Tag: Peter von Hippel

Probing the Nucleic Acid Binding Properties of the Single-stranded DNA Binding Protein of bacteriophage T4 Replication Complex at Single Nucleotide Resolution

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.

Mapping Interactions Of Single-Stranded (Ss) DNA With the Ss-DNA Binding Protein (Gp32) of the T4 DNA Replication Complex at Specific Nucleotide Residue Positions

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.

Investigating Gp32 Binding Behavior on Single-Stranded DNA With Different Polarity And Length Using Microsecond Resolution smFRET Measurements

Presenter(s): Anson Dang

Faculty Mentor(s): Andrew Marcus & Peter von Hippel

Poster 67

Session: Sciences

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. The T4 replication system serves as an excellent model for higher organisms as it contains all the essential components for DNA replication. This project aims to investigate how polarity and length of the ssDNA affect gp32 DNA binding. We perform microseconds resolution single-molecule FRET (smFRET) measurements on four primer templates of 14-15 base pairs and different polarities. Data are analyzed using both second- and fourth- order time correlation functions. At the current stage of this project, our results indicate at least three different conformational stages for gp32 binding. Further analysis is required to compare if and how gp32 dimer bind differently on the different constructs.