Automate SDM data acquisition and analysis
. SDM automation will be required for genome-wide analyses and will greatly increase the practicality of the method. The goal of this aim is to fullyeliminate the need for user input, once a micro-flow cell has been manually mounted to the stage.
Determine the unzipping signature for RNA Polymerase II (Pol II) transcription complexes
. Inthis aim, we will determine the in vitro Pol II unzipping signature for use in identifying native complexes inAim 3. Furthermore, new structural information about in vitro transcription complexes will be obtained.2.1
Unzip stalled Pol II in vitro transcription complexes
. Stalled complexes provided by theAdelman laboratory will be unzipped from both upstream and downstream directions. Individual
traces will be aligned and two reference ―signatures‖ will be produced: one for each sense or
antisense orientation of transcription.2.2
Atomistically model unzipping of protein-DNA complexes
. Molecular dynamics simulationsof the unzipping of naked DNA and a model protein-DNA system will be carried out. Results willguide structural interpretation of Pol II data from aim 2.1 and will be used to initiate a separate projectwith the long-term goal of full atomistic simulations of nucleosome and Pol II unzipping.
Map native yeast chromatin by single-molecule unzipping
. The ultimate goal of this proposal ishigh-resolution, single-molecule, site-specific mapping of nucleosome and polymerase positions on nativechromatin. Various independent approaches will be taken towards native chromatin unzipping in this aimand significant progress towards the overall goal will be achieved, independent of success of the other aims.3.1
Unzipping of yeast plasmid chromatin
. Yeast plasmid chromatin
will be isolated with andwithout induction of the
5 gene encoded in the plasmid
and ligated to unzipping constructs.Single-molecule unzipping will be used to determine the locations of the nucleosomes and comparedwith well-known locations from ensemble measurements.3.2
Unzipping of native yeast chromatin from a single genomic site
. Genomic chromatincontaining the YLR454 gene will be digested with I-SceI and ligated onto unzipping constructs.Nucleosome and polymerase positions will be measured by single-molecule unzipping. Chromatinstructure will be mapped in wild type, an spt16 mutant, a mutant deficient in histone H2Bubiquitylation, and the double mutant
. Correlated nucleosome and polymerase occupancy onindividual chromatin fibers will be measured.3.3
Shotgun chromatin mapping
. Yeast native chromatin will be digested with XhoI and randomfragments will be ligated onto unzipping constructs. Shotgun DNA mapping will be used to identifyindividual fragments. Nucleosome and polymerase positions will be identified at high resolution oneach individual fiber. High throughput experiments will reveal genome-wide correlations betweenpolymerases and histones and also identify divergent and cryptically initiated polymerases in wildtype, spt16 mutants, H2B-ubiquitylation mutants, and double mutants
New biophysical tools are needed for single-molecule analysis of native chromatin.
Success in thisproject will develop such tools and will answer fundamental open questions in eukaryotic transcription.Furthermore,
we will have developed a unique and powerful single-molecule genetics technique
that will enable many future high-impact research avenues. These may include studying single-moleculemapping of epigenetic chromatin marks, DNA damage repair and replication, structural genome mappingby unzipping, single-molecule telomere mapping, and studies of alternative splicing by single-moleculecDNA unzipping. Given this wealth of possibilities and the strong collaborations formed, this CAREERproject will lay a solid foundation for a successful research career.
CAREER: Single-Molecule Analysis of Genomic DNA and Chromatin in Eukaryotic TranscriptionProject Description, Page 2 of 15