Testing Polymerase Activity and Fidelity through Fluorescent Reporters

1. Testing Polymerase Activity and Fidelity through Fluorescent Reporters Camille Vasquez DNA Damage Tolerance Lab Mentor: Dr. Penny Beuning December 18, 2018

2. Causes and types of DNA damage • DNA damage occurs from exogenous and endogenous factors • Air pollution, UV light, respiration • Cause reactive oxygen species (ROS) • Damage and replication • 8-oxoG mutagenic lesion • Interaction between ROS and guanine • Causes misincorporation of A instead of C Guanine-cytosine pairing 8-oxoG-adenine pairing Aguiar, P. et al. “Oxidative Stress and DNA Lesions: The Role of 8-Oxoguanine Lesions in Trypanosoma cruzi Cell Viability”. Neglected Tropical Diseases. (2013). 7(6): 2279 • Aseervatham, G. et al. “Environmental factors and unhealthy lifestyle influence oxidative stress in humans—an overview”. Environmental Science and Pollution Research. (2013). 20(7): 4356-4369

3. Engineering Taq Polymerase III for lesion bypass • Thermus aquaticus • Thermophile • C-Family Polymerases • 2 types of polymerases • TLS-capable • Replicative Waters, L., Minesinger, B., Wiltrout, M, S. D'Souza, S., Woodruff, R. and G. C. Walker. “Eukaryotic Translesion Polymerases and Their Roles and Regulation in DNA Damage Tolerance”. Microbiology and Molecular Biology Reviews (2009). 73: 134-154. McCulloch, S. and T. A. Kunkel. “The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases. ” Cell Res. (2008). 18: 148-161.

4. DNA at a crime scene is oxidatively damaged, thus difficult to analyze Goal: Engineer a Taq Pol III that can replicate accurately and efficiently over 8-oxoG damage Taq Engineering Project • Taq Expression • Fidelity Assay • FQ Assay

5. Fidelity Assay: Low Throughput Competitor Strand LacZ Correct LacZ Tindall, K.R. & Kunkel, T.A.. Fidelity of DNA synthesis by the Thermusaquaticus DNA polymerase. Biochemistry. 1988; 27: 6008-6013 Jozwiakowski SK, Connolly BA. Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase. Nucleic Acids Research. 2009;37(15). Pol Transformation Pol • Nick LacZ • Introduce • Competitor strand • Pol of interest • Transformation • Count Blue:White colony ratio Functional LacZ: Blue Colonies Incorrect LacZ Transformation Non-Functional LacZ: White Colonies Tindall, K.R., and T.A. Kunkel. “Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase.” Biochemistry. (1988). 27: 6008-6013 Jozwiakowski S.K., and B.A. Connolly. “Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase.” Nucleic Acids Research. (2009). 37(15).

6. Fidelity Assay using GFP and mCherry Competitor Strand • Insert fluorescent genes (mCherry and GFP) • Test Pol for normal gapped • Test Pol for 8-oxoG damage Nicked mCherry mCherry GFP Full mCherry Increase Heat Pol Competitor Strand Competitor Strand Nicked mCherry mCherry Nicked mCherry Full mCherry GFP x x 8-oxoG oligonucleotides x 8-oxoG 8-oxoG 8-oxoG Increase Heat Pol

7. Testing fidelity in a high-throughput way • GFP acts as internal control • Highest Fidelity = 1:1 signaling • Unsuccessful • Successful RFU RT-PCR Transformation Induce GFP mCherry RFU Transformation RT-PCR Induce GFP mCherry

8. Fidelity Assay: Site-Directed Mutagenesis • Mutations using QuikChange Protocol • Silent mutation in mCherry (c→t) • Used as nicking site • Success! • mCherry-Stop • Negative control • Success (produces white colonies) • Subcloning into pETduet • In progress

9. Using Template/Quencher to Test Activity Step 4 Step 3 Step 1 Step 2 Q F Increase temp Q Active = Fluorescence! F F Decrease temp Q ≈ F Active Polymerase = Fluorescence! Or Q Inactive= No Fluorescence F Inactive= No Fluorescence

10. Determining optimal FQ assay conditions • RT- PCR • No Pol Test • Mixing template and quencher • Raise and lower temp (increase and decrease fluorescence) • Find melting/annealing temp • Determining optimal concentrations • LB vs. SF water • Dpo4 Q F Increase temp Q F Increase temp Q F Decrease temp Q F Larsen, Andrew C., et al. "A general strategy for expanding polymerase function by droplet microfluidics." Nature communications 7 (2016): 11235.

11. Determining how various samples affect fluorescence • Comparing fluorescent signaling to quenched

12. Fluorescence throughout melting curves with quencher • Increasing 1.5x Iowa dampens signal • Added Dpo4 • Did not remain fluorescence = Unsuccessful in amplifying

13. Future Steps of the Project • Subcloning • Finish GFP + mCherry into pETduet • Introduce 8-oxoG lesion • Create ssDNA competitor strand • F-Q Assay • Test on different active polymerases • Test on Taq Polymerase III • Determine extension times • Determine optimal temperature

14. Skills I have developed on Co-op • Make buffers • Design and prepare primers • Use a spectrometer to measure optical density • Modify protocols to improve results • RT-PCR to detect fluorescence • Accurately document data in lab notebook • Present scientific data • Read scientific papers and extract essential information • Pipetting • Streak bacteria on plates • Transformations • Make overnight cultures • Miniprep to extract DNA • Site-directed mutagenesis • PCR • Make frozen stocks • Sequence DNA • Make agarose gels • Run gel-electrophoresis

15. Acknowledgements • Jim McIsaac • Sam Watson • Melissa Liriano • Sarah Fields • Israel Adam • Lura Slowinski • Dr. AkramAlshawabkeh • ROUTES Program • Dr. Penny Beuning • Lisa Ngu, Ph.D • Caitlyn Mills, Ph.D • Jenifer Winters, Ph.D • Tim Coulther • Lydia Ruffner • Hannah Stern • Amy Blondin Hotchkiss