VISRA Talk

EXPRESSION AND CHARACTERIZATION OF THE C-TERMINAL DOMAIN OF THE YEAST PRIMASE REGULATORY SUBUNIT

Durbis Javier Castillo-Pazos

Vanderbilt International Summer Research Academy 2016Walter Chazin’s Lab

VANDERBILT UNIVERSITYBIOCHEMISTRY DEPARTMENTCENTER FOR STRUCTURAL BIOLOGY

05/01/2023 2

DNA REPLICATION IS ESSENTIAL FOR LIFEReplication fork

Initiation, elongation, termination

Pol α –Primase synthesizes primer

Pol-ε or Pol-δ elongateVISRA SYMPOSIUM | DURBIS JAVIER CASTILLO PAZOS | SUMMER 2016

Burgers, 2008

05/01/2023VISRA SYMPOSIUM | DURBIS JAVIER CASTILLO PAZOS | SUMMER 2016 3

PRIMASE IS AN RNA POLYMERASE

Replicative polymerases need a primer

Primase synthesizes an 8-10 NT primer

Cluster [4Fe-4S]

p58C yeast subdomainImage from Lauren Salay


PRIMASE CAN ENGAGE IN DNA CHARGE TRANSPORT

PDB ID: 309Q

Service, 2014


WHAT IS A CHEMIST DOING HERE?

Generate yeast p58C in E. coliMutate homologous tyrosinesCharacterize and validate mutants

Learn about structural biology


GENERAL WORKFLOW

Cloning

Mutation

Expression and purification

Characterization


SUCCESSFUL GENERATION OF YEAST p58C

Cloning

MutationExpression and purification

Characterization PCR Amplicon


SUCCESSFUL MUTAGENESIS OF HOMOLOGOUS TYROSINES

Cloning

Mutation

Expression and purification

Characterization

Y397F

Y395F


p58C PURIFICATION SCHEME

NiNTA colum

nReflo

w

Heparin

column

Cloning


Characterization

05/01/2023 10

INDICATIONS OF PURIFIED PROTEIN – NiNTA STEPS

VISRA SYMPOSIUM | DURBIS JAVIER CASTILLO PAZOS | SUMMER 2016

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1) Ladder 2) Lysate WT3) Flow-through WT4) Wash WT5) Elution WT6) Repass FT WT7) Repass E WT8) Flow-through 3959) Wash 39510) Elution 39511) Repass FT 39512) Repass E 39513) Flow-through 39714) Wash 39715) Elution 39716) Repass FT 39717) Repass E 397


FPLC FRACTIONS OF WT AND MUTANTSWild Type Y395F

Ladd

er

Y397F

Ladd

er


SECONDARY STRUCTURE ANALYSIS BY CIRCULAR DICHROISM

University of Leeds Aarhus University


WT AND MUTANTS ARE PRIMARILY HELICAL

Cloning


Characterization


FLUORESCENCE ANISOTROPY IS USED TO MEASURE INTERACTIONS

Toseland, 2014


MUTANTS BIND DNA SIMILARLY TO WT

Cloning


Characterization


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 distributionThe mutations do not grossly alter the DNA-binding abilities

Proteins will be sent to the Barton Group for electrochemical analysis


ACKNOWLEDGEMENTS

Walter Chazin, Lauren SalayChazin Lab MembersKathy, Leslie, and BethVISRA and VISP studentsDr. Kathy Friedman


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.


YEAST p58C IS HOMOLOGOUS TO HUMAN p58C

VISRA Talk

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