DNA supercoiling is involved with virtually all DNA-related biochemical processes, therefore physical chemical and structural properties of supercoiled DNA have been important areas of DNA biophysics over last two decades. Recent experimental studies and advanced theoretical analyses suggest that DNA dynamics rather than static DNA structure plays a critical role in the control of gene expression, DNA replication, recombination, and repair. However, progress in experimental studies in this important area of DNA biophysics has been very low due to the lack of appropriate experimental techniques. Recent advances in single molecule studies, allowing the observation of structural dynamics of individual molecules, showed that the single molecule approaches could provide unique information on the structure and dynamics of biological molecules. Recent studies showed that local and global DNA conformations are in a dynamic equilibrium. Competing transitions between different local structures accompanied by dynamic changes of global DNA conformations may have a profound role in various DNA functions. This proposal utilizing single molecule AFM and fluorescence microscopy tests a number of critical hypotheses and provides a realistic means for breaking new ground in this undeveloped area of molecular biology. (1) The hypothesis that the formation of cruciforms and H-DNA can limit global DNA mobility affecting the juxtaposition of distantly separated DNA regions will be tested. (2) The hypothesis that conversion of one local structure into another is an alternative molecular switch mechanism for the global DNA structure and dynamics (3) The mechanism of binding of the restriction enzyme SfiI involving the interaction of distant DNA regions will be analyzed and the hypothesis that the formation of local structures may regulate the enzyme activity will be tested. In addition to the hypotheses testing, novel DNA immobilization and labeling techniques for DNA dynamics studies will be developed. Altogether, the proposed studies are the first attempt to look at the dynamics of DNA at various levels providing spatial and temporal information about the fundamental and functional properties of DNA. Advances in sample preparation techniques combined with corresponding instrument development will provide structural biologists pursuing single molecule approaches with novel tools for applications, ramifications considerably wider than the objectives outlined in this proposal.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM062235-04
Application #
6720889
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lewis, Catherine D
Project Start
2000-12-18
Project End
2004-06-30
Budget Start
2004-04-01
Budget End
2004-06-30
Support Year
4
Fiscal Year
2004
Total Cost
$64,107
Indirect Cost
Name
Arizona State University-Tempe Campus
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
943360412
City
Tempe
State
AZ
Country
United States
Zip Code
85287
Lyubchenko, Yuri L; Shlyakhtenko, Luda S; Ando, Toshio (2011) Imaging of nucleic acids with atomic force microscopy. Methods 54:274-83
Lyubchenko, Yuri L (2011) Preparation of DNA and nucleoprotein samples for AFM imaging. Micron 42:196-206
Zaremba, Mindaugas; Owsicka, Amelia; Tamulaitis, Gintautas et al. (2010) DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme. Nucleic Acids Res 38:7142-54
Gilmore, Jamie L; Suzuki, Yuki; Tamulaitis, Gintautas et al. (2009) Single-molecule dynamics of the DNA-EcoRII protein complexes revealed with high-speed atomic force microscopy. Biochemistry 48:10492-8
Shlyakhtenko, Luda S; Lushnikov, Alexander Y; Lyubchenko, Yuri L (2009) Dynamics of nucleosomes revealed by time-lapse atomic force microscopy. Biochemistry 48:7842-8
Lyubchenko, Yuri L; Shlyakhtenko, Luda S (2009) AFM for analysis of structure and dynamics of DNA and protein-DNA complexes. Methods 47:206-13
Shlyakhtenko, Luda S; Gilmore, Jamie; Kriatchko, Aleksei N et al. (2009) Molecular mechanism underlying RAG1/RAG2 synaptic complex formation. J Biol Chem 284:20956-65
Karymov, Mikhail A; Chinnaraj, Mathivanan; Bogdanov, Aleksey et al. (2008) Structure, dynamics, and branch migration of a DNA Holliday junction: a single-molecule fluorescence and modeling study. Biophys J 95:4372-83
Shlyakhtenko, Luda S; Yuan, Bin; Emadi, Sharareh et al. (2007) Single-molecule selection and recovery of structure-specific antibodies using atomic force microscopy. Nanomedicine 3:192-7
Shlyakhtenko, Luda S; Gilmore, Jamie; Portillo, Alex et al. (2007) Direct visualization of the EcoRII-DNA triple synaptic complex by atomic force microscopy. Biochemistry 46:11128-36

Showing the most recent 10 out of 34 publications