CAREER: DNA Binding Ligands and the Helix-Coil Transition of Single DNA Molecules

Northeastern University, Boston MA

Investigators

Abstract

This CAREER project combines research and education in the design and use of optical tweezers instruments and in the study of the biophysics of DNA. Optical tweezers instruments will be used to measure the forces required to extend single DNA molecules beyond their natural contour length in the presence of DNA binding ligands. Three major systems will be examined to elucidate the mechanism by which ligands that bind preferentially to single-stranded DNA alter the helix-coil transition of double-stranded DNA molecules. The DNA helix-coil transition at low pH will first be examined. Since the proton is the simplest charged single-stranded DNA binding ligand, a study of the effect of pH on DNA overstretching will provide a firm basis for understanding more complex ligands. It has been shown that HIV-1 nucleocapsid protein functions by altering the DNA helix-coil transition. To determine the entropic or enthalpic nature of this altered cooperativity, this work will examine the temperature dependence of this effect for wild-type and mutant proteins. Bacteriophage T4 Gene 32 protein (gp32) is a DNA binding protein essential for DNA replication, recombination, and repair. Measurements of the dependence of DNA helix-destabilization by this protein on temperature, ionic strength, and protein concentration will be used to elucidate the nature of gp32's interactions with DNA. By studying force-induced melting of single DNA molecules in the presence of DNA binding ligands, single molecule manipulation techniques will be established as a quantitative tool for elucidating the role of DNA binding proteins in biological processes such as retroviral reverse transcription and DNA replication. This work will have a strong educational impact by training students in an entirely new interdisciplinary area. Undergraduate and graduate students will be trained in an exciting new field at the interface of physics, chemistry, and biology. It will also have a strong scientific impact by developing a new technique for studying important biological interactions. The PI will also lead the development of a new Advanced Physics Lab class that will train advanced undergraduate physics majors at Northeastern University to design and build physics experiments, including the construction of single beam optical tweezers instrument. This project is supported by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Division of Physics in the Mathematical and Physical Sciences Directorate.