We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.
We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5’end and a Dabcyl moiety conjugated to the 3′ end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.
1. Molecular Beacon Design
1.1 Designing and ordering your molecular beacon
The design of your molecular beacon must consider the following:
1.2 Annealing your molecular beacon
1.3 Quality assessment of your molecular beacon
2. Nuclear Lysate Preparation
2.1 Base Excision Repair reaction buffer5 preparation
2.2 Cell culture and cell isolation
2.3 Nuclear lysate preparation
2.4 Dialysis, protein quantification and aliquoting
3. Molecular Beacon Assay
3.1 Equipment requirements
3.2 Buffers and reagents
3.3 Assay set-up (Table 5)
3.4 Run assay
4. Molecular Beacon Assay Analysis
We used Microsoft Excel software to perform these analyses and calculations. We recommend establishing templates and macros that will allow easy and quick analysis and graph displays based on data derived from standard 96-well formats.
4.1 Removal of Background
4.2 Fl(Tmax) normalization
To address the variability in pipetting and fluorescence detection in different wells, we normalize the data to the maximal fluorescence values (that correlate with beacon input).
4.3 Plot
5. Representative Results
We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons (Figure 1). Once annealed, we suggest performing a melt curve analysis (Table 1) as a quality assessment of your molecular beacon (Figure 2). Nuclear lysates can then be prepared, using the buffer components listed in Table 2. Dialyze the lysate in 0.5 L pre-chilled dialysis buffer for 90 minutes at 4 °C with gentle stirring using a PIERCE Slide-A-Lyzer Dialysis cassette against the dialysis buffer listed in Table 3. Collect the dialyzed nuclear protein solution from the dialysis cassette using a syringe. Use a different gasket than used previously to inject the protein into the dialysis cassette. Determine the concentration of the dialyzed protein using standard protocols. Examples of expected nuclear lysate concentrations are shown in Table 4. Run the reaction as indicated above (step 3.4), using the plate layout as shown in Table 5. A representative analysis of two tumor cell lysates: LN428, a glioma cell line with undetectable levels of methyl purine DNA glycosylase (MPG) and LN428/MPG, the same cell line engineered for elevated expression of MPG; both cell lines have equivalent levels of AP endonuclease 1 (APE1)1,2. Each were probed for APE1 activity and MPG activity using the BER THF Molecular Beacon (Figure 3) and the BER Hx Molecular Beacon (Figure 4), where THF = tetrahydrofuran (a substrate for APE1) and Hx = hypoxanthine (a substrate for MPG). Finally, using a BER dU/A Molecular Beacon that reports on uracil glycosylase activity, we show that signal output or signal strength is maximal at 10μg of protein and decreases to an undetectable level at 0.62 μg of protein (Figure 5).
Figure 1. Overall structure of the BER Molecular Beacon. Shown is the sequence and overall design of the BER Molecular Beacons used herein as a single-stranded molecule, after annealing and again after melting. The Bolded, Italics portion of the sequence is the stem (15 bases) and the underlined portion of the sequence is the loop (13 bases). FAM = the 5′ fluorescent dye 6-Carboxyfluorescein [on the 5’end; Abmax = 495 nm, Emmax = 520 nm, Extinction Coefficient (260 nm): 20,960; Extinction Coefficient (at absorbance max): 75,000] and Dabcyl = the 3′ quenching agent Dabcyl [on the 3’end; Abmax = 453 nm; Quenching Range = 425-520 nm]. X = the lesion (varies depending on the research question) and Y = the base opposite the lesion (varies depending on the research question).
Figure 2. Melt Curve. A representative melting curve is shown for the BER Molecular Beacon Control (A), and the BER Molecular Beacons containing the tetrahydrofuran (THF) moiety (B) or the Hypoxanthine (Hx) moiety (C). Oligonucleotide concentrations ranged from 0 nM (dark blue), 8 nM (red), 16 nM (green), 32 nM (purple), 64 nM blue to 128 nM (orange). As described in Step 1.3, the annealed oligonucleotides were heated from 25 °C to 95 °C in 0.5 °C intervals and the StepOnePlus system is programmed to measure FAM fluorescence at each 0.5 °C interval.
