CAREER: Conformational dynamics of DNA and nucleosomes: A quantitative single-molecule study

Arizona State University, Scottsdale AZ

Investigators

Abstract

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.