Our research is investigating chromatin structure and function as revealed by single molecule approaches. Single molecule approaches (e.g. atomic force microscopy AFM, optical tweezers) can answer particular questions that are difficult (if not impossible) to answer by population-ensemble experiments (e.g. gel electrophoresis etc.). Our interests in chromatin are (i) linker and core histones (variants, stoichiometry) and their contributions to fiber structure (ii) post- translational modifications (acetylation, phosphorylation, ADP- ribosylation etc.) of histones affecting structure, (iii) effect of DNA methylation on fiber structure and (iv) interactions of sequence specific chromatin fibers or single nucleosomes with biological processes (e.g. polymerases and other chromatin remodeling factors).With AFM our approach is twofold: on one side there is the imaging of protein/DNA complexes (chromatin fibers of various composition, stoichiometry and post-translational modifications), and on the other side there is the direct manipulation with the AFM tip to probe the forces holding the chromatin fiber together. We have initiated the application of a new technique to study chromatin fibers - directly measuring the force of stretching single chromatin fibers with the atomic force microscope. Force is a factor in many processes involving chromatin and chromosome structural reorganizations during the life of any eukaryotic cell. Additionally, there is a need for force to clear histones from the DNA for biological processes such as transcription, replication and repair. Force generation and application to biological structure is a major component of dynamic biological processes, but forces governing chromatin structure and function have not been experimentally approached up to now. Pulling of synthetic chromatin fiber causes stretching of the linker DNA, but the nucleosomes stay on the fiber, even when forces of several hundred piconewtons are applied. Such forces are well beyond the capability of known molecular motors, which implies that chromatin structure must be weakened, possibly by chromatin remodeling factors, before nucleosomes can be removed. Our interests in the effect of force applied to chromatin fibers has lead us to collaborate with researchers at the University of Twente (The Netherlands) on applying optical tweezers to chromatin fibers. With the optical tweezers we can probe the lower region of forces 1-150 piconewtons whereas with the AFM we can probe forces above 100 piconewtons. In these optical tweezers experiments, we first attach a piece of DNA between two beads, demonstrate it is an intact single molecule of DNA that can undergo the well known B-DNA to Z-DNA transition, and then assemble histones onto DNA by injecting a nuclear assembly extract into the liquid cell of the instrument. These kinds of experiments open a whole new approach because with the nuclear extract it is possible to assemble chromatin with various complements of histones (i.e., only core histone H3/H4 tetramers, fluorescently modified histones, with or without linker histone subtypes, etc.). Preliminary results suggest that this system is sensitive enough to detect the disruption of single nucleosomes among the ~250 nucleosomes assembled on the 48,502 bp of lambda DNA. - Atomic force microscopy, single molecule detection and manipulation, chromatin structure, function and dynamics,

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
United States
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Zlatanova, J; Lindsay, S M; Leuba, S H (2000) Single molecule force spectroscopy in biology using the atomic force microscope. Prog Biophys Mol Biol 74:37-61
Leuba, S H; Bustamante, C (1999) Analysis of chromatin by scanning force microscopy. Methods Mol Biol 119:143-60
Zlatanova, J; Leuba, S H; van Holde, K (1999) Chromatin structure revisited. Crit Rev Eukaryot Gene Expr 9:245-55