Biochemical and Biophysical Analyses of the DNA Packaging Protein Machine the Bacteriophage HK97

Packaging of DNA into the phage head requires the action of a terminase complex, which is comprised of a small terminase (TerS in HK97) subunit and a large terminase subunit (TerL in HK97). TerS binds the phage DNA and regulates the activity of TerL. TerL possesses nuclease and ATPase activities needed to generate a genome-length unit of phage DNA from a DNA concatemer and to translocate the mature DNA into the phage head. In the bacteriophage HK97, an HNH endonuclease, gp74, is also involved in phage head packaging. HNH endonucleases are proteins which bind and digest DNA in the presence of divalent metal cofactors. Gp74 enhances the activity of the terminase enzymes toward the phage DNA substrate, and this activity is dependent on an intact HNH motif. Mutation of the metal binding residue in the HNH motif abrogates the gp74-mediated enhancement of terminase function and inhibits phage formation. Work in this thesis addresses the molecular mechanism by which gp74 enhances terminase activity. Using NMR spectroscopy, I have shown that gp74 possesses a helical insertion in its HNH motif that affects its metal binding capability. Further, I have shown that the helical insert is involved in gp74-mediated enhancement of DNA digestion by the terminase enzymes. Finally, the helical insert is also necessary for phage formation. The biochemical experiments examining how wild type and gp74 mutants affect terminase functions required purified samples of the individual terminase subunits. Thus, part of this thesis also describes methods I developed to purify HK97 terminase proteins such that they are soluble in solution and functional. My studies involving gp74, TerS, and TerL have enhanced our understanding regarding the molecular basis by which cofactors (e.g. gp74) regulate the activities of terminases that are needed for DNA packaging and phage maturation.Ph.D.2021-10-25 00:00:0