysis. (2) Determine how interactions of T4 Gp59 protein with replication fork DNA control helicase assembly and polymerase blockage. We will test a model in which cooperative binding of Gp32 to lagging-strand ssDNA converts Gp59 from a polymerase-blocking to a helicase-loading conformation that recruits Gp41 helicase to the replication fork. (3) Determine how interactions with Gp32 modulate the DNA helicase functions of T4 Dda protein. We will test a model in which Dda-Gp32 protein-protein interactions promote the oligomerization of Dda and enhance its DNA unwinding properties in both replication and recombination transactions. Understanding how helicase and recombinase enzymes are correctly assembled onto ssDNA is fundamental to understanding DNA replication, recombination, and repair mechanisms that are conserved in all organisms. There are clear links between errors in DNA replication/recombination/repair machineries and human disease states including cancer. Understanding how recombinase- and helicase- ssDNA complexes are correctly assembled and activated may therefore aid in the prevention, diagnosis, and treatment of cancer.

Public Health Relevance

Proper assembly of enzyme-ssDNA complexes is critical for genome replication and maintenance in all organisms. Defects in enzyme-ssDNA assembly processes are implicated in cancer and other human disease states. Enzyme-ssDNA assembly pathways are also potential targets for new classes of antibiotic and antitumor drugs. Our studies of enzyme-ssDNA assembly mechanisms may therefore shed light on how tumors develop and how infectious diseases progress, and may also suggest new treatment strategies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Reddy, Michael K
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University of Vermont & St Agric College
Schools of Medicine
United States
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Morrical, Scott W (2015) DNA-pairing and annealing processes in homologous recombination and homology-directed repair. Cold Spring Harb Perspect Biol 7:a016444
Jordan, Christian S; Morrical, Scott W (2015) Regulation of the bacteriophage T4 Dda helicase by Gp32 single-stranded DNA-binding protein. DNA Repair (Amst) 25:41-53
Branagan, Amy M; Klein, Jenny A; Jordan, Christian S et al. (2014) Control of helicase loading in the coupled DNA replication and recombination systems of bacteriophage T4. J Biol Chem 289:3040-54
Liu, Jie; Berger, Christopher L; Morrical, Scott W (2013) Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein. Biochemistry 52:7878-89
Branagan, Amy M; Maher, Robyn L; Morrical, Scott W (2012) 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 287:19070-81
Maher, Robyn L; Branagan, Amy M; Morrical, Scott W (2011) Coordination of DNA replication and recombination activities in the maintenance of genome stability. J Cell Biochem 112:2672-82
Liu, Jie; Ehmsen, Kirk T; Heyer, Wolf-Dietrich et al. (2011) Presynaptic filament dynamics in homologous recombination and DNA repair. Crit Rev Biochem Mol Biol 46:240-70
Xu, Hang; Beernink, Hans T H; Morrical, Scott W (2010) DNA-binding properties of T4 UvsY recombination mediator protein: polynucleotide wrapping promotes high-affinity binding to single-stranded DNA. Nucleic Acids Res 38:4821-33
Bleuit, J S; Xu, H; Ma, Y et al. (2001) Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair. Proc Natl Acad Sci U S A 98:8298-305