Single-molecule spectroscopy of chromatin & molecular motors

Since the discovery of the nucleosome in the early 1970's, scientists have sought to correlate chromatin structure and dynamics with biological function. More recently, we have learned that nucleosomes and chromatin play a critical role in the regulation of transcription, replication, recombination, and repair (Zlatanova and Leuba, 2004);. Our laboratory uses an interdisciplinary approach combining the disciplines of molecular biology, biochemistry, engineering, and physics to try to understand at the single nucleosome and single chromatin fiber level how chromatin structure and dynamics regulate biological processes that use DNA as a template. To this end, we are applying several single-molecule approaches such as atomic force microscopy (AFM);, magnetic tweezers, optical tweezers and single-pair fluorescence resonance energy transfer (spFRET); to native or reconstituted chromatin fibers of different protein compositions with the latter three methods using homebuilt instrumentation. Single-molecule techniques provide the sensitivity to detect and to elucidate small, yet physiologically relevant, changes in chromatin structure and dynamics. Recent examples of what we have been able to discover include the following:

• We have been able to use AFM to detect conformational changes in chrmatin fiber structure due to the presence of 24 methyl groups per nucleosome (Karymov et al., 2001); implying that the combined action of the DNA methylation and linker histone binding required to compact chromatin may affect the transcription of large chromatin domains.
• We also used AFM to investigate the role of histone variants in chromatin fiber structure (Tomschik et al., 2001);. Eukaryl and archaeal organisms have similar fiber structure with differences likely related to the more complex needs of eukaryl organisms to regulate transcription.
• We have used optical tweezers to determine the piconewton forces necessary to unravel individual nucleosomes in a fiber context (Bennink et al., 2001); and found that the measured forces for individual nucleosome disruptions are in the same range of forces reported to be exerted by RNA- and DNA-polymerases.
• We have used magnetic tweezers to observe a dynamic equilibrium between force dependent nucleosomal assembly and disassembly on a single DNA molecule in real time (Leuba et al., 2003); as a model of what happens to nucleosomes when a transcribing polymerase passes through the region where they are located.
• We have used spFRET to demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed); with the DNA wrapped around the histone core, and open, with the DNA significantly unraveled from the histone octamer (Tomschik et al., 2005);, implying that most of the DNA on the nucleosome can be sporadically accessible to regulatory proteins and proteins that track the DNA double helix.
• In collaboration with Saleem Khan (Microbiology and Molecular Genetics), we have used spFRET to demonstrate that PcrA DNA helicase displaces RecA from both ssDNA as well as dsDNA as a model for regulation of homologous recombination (Anand et al., 2007).

Our future goals are to build combination single-molecule instruments to image and manipulate intramolecular nanometer movements in submillisecond real-time with piconewton force sensitivity (e.g., we want to observe directly what happens to the histones in a nucleosome in the path of a transcribing polymerase);. We want to observe what changes in superhelicity occur upon nucleosome formation, nucleosome by nucleosome. We hope to resolve whether the positive supercoils generated by a transcribing polymerase are sufficient to displace histone octamers. In addition to chromatin, we are studying the mechanism of action of individual helicases unwinding DNA. We are also working on the capability to observe in real time single nucleosome dynamics in living cells.

Education

PhD 1993, Oregon State University

Postdoctoral Training

1994-1997, Institute of Molecular Biology, University of Oregon
1997-1998, Arizona State University
1998-2002, Laboratory of Receptor Biology & Gene Expression, NIH, NCI 

Department of Cell Biology and Physiology
University of Pittsburgh 
2.26g Hillman Cancer Center
UPCI Research Pavilion
5117 Centre Avenue
Pittsburgh, PA 15213-1863

Phone: (412) 624-8770
Fax: (412) 623-4840

E-mail: leuba@pitt.edu 

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