This project is directed toward achieving a quantitative understanding of the dynamical processes that occur in DNA and nucleosomes. Nucleosomes are the first and most fundamental packing level of genomic DNA in eukaryotes, which consist of a complex of eight histone proteins around which 147 base pairs of DNA are wrapped in approximately two superhelical turns. Nucleosomal organization protects genomic DNA from enzymatic degradation, but it also makes most of the genome inaccessible to the many proteins that must bind to it for gene regulation, transcription, replication, recombination, and repair. The elucidation of the mechanisms and rates of the spontaneous conformational changes in nucleosomal DNA is critical to understand how such proteins can gain access to their target sites without requiring the complete dissociation of the histone octamer.
The specific aims of the project seek to gain a deeper quantitative understanding of the simplest biological components of chromatin: double stranded DNA and nucleosomes. A new experimental approach based on fluorescence fluctuation spectroscopy will allow the determination of the rates of conformational changes in biopolymers in a broad range of timescales. These, and other methods of analysis, will be applied to the study of DNA flexibility and dynamics in naked DNA and nucleosomes. The proposed approach is novel, in that it has a strong emphasis in the application and development of new methods of data acquisition and analysis that will advance the single-molecule field towards a more quantitative era. The successful completion of these studies is expected to establish the foundation necessary to undertake the study of the more complex biological systems such as native chromatin.
This proposal involves research and teaching activities that aim to stress the importance of quantitative research. The lack of basic numerical skills, and the negative attitude that many students hold about mathematics, conflicts with the demands of today's interdisciplinary research and seriously limits students in their long-term opportunities. The PI proposes to develop a course intended to act as a bridge between the traditional math classes and the more quantitative science courses. The PI proposes to create a website that will gather testimonials and information of faculty members around the nation illustrating 1) the need of learning the quantitative sciences in today's biologically-oriented disciplines, and 2) the challenges of being a minority in science. The latter will be bilingual English/Spanish, and will be written in lay terms with the goal of reaching not only students, but also their families and the general public.
DNA is the material that contains the code that determines most physical characteristics of every living creature. In our cells, DNA is part of a complex architecture called chromatin, which despite decades of research remains poorly understood. The building block of chromatin is the nucleosome, a complex formed when DNA wraps around a group of proteins to form a spool-like structure. This allows very long DNA molecules (about 6 feet in length) to be densely packed into the nucleus of cells—an area that is about a millionth of its size. Understanding nucleosomes is critical to answer fundamental questions about the underlying mechanisms by which genes are turned ‘on’ and ‘off’. This is required by all cells during normal function, and is also related to disease. The structure of nucleosomes has been known for many decades, but scientists have long recognized that its structure alone did not provide insights into how they function. In the nucleosome, DNA is tightly wrapped around proteins, and is mostly inaccessible to the many proteins that need to bind to it in order for the cell to function. This led researchers to hypothesize that nucleosomes are not static structures, but instead, DNA had to be dynamic so that transient unwrapping events provide the window of opportunity proteins need to find their targets and bind to them. The study of dynamical conformational changes in nucleosomes, and more broadly in any biological molecule, has been hampered by the lack of experimental approaches that allow such measurements. The goal of this research was to develop methodologies that can be generally used to investigate conformational dynamics in biological molecules, and to use these methodologies to learn about the dynamics of DNA in nucleosomes. Measuring dynamics is a difficult task because until recently, all experimental methods dealt with measurements involving huge numbers of molecules. To put this in perspective, a small drop of water contains more molecules than people live on earth. If we wanted to learn about how humans walk by measuring the average distance between the feel of all the individuals in this planet we would not get too far. Instead, if we could look at the feet of many different individuals, but one by one, we would be able to understand the diversity that exists among different people, and the dynamics of how humans move. A similar situation occurs when looking at molecules. We need new techniques that allow researchers to look at individual molecules one by one. The signals we get from individual molecules are tiny, and that is why these experiments could not be done decades ago. In order to observe and characterize the dynamic behavior of nucleosomes, we relied on an imaging method known as Fluorescence Resonance Energy Transfer or FRET. The technique allowed us to look at a pair of fluorescent molecules or fluorophores, one of which is attached to the end of the exposed DNA strand, the other, to one of the proteins around which the DNA is coiled. The unwrapping of the DNA translates into a larger distance between these reporter fluorophores, which we can detect as a change in the number of photons being emitted by the sample. In order to achieve this goal we had to devote an important amount of time developing new methods of measurement and analysis. Although our main focus in this project was on nucleosome dynamics, these methods are useful to look at the dynamics of many other systems of interest in the biological sciences. The results of our studies with nucleosomes showed that for base pair sequences close to the end of the nucleosome, spontaneous DNA unwrapping occurs at a rate of about 4 times per second. This corresponds to a period of only 250 milliseconds during which this region of DNA remains fully wrapped and occluded by the protein complex. Once unwrapped, the DNA remains exposed for 10-50 milliseconds. These findings present a possible mechanism that allows protein to bind to unwrapped DNA. Instead, sequences of DNA that are closer to the nucleosome’s center are less dynamic. The results of these studies indicate that the position of different DNA sequences within the nucleosome influences its likelihood to be exposed and become accessible for protein binding. The research funded by this grant had also an important impact in the educational and outreach activities of Prof. Levitus. During the period covered by this grant, Prof. Levitus designed and taught "Mathematical Methods in Chemistry" (an undergraduate course) and "Quantitative Foundations of Modern Biochemistry" (a graduate course). Both courses focused on improving the quantitative skills of chemistry and biochemistry majors. In addition, Prof. Levitus has been involved in several activities designed to increase the participation of underrepresented minorities in the physical sciences, and she mentored five graduate students that obtained PhD and MS degrees under her supervision.
