Pimpley Manasi R, Foley Megan L, Latimer Jean J
Department of Pharmaceutical Sciences, Nova Southeastern University and AutoNation Breast Cancer Institute, Fort Lauderdale, FL, USA.
Methods Mol Biol. 2020;2102:483-507. doi: 10.1007/978-1-0716-0223-2_27.
The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C (254 nm) irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results have been used to clinically diagnose human DNA repair deficiency disorders, and provide a basis for investigation of repair deficiency in human tissues or tumors. Genomic sequencing to establish the presence of specific mutations is also used now for clinical diagnosis of DNA repair deficiency syndromes. Few functional assays are available which directly measure the capacity to perform NER on the entire genome. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR). As discussed in Chap. 28 is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. Our laboratory also explored the fluorescent label-based Click-iT assay that uses EdU as the label, rather than H thymidine. Despite emerging studies in the literature finding this assay to be useful for other purposes, we found that the EdU-based UDS assay was not consistent or reproducible compared with the H thymidine-based assay.
非计划DNA合成（UDS）检测可测量细胞进行全基因组核苷酸切除修复（NER）的能力。本章介绍了通过使用UV-C（254nm）照射产生6-4光产物和嘧啶二聚体来应用该技术的方法。此程序专门设计用于定量6-4光产物。修复通过损伤后修复合成过程中掺入的放射性胸苷量进行定量，放射性通过放射自显影后的颗粒计数进行评估。这些结果已用于临床诊断人类DNA修复缺陷疾病，并为研究人类组织或肿瘤中的修复缺陷提供依据。现在也使用基因组测序来确定特定突变的存在，以用于DNA修复缺陷综合征的临床诊断。很少有功能检测可直接测量对整个基因组进行NER的能力。由于该检测需要活细胞，因此必须预先建立外植体培养技术。宿主细胞再激活（HCR）。如第28章所述，它不是一种等效技术，因为它仅测量活跃基因处的转录偶联修复（TCR），这只是总NER的一小部分。我们实验室还探索了基于荧光标记的Click-iT检测，该检测使用EdU作为标记，而不是H胸苷。尽管文献中有新的研究发现该检测对其他目的有用，但我们发现基于EdU的UDS检测与基于H胸苷的检测相比不一致且不可重复。
