Download REPSA-Directed Identification of DNA

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REPSA-Directed Identification of DNA-Binding Specificity
for Orphan Transcription Factors
Kamir Hiam
Van Dyke Lab
Dept. of Biochemistry
KSU Symposium of Student Scholars 2015
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The Power of Modern Genetics
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Genetic Sequencing  Raw Data
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Series of bases ≠ cellular function
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Further experimentation &
data analysis needed
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Central Dogma of Biology
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DNARNAProtein
Proteins responsible for the
majority of cellular activities
Transcription Factors are proteins
that bind to DNA to regulate
expression
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The Incomplete Tale of E. coli
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Complete genome sequenced
in 1997 (4.6+ Mbp)
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4290 Open Reading Frames
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240 potential TFs
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Detailed binding profiles for
only 68
Understanding orphan
regulatory proteins can
improve public health
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Microbial disease
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Human microbiome
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REPSA and Combinatorial Methods
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All combinatorial methods use large
pools of randomized oligonucleotides
difference in selection
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(A) CASTing involves physical
separation of bound protein-DNA
complex
requires knowledge of protein
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(B) REPSA involves protein protection
of enzymatic cleavage
no prior knowledge of protein needed
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REPSA optimal technique for
transcription factor discovery
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A Closer Look at REPSA Selection
Type IIS Restriction Enzymes cleave DNA at a defined distance from their recognition sites
Selection Template (68 bp)
5’ - CTAGGAATTCGTGCAGAGGTGAAT NNNNNNNNNNNNNNNNNNNN TTA CATCC CTCCAG AAGCTTGGAC –
3’
3’ - GATCCTTAAGCACGTCTCCACTTA NNNNNNNNNNNNNNNNNNNN AAT GTAGG GAGGTC TTCGAACCTG –
BsgI HphI
FokI
BpmI
5’
+ Orphan TF Specificity in the Van Dyke Lab
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General Experimental Procedure
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Clone bacteria (E. coli K12) with gene of interest
Induce protein expression
Perform combinatorial selection for sequence
specificity (REPSA)
Sequence final pool of DNA
Analyze data for consensus sequence
Verify, Hypothesize, & Publish
Repeat!
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LexA as model protein for REPSA
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Previously characterized repressor protein
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SOS response regulator
Known consensus binding sequence
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DNase I protection
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EMSA
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Weight Matrix Studies
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Combinatorial methods never used
Ideal candidate for REPSA protocol
optimization
Overall structure of E. coli LexA-DNA complex
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Representative Round of REPSA
Protein/
No Enzyme
No Protein/
Enzyme
Protein/
Enzyme
6 Rounds
PCR
9 Rounds
PCR
12 Rounds
PCR
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Unexpected Consensus Sequences
Round 7: 553/1000 sequences
e=4.0e-290
• What are structural features of
T-rich sequences?
• Why would this inhibit cleavage?
Round 14: 923/1000 sequences
e=9.3e-2093
• Doesn’t this sequence look
familiar?
• Why would this inhibit cleavage?
According to previous literature, FokI cleaves DNA without regard for sequence
specificity… Yet we appear to have found violations of this rule.
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Future Direction
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LexA Specificity Studies
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Examine Orphan Transcription Factors
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Work out issues with protein binding
In E. coli and other organisms such as extremophiles
Mixtures of Transcription Factors
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Develop REPSA techniques to handle multiple TFs at one time
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Ultimate goal to work with less pure cellular extracts
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Acknowledgements
Van Dyke Lab Members
NSF STEM Scholarship
LSAMP Scholarship
OVPR
KSU Foundation
NIH