定量，实时分析，利用分子信标病变特定的细胞裂解液中的碱基切除修复活动
1Department of Pharmacology & Chemical Biology,University of Pittsburgh School of Medicine, 2Hillman Cancer Center,University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy,The Netherlands Cancer Institute, 4Department of Human Genetics,University of Pittsburgh School of Public Health
Summary
我们描述了DNA糖基化酶和AP内切酶的活动，在核细胞裂解定量，实时测量的方法。该法产生服从动力学分析DNA修复活动率，是适应组织和肿瘤裂解物或纯化蛋白质的DNA修复能力的量化。
Abstract
我们描述了一个定量，实时测量碱基切除修复（BER）的分子信标DNA糖基化酶和AP内切酶的活动，在核细胞裂解方法。基板（灯塔）是由deoxyoligonucleotide包含单碱基损伤与6 – 羧基荧光素（6-FAM）基团结合到5'端和DABCYL基的共轭寡核苷酸的3'结束。 BER分子信标是43基地在长度和序列的目的是为了促进与13环和15个碱基对中干1,2个核苷酸的茎环结构的形成。在此配置折叠时6-FAM基淬火DABCYL非荧光的方式通过福斯特共振能量转移（FRET）3,4。病变位置等基地，以下病灶切除和链的断裂，其余5个基地的寡核苷酸，含6-FAM基干公布。释放第二支队从猝灭（DABCYL）荧光的增加，DNA修复水平是相称的结果。通过收集多个读取的荧光值，实时误码活动的评估是可能的。标准定量实时PCR仪器的使用，使许多样品同时分析。这些BER分子信标设计，用一个单一的基础病变，是适合进行动力学分析，误码率的量化和抑制剂验证，是适应组织和肿瘤细胞裂解物中的DNA修复能力的量化或用纯化的蛋白质。在肿瘤裂解物或使用这些分子信标组织抽吸的BER活动的分析，可能是适用于功能性的生物标志物的测量。此外，使用这种定量检测与纯化蛋白质的BER活动分析，误码率抑制剂的发现和验证提供了一种快速，高通量的方法。
Protocol
1。分子信标设计 1.1设计和分子信标订购您的分子信标设计必须考虑以下几点： 我们有43-MER DNA寡核苷酸，有良好的效果。 GC含量应大于32％。排除在G碱基的5'端。 6-FAM（6 – 羧基）共轭上的5'端和“修改为3 DABCYL请求。 病变的位置，应该是基地，从5'端＃6。 干或发夹双工的长度应该是至少15个基点。 建…
Discussion
有超过600个使用“分子信标”检测和量化许多分子和酶的活动，包括解旋酶的活性，DNA聚合酶活性7,8，DNA连接酶的活动9，10端粒酶活性，DNA光解酶的活性11和DNA /引用许多人的RNA之间的杂交种12。除了 利用分子信标测量DNA修复上面列出的活动，我们和其他人已经证明误码活动1,2,13-15分析这些探头的效用。
实时BER活性测定，我们描述…
Disclosures
The authors have nothing to disclose.
Acknowledgements
从匹兹堡基金会和国家卫生研究院（NIH）的[GM087798; CA148629; ES019498] RWS的赠款，以支持这项工作。 UPCI慢病毒设施的圣淘沙名胜世界提供了支持，从国家卫生研究院癌症研究中心支援津贴[P30的CA047904]。由美国匹兹堡大学的药理学部化学生物学的DS也提供了支持。也提供了支持ESTRO到CV。
Materials
| 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 |
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Tags
Quantitative AnalysisReal-time MeasurementBase Excision RepairDNA GlycosylaseAP EndonucleaseMolecular BeaconsSubstrateDeoxyoligonucleotideCarboxyfluorescein (6-FAM)DabcylStem-loop StructureNucleotidesBase PairsFörster Resonance Energy Transfer (FRET)FluorescenceDNA RepairFluorescence ValuesReal-time AssessmentPCR Instruments
Cite This Article
Svilar, D., Vens, C., Sobol, R. W. Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons. J. Vis. Exp. (66), e4168, doi:10.3791/4168 (2012).