In this project, Dr. Harrington proposes a novel approach based upon a recent major advance in scanning force microscopy (SFM) to investigate a number of specific DNA sequence elements that appear with high frequency in genomic regulatory regions and which recent evidence suggests may bend or flex. It is now widely accepted that sequence-directed bending and flexibility in DNA play important roles in chromatin structure, specific recognition by regulatory proteins and a variety of additional site-regulated transactional events including recombination. Although static bending in DNA due to d(A)n tracts has been studied for over a decade, more recent evidence has implicated additional sequence elements in static bending and has suggested that site-directed anisotropic flexibility, or dynamic bending, may also be an important factor in DNA function. In addition, specific ion, ionic strength and sequence context effects appear to complicate both the static and dynamic bending phenomena. Although much progress has been made, our understanding of the structural and physical chemical factors that govern static and dynamic bending is still severely limited. In the past, experimentally differentiating static from dynamic bending in a DNA sequence has never been unambiguous, and the quantitative study of dynamic bending in a DNA sequence has proved especially elusive. The proposed experiments will address the static/dynamic bending characteristics of sequence elements d(CA), d(TA)m, d(AG) and copolymer expansions d(CAC), d(CACA), d(TAT), d(TATA), d(AGA), d(AGAG), as well as static/dynamic bending sequences d(G)n(C)m(n,m = 1 to ~4) and d(A)n(n ~5), and effects of specific ions, ionic strengths and sequence context on these under fully hydrated conditions in solution. Concurrently with this, a number of specific protein binding sites that contain these sequence elements will be similarly investigated, both in the presence and absence of bound protein. The experimental approach will utilize a number of biochemical methods including polyacrylamide gel mobility, cyclization or ring closure studies and chemical probe methods, along with direct visualization of linear and axially strained and unstrained topologically flat ring oligomers containing these sequence elements using a remarkable new non-contact, high resolution scanning force microscope (MacMode SFM). Dr. Harrington has recently shown that adequate resolution is obtainable with this technology to distinguish abrupt bends and kinks in short DNA oligonucleotides and in axially strained microcircles with considerable latitude in specific ion and ionic strength conditions. Although initial experiments focus on DNA oligomers, the principal goal of the project is to expand the understanding of indirect recognition in specific nucleoprotein complexes. Thus, Dr. Harrington will also study the conformational behavior of these sequence elements in naturally occurring recognition sequences used by important regulatory proteins in nature.
| Balagurumoorthy, P; Lindsay, Stuart M; Harrington, Rodney E (2002) Atomic force microscopy reveals kinks in the p53 response element DNA. Biophys Chem 101-102:611-23 |
| Feng, X Z; Bash, R; Balagurumoorthy, P et al. (2000) Conformational transition in DNA on a cold surface. Nucleic Acids Res 28:593-6 |
| Nagaich, A K; Zhurkin, V B; Durell, S R et al. (1999) p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting. Proc Natl Acad Sci U S A 96:1875-80 |
| Dlakic, M; Harrington, R E (1998) DIAMOD: display and modeling of DNA bending. Bioinformatics 14:326-31 |
| Dlakic, M; Harrington, R E (1998) Unconventional helical phasing of repetitive DNA motifs reveals their relative bending contributions. Nucleic Acids Res 26:4274-9 |
