Explore POLQ in the DNA Damage Response (DDR) in this Free eBook

POLQ Helicase ATPase Assay System

The POLQ Helicase ATPase Assay System includes POLQ helicase (DNA polymerase theta) enzyme, substrate, assay plates, and buffers required for a POLQ enzymatic reaction. The Transcreener ADP Assay (sold separately), a far-red competitive assay designed for HTS and inhibitor dose-response measurements, measures ADP produced by ATP hydrolysis to determine POLQ activity. 

POLQ is synthetically lethal with BRCA-1 and ATM mutations, and both the polymerase and ATPase activities are being targeted with small molecules for anti-cancer drug discovery. The POLQ Helicase ATPase Assay System, when used with the Transcreener ADP Assay Kit, provides all reagents required to screen and profile POLQ inhibitors.

The Transcreener ADP Assay is not included in the assay system but is available for separate purchase through BellBrook Labs. For ADP assay ordering information, please view Transcreener ADP Assay. The Transcreener ADP Assay is available with an FP, FI, or TR-FRET configuration. When purchasing the POLQ helicase assay system, it is important to choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener ADP Assay Kit.

The Comprehensive Approach to Measure POLQ Enzymatic Activity

POLQ Assay System Schematic

Assay System includes enzyme, substrate, & buffers for the POLQ reaction. Transcreener's ADP Assay measures corresponding POLQ activity.

An Outline of the Complete Protocol

The POLQ enzyme reaction is initiated by the addition of DNA substrate and ATP. After the enzyme reaction is completed, ADP detection reagents are added (Transcreener ADP2 Antibody and Tracer).

POLQ Protocol Outline

Robust Assay Yields Quality Data

The POLQ Helicase ATPase Assay System used with the Transcreener ADP assay produces robust results amenable to HTS. Z' measurement using optimized POLQ assay conditions (n=12). Z' = 0.93.

Z' for POLQ Assay System

Detection of POLQ Under Initial Velocity - FP Readout

Conversion of raw data to ADP formed using a standard curve demonstrates assay linearity. Here, we use 50 µM ATP with 100 nM POLQ ssDNA substrate. The enzyme buffer included 50 mM Tris (pH 7.5), 1 mM MgCl2, and 0.01% Triton. The POLQ enzyme reaction took place for 60 minutes at 30°C. The ADP Detection Mix included 1X Stop & Detect Buffer B, 4 nM ADP2 Alexa Fluor 633 Tracer, and 55 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature.

POLQ Enzyme Titration

POLQ Assay System Enzyme Titration

Linear Response

POLQ Linear Response Curve

Screen for Inhibitors & Perform SAR

Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow-up SAR can also be performed using the assay to determine inhibitor potency with ease. Here, 1280 compounds were screened from the Tocris 2.0 Library set using the Transcreener ADP2 FP Assay. Assay conditions included 0.5 nM POLQ helicase, 50 μM ATP, 100 nM 30-bp-ssDNA oligomer, 50 mM Tris, 1 mM MgCl2, 0.01% Triton (pH 7.5), 1X Stop & Detect Buffer B, 4 nM ADP2 AlexaFluor 633 Tracer, and 55 μg/mL ADP2 Antibody.

Pilot & Interference Screen

POLQ Pilot and Interference Screen

Dose Response Curve

POLQ Dose Response with CBA and Suramin

IC50 [CBA] = 1.56

IC50 [Suramin] = 0.92

Three Fluorescent Configurations Available

Choose the readout configuration that is the best fit for your lab based on preference and plate reader compatibility. 

Fluorescent Intensity

POLQ Assay System Titration FI Readout

FI Detection Mix (10 μL): 1X Stop & Detect Buffer B, 50 μg/mL ADP2 Antibody-IRDye QC-1, 8 nM ADP2 AlexaFluor 594 Tracer.

TR-FRET

POLQ Assay System Titration TR-FRET Data

TR-FRET Detection Mix (10 μL): 1X Stop & Detect Buffer C, 8 nM ADP2 Antibody-Terbium Conjugate, and 500 nM ADP HiLyte647 Tracer.

Overview of the Transcreener ADP Assay (Sold Separately)

ADP Assay Schematic

The Transcreener® ADP2 Assay is a far-red, competitive assay that measures ADP production to determine enzymatic activity. The technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer, changing the fluorescent properties and providing a fluorescent readout.

The Transcreener® assay is designed specifically for high-throughput screening (HTS), with a single addition, mix-and-read format. It offers reagent stability and compatibility with commonly used multimode plate readers. The assay is available as an FP, FI, or TR-FRET configuration.

BellBrook's Recombinant POLQ Helicase Enzyme

Active, human DNA polymerase theta enzyme (AA 67-894), C-terminal 6xHis-tagged protein expressed and purified from insect cells. The enzyme has been thoroughly validated with the Transcreener ADP Assay Kit. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >90% in a composition of 20 mM HEPES, 5 mM NaCl, 5% Glycerol, and 0.5 mM TCEP (pH 7.5). Currently used with the Transcreener assay at a concentration of 0.42 nM in a 10 μL reaction.

POLQ Helicase Enzyme Graphic

POLQ Helicase Assay Services

Interested in moving your program forward with BellBrook's POLQ assay services? BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your POLQ discovery program.

Scientist Studying DDX41

ATPase Profiling Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements Determination of koff using 'jump dilution' enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.
  • Triaging Non-stoichiometric Inhibitors – Run assays under different conditions such as varying detergent and enzyme concentrations.
  • Evaluate Compound-Target Binding – Run thermal shift assays to confirm compound-target engagement via shifts in melting temperature.

Contact Us to Learn More

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What's Included

What You Will Need

POLQ Helicase ATPase Assay System

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Please choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener ADP Assay Kit.

Transcreener ADP Assay Kits

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Related Resources

Interconnections Between the DNA Damage Response and Innate Immunity

There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.

In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets. The enzyme targets discussed include:

DDR

  • POLQ
  • WRN
  • PARP1
  • PARG
  • DNA PK

Innate Immunity

  • cGAS
  • TREX1
  • TBK1
  • IKKβ
  • IRAK4
  • ENPP1
  • CD38
  • OAS-1
  • RIG-1/MDA5
  • DDX3
  • CD39
  • CD73
DNA Damage Response and Innate Immunity Preview

POLQ As a Therapeutic Target

POLQ helicase functions as an ssDNA-dependent ATPase that can unwind short DNA duplexes and displace replication protein A (RPA) from single-stranded DNA. It also acts as a scanning motor protein that searches for microhomologies in the break region, using the ssDNA break on the opposite side of the break as a template for DNA extension to fill the gap. 

Normal adult tissues express low or no POLQ. However, it overexpresses in lung, stomach, breast, ovarian, small intestine, and colorectal cancers. In each of these tumor types, high expression of POLQ tightly correlates with decreased survival. Since an expanding range of cancers seems to become addicted to POLQ in response to the loss of more accurate DSB repair systems, there is an opportunity to extinguish such cancers by targeting POLQ. Clinical trials began with promising candidate therapeutics in early 2022.

Read more in this article from BellBrook Labs: Looking Ahead: POLQ in the Future of Immuno-Oncology

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