DNA Polymerase Theta Activity Assay Kit

Background

DNA polymerase theta (Pol θ) is involved in an end-joining pathway of DNA double-strand breaks. Over expression of Pol θ is found in many cancers, including stomach, colon, breast and lung cancers, and correlated with poorer patient survival. Because suppression of gene expression of Pol θ results in sensitivity of cells to ionizing radiation and some DSB-inducing drugs, Pol θ is a validated anti-cancer drug target. Description The Aurora DNA Polymerase Theta activity assay kit is a homogeneous fluorescence-based assay for screening inhibitors that block DNA polymerase activity of DNA Pol θ. The assay is fast, convenient, and requires just two steps. In the first step, the DNA Pol θ enzyme synthesizes double-stranded DNA using a DNA template in the presence of dNTP. In the second step, a dye that binds to double-stranded DNA is added to the solution resulting in the increase of fluorescence, intensity of which can be measured with a fluorescent plate reader at the excitation wavelengths of 495 nm and emission wavelengths of 525 nm. Materials supplied Catalogue Number 362201 362204 4687 362003 4930 362202 Item 2X Assay Buffer 20 µM DNA template 10 mM dNTP Recombinant DNA Pol θ CTD Dye solution Stop solution Black low binding 96 well plate Amount 25 mL 7 µL 5 µL 5 µL 15 µL 3 mL 1 Storage -20°C -20°C -20°C -80°C -20°C -20°C RT Materials Needed but not supplied A microplate reader capable of measuring fluorescence at excitation wavelengths of 495 nm and emission wavelengths of 525 nm. 1. 0.5 M DTT 2. Adjustable micro-pipettor 3. Sterile Tips Stability 12 months if stored under the indicated conditions. Aurora Biolabs LLC, San Diego, CA 92121, USA. www.aurorabiolabs.com, 1

Materials Not Supplied

• 0.5 M DTT
• Adjustable micro-pipettor
• Sterile Tips
• Prepare 1X buffer containing 1 mM DTT.
• Prepare the inhibitor compound solution.
• Prepare DNA Pol Theta solution.
• Add the inhibitor solution
• Incubate at room temperature for 30 minutes (optional).
• Prepare substrate solution
• Incubate the plate at 30°C for 1 hour.
• Prepare dye solution
• Incubate at room temperature for 30 minutes.
• Measure the fluorescent intensity

Assay Protocol

1. Step 1. Prepare the inhibitor compound solution. If the inhibitor compound is dissolved in water, make a solution of the compound 5-fold higher than the final concentration in 1X assay buffer (since you will add 5 µl to the 25 µl reaction). Then make a series of dilutions in 1X assay buffer from this solution to your preferred concentrations. If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 20-fold dilution in 1X assay buffer (at this step, the compound concentration is 5-fold higher than the final concentration and the DMSO concentration is 5%). Then make a series of dilutions in 5% of DMSO from this solution to your preferred concentrations. Since 5 µl of the compound solution will be added to the 25 µl reaction, the final concentration of DMSO in all of reactions is 1%.
2. Step 2. Prepare DNA Pol Theta solution. Thaw DNA Pol θ CTD enzyme (catalogue number 362003) on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: DNA Pol θ CTD enzyme is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted enzyme. Dilute DNA Pol Theta enzyme 650-fold (1:650) in 1X assay buffer with 1 mM DTT. Add 10 µl of diluted enzyme solution to each of positive control well and inhibitor test well. Add 1X buffer to each of background well.
3. Step 3. Add the inhibitor solution Add 5 µl of 1X assay buffer to each background well and positive control well if the inhibitor is diluted in 1X buffer. Add 5 µl of 1X assay buffer with 5% DMSO to each of background well and positive control well if the inhibitor is diluted in 1X buffer with 10% DMSO. Add 5 µl of diluted inhibitor solution from Step 2 to each of the inhibitor test well.
4. Step 4. Incubate at room temperature for 30 minutes (optional).
5. Step 5. Prepare substrate solution During the incubation of the enzyme and the inhibitor solution, prepare substrate solution containing 0.125 µM DNA template (dilute from 20 µM DNA template, catalogue number 362004) and 25 µM dNTP (dilute from 10 mM dNTP) in 1X assay buffer. Make only enough solution as need for the assay. Store the remaining 20 µM DNA template and 10 mM dNTP solution to -20°C. Add 10 µl of the substrate solution to each of well, including background wells, positive control wells and the inhibitor test wells.
6. Step 6. Incubate the plate at 30°C for 1 hour.
7. Step 7. Prepare dye solution Dilute the Dye solution 200-fold in Stop solution (catalogue number 362202). Make only enough solution as need for the assay. Store the remaining Dye solution to -20°C. Add 25 µl the Dye solution to each well.
8. Step 8. Incubate at room temperature for 30 minutes.
9. Step 9. Measure the fluorescent intensity Measure the fluorescent intensity at the excitation wavelengths of 495 nm and the emission wavelengths of 525 nm. Positive Control Inhibitor Test

Data Analysis

Calculate percentage activity of the enzyme % Activity= (Fp – Fb) – (Fi-Fb) Fp - Fb X 100 Where Fp refers to fluorescent intensity of the positive control, Fb refers to fluorescent intensity of background, and Fi refers to fluorescent intensity of the inhibitor. Graph the percentage activity as a function of the inhibitor concentration to determine the IC50 of the test inhibitor. No CPD refers to no compound control (compound vehicle only).

Assay Validation

Biological Pathway / Process

DNA Double-Strand Break Repair (End-Joining TMEJ)

Therapeutic / Disease Area

Oncology (POLQ-overexpressing cancers: breast; lung; colon; stomach)