Presenter(s): Anson Dang − Biochemistry
Faculty Mentor(s): Pete von Hippel
Poster 19
Research Area: Natural Science, Biochemistry
Funding: Dreyfus Undergraduate Mentorships
The single-stranded (ss)DNA binding protein (gp32) of bacteriophage T4 plays a central role in regulating the functions and integration of the helicase, polymerase and primase components of the T4 DNA replication system. To understand how gp32 interacts with itself and with the other regulatory proteins and sub-assemblies of the T4 replication complex, we must first understand the structural details of how this protein binds to ssDNA lattices, both as isolated monomer subunits and as cooperatively bound gp32 clusters. We have explored these issues by monitoring differences in the fluorescence and circular dichroism (CD) spectra of site-specifically positioned monomers and dimer-pairs of 2-aminopurine (2-AP) probes located at various ssDNA positions within the binding site. In its cooperatively bound form gp32 spans 7 nucleotide residues per protein subunit, and by mapping spectral changes on binding to ssDNA lattices that are exact multiples of 7 residues in length we have been able to characterize interactions at defined positions within the gp32 binding cleft. We have extended these studies using acrylamide quenching and permanganate foot-printing assays to monitor degrees of base exposure at various lattice positions. Our results show that gp32 binds randomly at low concentrations, and then shifts toward preferential binding at the 5’-ends of the lattice as cooperatively bound gp32 clusters form at higher gp32 concentrations. Bases located near the middle of a gp32 binding site display lower solvent accessibility than those near the ends of the site. These differences in base ‘shielding’ may reflect deeper burial of the middle bases within the electropositive binding cleft, while bases at the ends may be made more accessible by fluctuations of the C- and N-terminal regulatory sub-domains of the protein. Insights into gp32- ssDNA interactions involved in controlling the functions of the T4 DNA replication complex that result from these studies will be discussed.