Calander Home 2001-2002 2002-2003 Ioulia Rouzina Studying DNA duplex stability by pulling on single DNA molecules Date: December 2 Time: Noon to 1 Place: BSBE 4-101 Recently advancement of technology made possible mechanical manipulation of the individual molecules. Pulling on single macro molecules became a powerful technique for studying their structure, stability and kinetics. This method was successfully used to study flexibility of the polymers, stability of the secondary and tertiary structures of proteins, motion of the molecular motors, etc. I will discuss stretching of the single long double stranded DNA molecule by its opposite ends. First, DNA molecule stretches to its contour length, yielding information on its flexibility. Upon further extension the molecule becomes about twice longer without any increase in the applied force. Such a peculiar behavior signifies the cooperative phase transition of the molecular structure, that we interpreted as a separation between the two strands comprising the DNA double helix, i.e. DNA melting. Based on the "melting" idea we predicted the behavior of the transition force on various solution conditions such as temperature, pH, solution ionic strength. Experiments performed in our lab verified all of these predictions. Thus measuring of the mechanical energy needed for the strand separation became the first direct method for studying the duplex stability. The advantage of this method over the traditionally used thermal melting, is that the duplex stability can be measured at the arbitrary temperature, including the physiological one. This opens the possibility to study the effect of DNA binding proteins on duplex stability. This novel technique was already used to explore the action of the proteins that are known to promote the double to single stranded DNA inter conversion, and chaperone the restructuring the DNA and RNA helices. I will discuss several very resent results on the HIV-1 nucleocapsid protein and gp32 single stranded DNA binding protein. I will also describe the new challenges for both theory and experiment in analysis of stretching data for the very slow DNA binding proteins.