DNA damage repair is a critical process that all cells need to survive. NER is one of many DNA damage repair processes that attempts to remove UV or chemical-induced DNA damage so that replication or transcription can proceed. Xeroderma pigmentosum and Cockayne syndrome are autosomal recessive disorders caused by mutations in genes involved in the NER process. These patients are highly susceptible to melanomas and other health problems due to their inability to repair DNA damage caused by the sun or other chemical insults. Current assays for determining DNA repair activity take multiple days to complete and require the use of radioactively labeled 3H-thymidine or bromouridine (BrdU), which are incorporated into DNA during S phase. Each of these molecules has a similar structure to a DNA base, so when they are added to cells that have been irradiated, they become incorporated into the DNA. In order to measure the amount of incorporated molecules, an autoradiograph or scintillation counter must be used. These assays are not amenable to a high-throughput format. Additionally, these assays are critical for patients with mutations in NER genes to determine which genes are mutated in order to determine prognosis. The authors of this paper report a protocol that uses 5-ethynyl-2′-deoxyuridine (EdU) or 5-ethynyluridine (EU) for assessment of DNA damage and repair. These molecules are conjugated to an alkyne, which can be added to cells and incorporated into DNA during S phase just as in the older assays using BrdU. However, the addition of a fluorescent azide dye plus copper catalyzes an alkyne-azide-coupling click-chemistry reaction resulting in a fluorescent signal. This signal can be detected on any high content imaging instrument and is more sensitive and quantitative than BrdU. They define protocols to assess UDS and RRS, which can all be done in a 96-well plate over 3–5 days (figure above). Induction of DNA damage is followed by addition of EdU to allow for DNA incorporation. Plates are then fixed and blocked, and a fluorescent azide dye is added followed by DAPI staining. These plates can then be read on any high content reader so the entire protocol can be semi-automated. Additionally, they have outlined similar protocols that can be used to determine cell sensitivity after UV irradiation and virus complementation or RNAi assays to determine mutated genes in the NER pathway. Each of these protocols is described in great detail, making this paper an excellent resource for scientists who want to attempt an assay to assess DNA damage.
* Abstract from Nature Protocols 2015; DOI: 10.1038/nprot.2014.194
DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete.
Kelli M. Wilson is a contributor to ASSAY and Drug Development Technologies, published by Mary Ann Liebert, Inc. She is from Bethesda, Maryland.
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Nature Protocols titled “A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents.” Authors of the paper are Jia N, Nakazawa Y, Guo C, Shimada M, Sethi M, Takahashi Y, Ueda H, Nagayama Y, Ogi T.