Presenter: Benjamin Camel
Co-Presenters: Katherine Meze, Davis Jose, Peter von Hippel
Faculty Mentor: Davis Jose, Peter von Hippel
Presentation Type: Poster 51
Primary Research Area: Science
Major: Biochemistry
Funding Source: GM-15792, NIH, $350k/yr. (4 yrs.)
Previous studies have mapped the structural details and assembly properties of the single-stranded (ss)DNA binding protein (gp32) of bacteriophage T4 as it binds to various ssDNA lattices, both as isolated monomers and as cooperatively bound gp32 clusters. Building on previous studies, our work seeks to understand these binding interactions at single nucleotide resolution. We have utilized site-specifically positioned 2-aminopurine (2-AP) fluorescent base analogs of adenine incorporated into ssDNA lattices as either monomer or dimer-pair probes, to map the detailed interactions of gp32 with ssDNA lattices of various lengths. To this end we have employed changes in the fluorescent and circular dichroism (CD) spectra of these probes in order to determine how the binding site of the protein interacts with these site-specifically positioned probes. Our results demonstrate that gp32 binds at random at low concentrations, and then shifts to preferential binding at the 5’-end of the lattice as the proteins shift into cooperative, cluster-bound forms at higher gp32 concentrations. We have also used acrylamide quenching to monitor solvent exposure of the ssDNA bases at various lattice positions. These results provide new insights into the molecular mechanisms of the gp32-ssDNA interactions that are involved in controlling the functions of the T4 DNA replication complex.