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 chromatin 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
- 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
- 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).
- In collaboration with Pedro Rodriguez-Collazo (Cell Biology and Physiology), we have developed a method to isolate in one-step histones containing their native post-translational modifications (Rodriguez-Collazo et al., 2009). This method has also been patented and licensed.
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.
- Anand, S. P., Zheng, H., Bianco, P., Leuba, S. H. and Khan, S. A. DNA
helicase activity of PcrA is not required for the displacement of RecA from
DNA and inhibition of RecA-mediated strand exchange. J.
Bacteriology 189, 4502-4509, 2007.
- Bennink, ML,
SH Leuba, GH Leno, J Zlatanova, BG de Grooth & J Greve (2001)
Unfolding individual nucleosomes by stretching single chromatin fibers
with optical tweezers. Nature Struct. Biol. 8, 606-610.
- Karymov, MA,
M Tomschik, SH Leuba, P Caiafa & J Zlatanova (2001) DNA Methylation-dependent
chromatin fiber compaction in vivo and in vitro: Requirement for linker
histone. FASEB J. 15, 2631-2641.
- Leuba, SH, MA
Karymov, M Tomschik, R Ramjit, P Smith & J Zlatanova (2003) Assembly
of single chromatin fibers depends on the tension in the DNA molecule:
magnetic tweezers study. Proc. Natl. Acad. Sci. USA 100, 495-500.
- Tomschik, M,
MA Karymov, J Zlatanova & SH Leuba (2001) The archaeal histone-fold
protein HMf organizes DNA into bona fide chromatin fibers. Struct.
Fold. Des. 9, 1201-1211.
- Tomschik, M,
H Zheng, K van Holde, J Zlatanova & SH Leuba (2005) Fast, long-range,
reversible conformational fluctuations in nucleosomes revealed by
spFRET. Proc. Natl. Acad. Sci. USA 102, 3278-3283.
- Zlatanova, J
& SH Leuba, Eds. (2004) Chromatin Structure and Dynamics: State-of-the-Art.
New Comprehensive Biochemistry Vol. 39, Elsevier, Amsterdam, 507 pg.
- Rodriguez-Collazo P, Leuba SH, Zlatanova J. Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.Nucleic Acids Res. 2009 37:e81. Epub 2009 May 13.PMID: