Cell Biology Annual Report 2010-11 (FY 2011) - Department of Cell ...

CBP Faculty Research Summaries
Cell Biology and Physiology
Annual Report
(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 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 (Molecular Genetics and Biochemistry), we have used
spFRET to demonstrate that PcrA DNA helicase displaces RecA from both ssDNA as well as
dsDNA (Anand et al., 2007), as a model for regulation of homologous recombination.
- In collaboration with Pedro Rodriguez-Collazo (Cell Biology), 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.
- In collaboration with Michael Trakselis (Chemistry), we have used spFRET to demonstrate the
wrapping of DNA around the archaeal homohexameric MCM helicase from Sulfolobus
solfataricus (Graham et al., 2011), protecting the displaced single-stranded DNA tail and
preventing reannealing.
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- In collaboration with Paul Sammak (Cell Biology) we have developed methods for