Print

Xu, Hang

Biochemistry

2005

PhD

DNA recombination and replication in bacteriophage T4 are highly coordinated in an efficient system for recombination dependent replication (RDR), a pathway that mimics the double strand break repair (DSBR) pathway in eukaryotes. Single-stranded DNA and its associated enzymes play key roles in both recombination and replication. However in many cases, the assembly of an enzyme-ssDNA complex is inhibited by a single stranded DNA binding protein (SSB). A unique class of protein factors, the recombination and replication mediator proteins (RMPs), can modulate the inhibitory effects of SSBs on the formation of active enzyme complexes on ssDNA. The mediator activity is attributed to interactions between RMP, ssDNA, SSB, and enzymes. The two best characterized RMP proteins are both bacteriophage T4 proteins. The recombination mediator protein, UvsY, facilitates the assembly of UvsX recombinase onto ssDNA to form a presynaptic filament. Likewise, the replication mediator protein, Gp59, promotes the assembly of hexameric Gp41 helicase onto ssDNA at the lagging strand of replication forks. These two mediator proteins are considered the prototypes of RMP proteins, and the characterization of them will help us to understand the common functions of RMP proteins. Interactions between Gp59 and Gp32, the T4 SSB, are critical for helicase loading at replication forks. In this thesis work, the binding and hydrodynamic properties Gp59-Gp32 binary complexes are quantified. By monitoring the anisotropy change of rhodamine labeled Gp59 upon binding of truncated Gp32 species, the fundamental stoichiometry of Gp32-Gp59 interactions are determined to be 1: 1. The dissociation constants are in the low nM range. These results quantitatively confirm the very strong interaction between Gp32 and Gp59. Analytical ultracentrifugation studies further demonstrate the existence of Gp32 sp.-Gp59 heterodimeric complexes in solution. Combined with the binding affinity, it is implicated that Gp32-Gp59 complexes form a special subpopulation of Gp32 in the T4-infected E. coli cell, which represent the functional form of Gp59 in vivo. Another T4-encoded mediator protein, UvsY, stimulates the assembly of presynaptic filaments by nucleating and stabilizing UvsX recombinase-ssDNA interactions. To investigate the nucleation process, the DNA binding properties of UvsY are examined under conditions of low binding density by quantitative DNA cellulose chromatography. Results demonstrate that UvsY binds to ssDNA with approximately 300-fold higher affinity than dsDNA. The data support a model in which wrapping of ssDNA around a UvsY hexamer creates an optimal nucleation site for UvsX recombinase. In addition, the binding properties of UvsX to both ssDNA and dsDNA, with or without different nucleotide co factors, are examined. Except in the presence of ATP S, UvsX exhibits similar affinity to ssDNA and dsDNA, leading to a re-examination of the dsDNA-binding function of UvsX in recombination processes. In addition to biochemical characterizations of T4 proteins, X-ray crystallography studies were performed on UvsY and its complex with the oligonucleotide BrdU24, attempting to solve their atomic structures. High quality crystals and co-crystals were obtained, which diffract to high resolution, however phase information has not been obtained yet. Methods for future MIR and/or MAD phasing are developed in this thesis work and need further investigation.