A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents

Abstract

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.

DGCR8 Mediates Repair of UV-Induced DNA Damage Independently of RNA Processing

“…To confirm the involvement of DGCR8 in TC-NER, we utilized a recovery of RNA synthesis (RRS) assay (Jia et al, 2015) (Figure 4C and Figure S4D). DGCR8-depleted cells showed impaired RRS after UV radiation similarly to CSB-depleted cells.…”

“…RRS assays were done as described with some modifications (Jia et al, 2015; Nakazawa et al, 2010). U2OS cells stably expressing siRNA resistant FLAG-WT DGCR8, FLAG-S153A DGCR8 or FLAG-S153D-DGCR8 were transfected with siRNA against endogenous DGCR8.…”

“…U2OS cells stably expressing siRNA resistant FLAG-WT DGCR8, FLAG-S153A DGCR8 or FLAG-S153D-DGCR8 were transfected with siRNA against endogenous DGCR8. Forty eight hours after transfection of siRNAs, cells were irradiated with UVC (16 J/m 2 ) and immediately incubated with 5′-ethynyuridine (EU) (50 μM, Life Technologies) for 2 hours (Jia et al, 2015; Nakazawa et al, 2010). Then, cells were fixed and permeabilized at the indicated time points.…”

Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair

“…This procedure provides sensitive and reproducible information regarding NER performance, but the dependence on non-user-friendly tritiated thymidine and the fact that the assay is laborious and requires long autoradiographic exposure times, means that its application was limited to only a few laboratories worldwide. The recent replacement of radioactive labeled thymidine with EdU, which can be labeled using simple in situ Click-chemistry with azide-coupled fluorescent dyes of choice, makes the assay much more accessible, faster, and easier to quantify using fluorescence microscopy (19,46). In the present study, we developed an amplified UDS assay by combining the EdU-based UDS assay with a tyramide signal amplification.…”

Functional and clinical relevance of novel mutations in a large cohort of patients with Cockayne syndrome

“…In Pavia, cell fusion with known CS-A or CS-B cells using polyethylene glycol was used to establish complementation group 37. In Japan, complementation group was established by transduction with lentivirus expressing either ERCC6/CSB or ERCC8/CSA cDNA 38. Finally, the appropriate gene was sequenced using genomic DNA ( ERCC6 RefSeq NG_009442.1; ERCC8 RefSeq NG_009289.1) and/or cDNA ( ERCC6 NM_000124.3; ERCC8 RefSeq NM_000082.3).…”