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 &amp; Chemical Biology to DS. Support was also provided by ESTRO to CV.

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RWS is a Scientific Consultant to Trevigen, Inc. The rest of the authors declare that there are no conflicts of interest.

There are over 600 citations using the term &#x2018;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 ...

<table border="1">
<tbody>
 <tr>
 <td>Name of 			the reagent</td>
 <td>Company</td>
 <td>Catalog 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Potassium 			Chloride</td>
 <td>Fisher</td>
 <td>P217-500</td>
 <td>Molecular 			grade</td>
 </tr>
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 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP120-500</td>
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 <td>Glycerol</td>
 <td>Fisher</td>
 <td>BP229-1</td>
 <td>Molecular 			grade</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Fisher</td>
 <td>BP172-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES- 			Solution</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES-Salt</td>
 <td>Sigma</td>
 <td>H4034-500G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium 			Hydroxide</td>
 <td>Sigma</td>
 <td>17-8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22&mu;M 			filter</td>
 <td>Nalgene</td>
 <td>167-0020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Slide-A-Lyzer 			Dialysis Products</td>
 <td>Pierce</td>
 <td>66373</td>
 <td>MW 7000</td>
 </tr>
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 <td>Syringe</td>
 <td>Fisher</td>
 <td>309659</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>Fisher</td>
 <td>305196</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1.5 mL 			Microcentrifuge Tubes</td>
 <td>Fisher</td>
 <td>05-408-129</td>
 <td>Black </td>
 </tr>
 <tr>
 <td>15 mL Falcon 			Tubes</td>
 <td>Fisher</td>
 <td>352097</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NucBuster 			Protein Extraction Kit</td>
 <td>Calbiochem</td>
 <td>71183</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td>14190</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Molecular 			Beacons</td>
 <td>IDT</td>
 <td>&nbsp; </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BioRad 			Protein assay dye reagent concentrate</td>
 <td>BioRad</td>
 <td>Cat# 500-0006</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons

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 F&ouml;rster 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.

Watch this Scientific Journal Video about Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons at JoVE.com

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

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 ...

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13.5K Views.  University of Pittsburgh School of Medicine. 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.