Determination of the trajectory of linker DNA by fluorescence energy transfer distance measurements
Scherl, Dale S.
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Permalink
https://hdl.handle.net/2142/19478
Description
Title
Determination of the trajectory of linker DNA by fluorescence energy transfer distance measurements
Author(s)
Scherl, Dale S.
Issue Date
1993
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2011-05-07T12:08:47Z
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Language
eng
Abstract
We have used fluorescence energy transfer distance measurements to study the trajectory of linker DNA as it enters and exits the histone octamer in a nucleosome core particle. Measurement of the DNA-to-protein distances gives important structural information about the most fundamental level of chromosomal organization, and allows speculation about higher order compaction.
The implementation of this method required the reconstitution of a nucleosome core particle from natural and synthetic component molecules. The histone octamers are extracted and purified from chicken erythrocytes, specifically labeled with a fluorescent dye (one of a fluorescent energy transfer pair) and further purified. The DNA is produced using polymerase chain reaction (PCR) with fluorescently labeled and HPLC the purified synthetic primers, with template DNA that is extracted, cleaved with restriction enzymes, and HPLC purified from source DNA cloned into E. coli. This template DNA is the 256bp phasing sequence from the 5S RNA gene of Lytechinus variegatus, which allows reconstitution of phased nucleosome core particles. After reconstitution, characterization included fluorescence energy transfer distance measurements as well as HPLC, sedimentation, and enzymatic digests to insure structural integrity.
The distance between the protein and a linker DNA end is related to the distance between the attached dye molecules and can be determined by measurement of the energy transfer between this pair. Fluorescence lifetime, anisotropy, and quantum yield measurements are used to determine the magnitude of this transfer, which is proportional to the inverse 1/6$\sp{\rm th}$ power of the distance. These measurements, made as a function of the linker DNA length, indicate the linker DNA entrance and exit trajectory and directly relates to the global architecture of the initial steps in the condensation of a chromosome.
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