The objective of this project is to develop single molecule manipulation methods for the quantitative study of DNA-ligand interactions and to use these methods to investigate specific biologically important interactions. In addition to method development, this work will shed light on important problems ranging from the fundamental biophysics of DNA-small molecule interactions to the biophysical and biochemical mechanisms of complex protein interactions involved in DNA replication and recombination. The following three specific aims will be addressed to accomplish the above goals. 1. To probe the biophysical mechanism of DNA-small molecule interactions using DNA stretching. 2. To probe the binding mechanism of single-stranded DNA binding proteins, which are essential components of DNA replication. 3. To investigate the role of important DNA replication and recombination proteins from the bacteriophage T4 system.
The PI has developed innovative biophysical methods for examining DNA binding in exquisite detail using single molecule manipulation. The results will have a significant impact beyond this field; as such methods may be adapted to examine a wide variety of important biological systems. The PI also has a strong track record of training graduate and undergraduate students in interdisciplinary research at the interface of physics, chemistry, and biology, including active collaborations with minority-serving and undergraduate institutions. The broader impacts of this research also extend into the classroom, where the PI has developed a new Advanced Physics Laboratory course, which contains several advanced research level experiments, including the use of optical tweezers and atomic force microscopy. Experiments developed by the PI are made publicly available for use by scientists at other institutions. The PI also makes significant contributions to undergraduate teaching, bringing examples from his research into the classroom in courses such as Physics for Pharmacy and Physics for the Life Sciences. 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.
DNA is normally found as a double helix consisting of a sequence of base pairs, representing the genetic code. In order for this code to be read to create proteins (transcription and translation) or to make copies of the DNA (replication), the two strands of the double helix must be separated to expose the bases. The processes of replication and transcription are regulated by proteins that bind to DNA and alter the stability of the double helix. In our research we use optical tweezers instruments to apply very small forces to single DNA molecules. Measurement of these forces allows us to determine the stability of the DNA double helix and the extent to which various DNA binding proteins and small molecules alter the structure and stability of DNA. This approach provides unique insights into the function of these proteins in the cell. Using newly developed methods for quantifying DNA interactions, we examined three important classes of biological problems. First, we studied how small chemicals interact with DNA. We found that by stretching DNA, we could measure the time-dependent changes in DNA structure as these molecules bound to DNA. We used this method to demonstrate how the anti-cancer drug Actinomycin D preferentially binds to DNA that is estabilized by other proteins in the cell, and then becomes locked in place as these proteins leave the DNA. We also studied how the DNA binding of specific DNA replication proteins called SSBs is controlled by specific changes in protein structure, which in turn may be regulated by other replication processes. Finally, we developed methods to study how multiple proteins interact with DNA both in its helical, double-stranded form and when it is destabilized. We demonstrated how the protein UvsY modulates SSB binding to these different forms of DNA to regulate specific replication processes. Overall, we developed several new single molecule methods that allowed us to use the application of force to shed light on important biophysical interactions with DNA. These methods can be applied to many other systems, and will help us to understand how cellular transactions involving DNA are regulated at the molecular level.
