1. EXPRESSION AND CHARACTERIZATION OF THE C-TERMINAL
DOMAIN OF THE YEAST PRIMASE REGULATORY SUBUNIT
Durbis Javier Castillo-Pazos
Vanderbilt International Summer Research Academy 2016
Walter Chazin’s Lab
VANDERBILT UNIVERSITY
BIOCHEMISTRY DEPARTMENT
CENTER FOR STRUCTURAL BIOLOGY
2. DNA REPLICATION IS ESSENTIAL FOR LIFE
Replication fork
Initiation, elongation,
termination
Pol α –Primase synthesizes
primer
Pol-ε or Pol-δ elongate
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Burgers, 2008
3. PRIMASE IS AN RNA POLYMERASE
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Replicative polymerases need a
primer
Primase synthesizes an 8-10 NT
primer
Cluster [4Fe-4S]
p58C yeast subdomain
Image from Lauren
Salay
4. PRIMASE CAN ENGAGE IN DNA CHARGE TRANSPORT
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PDB ID: 309Q
Service, 2014
5. WHAT IS A CHEMIST DOING HERE?
Generate yeast p58C in E. coli
Mutate homologous tyrosines
Characterize and validate mutants
Learn about structural biology
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11. FPLC FRACTIONS OF WT AND MUTANTS
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Wild
Type
Y395F
Ladder
Y397F
Ladder
12. SECONDARY STRUCTURE ANALYSIS BY CIRCULAR DICHROISM
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University of Leeds
Aarhus University
13. WT AND MUTANTS ARE PRIMARILY HELICAL
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Cloning
Mutation
Expression and
purification
Characterization
14. FLUORESCENCE ANISOTROPY IS USED TO MEASURE INTERACTIONS
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Toseland, 2014
15. MUTANTS BIND DNA SIMILARLY TO WT
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Cloning
Mutation
Expression and
purification
Characterization
16. CONCLUSIONS AND FUTURE DIRECTIONS
We can express and purify wild type, Y395F, and Y397F mutants of
yeast p58C from E. coli
The mutations do not alter the secondary structure distribution
The mutations do not grossly alter the DNA-binding abilities
Proteins will be sent to the Barton Group for electrochemical
analysis
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17. ACKNOWLEDGEMENTS
Walter Chazin, Lauren Salay
Chazin Lab Members
Kathy, Leslie, and Beth
VISRA and VISP students
Dr. Kathy Friedman
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18. REFERENCES
Burgers, P.M.J., 2009. Polymerase dynamics at the eukaryotic DNA replication fork. Journal of Biological
Chemistry, 284(7), pp.4041–4045.
Garg, P. & Burgers, P.M.J., 2016. DNA Polymerases that Propagate the Eukaryotic DNA Replication Fork
DNA Polymerases that Propagate the Eukaryotic DNA Replication Fork. , 9238(June).
Grodick, M.A. et al., 2014. DNA-Mediated Signaling by Proteins with 4Fe − 4S Clusters Is Necessary for
Genomic Integrity.
Sauguet, L. et al., 2010. Shared Active Site Architecture between the Large Subunit of Eukaryotic Primase
and DNA Photolyase. , 5(4).
Weiner, B.E. et al., 2007. An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human
DNA primase. Journal of Biological Chemistry, 282(46), pp.33444–33451.
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19. YEAST p58C IS HOMOLOGOUS TO HUMAN p58C
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Editor's Notes
Cell process
In this animation
Here’s a static figure
Polymerases
Pol alpha primer de novo
Pol delta or épsilon elongate
My Project has been focused on primase
Primase is made out of two
Primase synthesizes
We’re specifically inyerested in
Something interesting is cluster
Dna charge transport is a process where redox species
Barton shown may play important role coordination dna processing proteins
This is a crystal structrure
Previous work has shown tyr residues
This is a gel image that shows the PCR product
Agarose gel weight
After we obtained amplification We performed digestión, ligation and further transformation
Here we see another gel image that shows us a succesful insertion
After this we performed site directed mutagenesis
We changed two tyr residues
We sent them for sequencing
We performed a three step purification
We passed our his tagged protein through
Once cleaved
Heparin column fractions collected
Optimized Pellegrini
This is a gel image SDS-PAGE
This is a gel image SDS PAGE run for
For the characterization stage
Here we can see a basic diagram of how CD Works
This is a plot with the basic shapes
This is the result of our cd analysis
We plotted elipticity and wavelength
As we can appreciate here mutants plot almost perfectly matches the pattern of wt
We appreciate that are primarily helical minima at 208 222 nm
Another technique that we used to make sure
We compare the interaction of light with a bound not bound species
Equilibrium plays a role in the signal we obtain
This is a fluorescence anisotropy plot, where the three lines plotted show a similar behavior
WT and 395 kd 17 397 23
All of these lead us to the conclusions of this summer are