Figure 3. Representative data using the BER THF Molecular Beacon. AP endonuclease activity specific for hydrolysis of the abasic site analog tetrahydrofuran (THF) was measured in nuclear lysates from the control cell line (LN428) and the MPG over-expression cell line (LN428/MPG). Each lysate was analyzed using either the BER Molecular Beacon Control (LN428, green; LN428/MPG, purple) or the BER THF Molecular Beacon (LN428, blue; LN428/MPG, red). Results are reported as (A) the mean fluorescence response units and (B) the same data normalized to beacon input as described in Section 4 above (THF = tetrahydrofuran).
Figure 4. Representative data using the BER Hx Molecular Beacon DNA glycosylase activity specific for removal of the MPG substrate hypoxanthine (Hx) was measured in nuclear lysates from the control cell line (LN428) and the MPG over-expression cell line (LN428/MPG). Each lysate was analyzed using either the BER Molecular Beacon Control (LN428, green; LN428/MPG, purple) or the BER Hx Molecular Beacon (LN428, blue; LN428/MPG, red). Results are reported as (A) the mean fluorescence response units and (B) the same data normalized as described in Section 4 above (Hx = hypoxanthine).
Figure 5. Representative data using the BER dU/A Molecular Beacon at different protein levels. DNA glycosylase (UNG) activity specific for removal of uracil (dU/A) was measured in nuclear lysates from T98G cells. The T98G lysate was analyzed using either the BER Molecular Beacon Control or the BER dU/A Molecular Beacon, at protein levels of 10, 5, 2.5, 1.25 or 0.62 μg, as indicated in the figure. Results are normalized data as described in Section 4 above.
Concentration of Beacon (nM) | 0 | 8 | 16 | 32 | 64 | 128 |
Molecular Beacon working solution (200nM) added (μL) | 0 | 1 | 2 | 4 | 8 | 16 |
BER reaction Buffer (w/o DTT) (μL) | 25 | 24 | 23 | 21 | 17 | 9 |
Table 1. Layout of a melt curve experiment.
1 L Total | Volume of Stock Solution needed | Con. of Stock Solution | Final Con. |
HEPES-KOH | 25 mL | 1M pH7.8 | 25 mM |
KCl | 75 mL | 2 M | 150 mM |
EDTA | 1 mL | 0.5M pH8.0 | 0.5 mM |
Glycerol | 10 mL | N/A | 1% |
DTT (add before use) | 0.5 mL | 1 M | 0.5 mM |
H2O | 888.5 mL | N/A | N/A |
Table 2. BER reaction buffer.
1 L Total | Volume of Stock Solution needed | Con. of Stock Solution | Final Con. |
HEPES-KOH | 50 mL | 1M pH7.5 | 50 mM |
KCl | 50 mL | 2 M | 100 mM |
EDTA | 1 mL | 0.5M pH8.0 | 0.5 mM |
Glycerol | 200 mL | N/A | 20% |
DTT (add before use) | 1 mL | 1 M | 1 mM |
H2O | 698 mL | N/A | N/A |
Table 3. Dialysis buffer.
Cell Line | Protein Concentration (μg/μL) |
LN428 | 5.34 |
LN428/MPG | 4.79 |
Table 4. Protein Determination results.
Negative Control 25μL BER Buffer | (Background Control) 20μL BER Buffer 5μL Con Beacon NO lysate |
(Test in Triplicate) 15μL BER Buffer 5μL Con Beacon 5μL LN428 Lysate |
(Test in Triplicate) 15μL BER Buffer 5μL Con Beacon 5μL LN428/MPG Lysate |
Negative Control 25μL BER Buffer | (Background Control) 20μL BER Buffer 5μL THF Beacon NO lysate |
(Test in Triplicate) 15μL BER Buffer 5μL THF Beacon 5μL LN428 Lysate |
(Test in Triplicate) 15μL BER Buffer 5μL THF Beacon 5μL LN428/MPG Lysate |
Negative Control 20μL BER Buffer 5μL lysate |
(Background Control) 20μL BER Buffer 5μL Hx Beacon NO lysate |
(Test in Triplicate) 15μL BER Buffer 5μL Hx Beacon 5μL LN428 Lysate |
(Test in Triplicate) 15μL BER Buffer 5μL Hx Beacon 5μL LN428/MPG Lysate |
Negative Control 20μL BER Buffer 5μL lysate |
Table 5. Molecular Beacon Assay set-up.
There are over 600 citations using the term ‘molecular beacon’ for detecting and quantifying numerous molecules and enzymatic activities, including helicase activity6, DNA polymerase activity7,8, DNA ligase activity9, telomerase activity10, DNA photolyase activity11 and DNA/RNA hybrids12, among many others. In addition to the use of molecular beacons for the measurement of the DNA repair activities listed above, we and others have demonstrated the utility of these probes for the analysis of BER activity1,2,13-15.
The real-time BER activity assay we describe herein incorporates a single modified base and allows for the quantitative assessment of BER rates for different lesions or alterations in the base opposite the lesion. Applications can range from molecular mechanistic studies to inhibitor screens. We demonstrated BER specificity by comparing lysates from MPG expressing cells with non-expressing cells and lack of activity in the control beacons. The assay is highly sensitive, allowing the detection of defects in expression of the BER proteins MPG2 or XRCC11 and is sufficient to detect minor changes in DNA repair kinetic parameters and the efficacy of DNA repair inhibitors [manuscript in preparation]. Multiple reads throughout the active repair time support calculated kinetic rates and the significance in any changes in those kinetic rates. The BER molecular beacon assay is therefore suitable for high-throughput studies to screen for DNA repair enzyme inhibitors (inhibitor screens) or for studies on the role of individual nuclear components. The analysis of nuclear lysates allows the assessment of DNA repair in the full context of the BER complex hence also depicting indirect alterations or influences on the BER machinery (e.g., post-translation modifications or mutations).
The protocol, as described, should yield highly reproducible and quantitative results. However, there are several details to consider to ensure proper analysis: (1) Background values of negative controls may be too high: This may be due to bad storage or manufacture of the Oligonucleotide. High background values in the lysates without beacons indicate confounding auto fluorescence (i.e. by contamination or cellular expression of exogenous fluorescent proteins). If expression of a fluorescent protein is unavoidable, the excitation/emission spectra for the reporter dye and fluorescent protein may not overlap; (2) Fl(Tmax) is low: DNA concentration is different than given. Check DNA concentration by spectrophotometry (OD260nm) for example, using the Nanodrop. High DNA concentration with low Fl(Tmax) indicates an insufficient FAM loading on the beacons. HPLC purification prevents contamination with unmodified DNA (see above); (3) Repair activity is poor: If the Fl(Tmax) values at the last cycles indicate sufficient beacon input and fluorescence detection, this might suggest that the nuclear extract preparation failed. Nuclear extract quality is crucial. Low temperatures should be assured throughout the procedure including long-term storage at -80°C and (4) Repair activity curves initiate with high values: Even very short exposure of the samples to room temperature prior to analysis initiates repair. Keep cool at all times.
This highly sensitive and quantitative BER assay is also applicable to the analysis of purified proteins, enabling studies on substrate specificity by altering the molecular beacons accordingly. Finally, the assay is applicable to analysis of tumor and tissue lysates, providing an opportunity to measure functional DNA Repair endpoints as biomarkers of response or therapeutic efficacy, as we have suggested for evaluating tumors for PARP1 inhibitor potentiation of the alkylating agent temozolomide2.
The authors have nothing to disclose.
This work was supported by grants from the Pittsburgh Foundation and the National Institutes of Health (NIH) [GM087798; CA148629; ES019498] to RWS. Support for the UPCI Lentiviral Facility was provided to RWS by the Cancer Center Support Grant from the National Institutes of Health [P30 CA047904]. Support was also provided by the University of Pittsburgh Department of Pharmacology & Chemical Biology to DS. Support was also provided by ESTRO to CV.
Name of the reagent | Company | Catalog number | Comments |
Potassium Chloride | Fisher | P217-500 | Molecular grade |
EDTA | Fisher | BP120-500 | |
Glycerol | Fisher | BP229-1 | Molecular grade |
DTT | Fisher | BP172-5 | |
HEPES- Solution | Invitrogen | 15630 | |
HEPES-Salt | Sigma | H4034-500G | |
Potassium Hydroxide | Sigma | 17-8 | |
0.22μM filter | Nalgene | 167-0020 | |
Slide-A-Lyzer Dialysis Products | Pierce | 66373 | MW 7000 |
Syringe | Fisher | 309659 | |
Needle | Fisher | 305196 | |
1.5 mL Microcentrifuge Tubes | Fisher | 05-408-129 | Black |
15 mL Falcon Tubes | Fisher | 352097 | |
NucBuster Protein Extraction Kit | Calbiochem | 71183 | |
PBS | Invitrogen | 14190 | |
Molecular Beacons | IDT | ||
BioRad Protein assay dye reagent concentrate | BioRad | Cat# 500-0006 |