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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.

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Figure 1
Figure 2
Figure 3: Typical 96-well plate formats.
Figure 4: Measurement of NER activity by UDS and RRS assays.
Figure 5: Experimental schemes.
Figure 6: Virus complementation experiments for UDS and RRS assays.
Figure 7: Virus complementation experiment for cell sensitivity assay.
Figure 8: RNA interference experiments for UDS and RRS assay.

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Acknowledgements

We are grateful to H. Niki for his support with the project. This work was supported by Special Coordination Funds for Promoting Science and Technology from Japan Science and Technology Agency, KAKENHI Grants-in-Aid for Young Scientists B (24790321) from the Japan Society for the Promotion of Science (JSPS), a cancer research grant from The Sagawa Foundation for Promotion of Cancer Research, the Sasakawa Scientific Research Grant from the Japan Science Society, a medical research grant from Mochida Memorial Founds for Medical and Pharmaceutical Research and a science research grant from Inamori Foundation to Y. Nakazawa; KAKENHI Grants-in-Aids for Young Scientists A (24681008) and for Exploratory Research (24659533) from JSPS, a grant for Health Labour Sciences Research (26310301) from The Ministry of Health Labour and Welfare, a research grant from The Kanae Foundation for the Promotion of Medical Science, a basic science research grant from The Sumitomo Foundation, a basic research grant from The Nakatomi Foundation, a research grant from Suzuken Memorial Foundation, a science research grant from Ube Industries Foundation for Promotion of Science, a science research grant from The Uehara Memorial Foundation to T.O.; KAKENHI Grants-in-Aid for Young Scientists B (25870534) from JSPS to C.G.; and the project is partly supported by Platform for Drug Discovery, Informatics and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Author information

Authors and Affiliations

Authors

Contributions

Y. Nakazawa and T.O. designed the study; N.J. and Y. Nakazawa optimized all the experimental protocols; N.J., Y. Nakazawa, C.G., M. Shimada and M. Sethi performed the experiments; Y.T., H.U., Y. Nagayama and T.O. coordinated the study; N.J. and T.O. wrote the manuscript; and all authors commented on the manuscript.

Corresponding author

Correspondence to Tomoo Ogi.

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Competing interests

The authors declare that they have one registered patent (JP5549908) and one pending patent (US12/656,408) associated with this manuscript.

Integrated supplementary information

Supplementary Figure 1 UDS and RRS assays for lymphoblastoid cell lines (LBLs).

(a) The experimental scheme of UDS and RRS assays for LBLs. (b) UDS and (c) RRS assays for normal 277 and XP-A-patient derived XPL15OS cells. RRS was normalised to activity measurement in non-irradiated cells; bars and error bars represent average fluorescent intensity of quintuplicate wells and standard deviations, respectively.

Supplementary Figure 2 Cell sensitivity assay after γ-irradiation using the VTI system.

(a) The experimental scheme of cell sensitivity assay after γ-irradiation. (b) Typical 96-well plate pattern for the assay and (c) cell sensitivity assay for normal 48BR and SCID-patients-derived NM-720 cells. Data in (c) were normalised to the EdU incorporation level measured in non-irradiated cells. Data points and error bars represent average fluorescent intensities of quadruplicate wells and standard deviations, respectively.

Supplementary Figure 3 Cell sensitivity assay after mitomycin C (MMC) treatment using the VTI system.

(a) The experimental scheme of cell sensitivity assay after MMC treatment. (b) Typical 96-well plate pattern for the assay and (c) cell sensitivity assay for normal 48BR, XP-CS-Fanconi-complex patients derived XPCS1CD and XP-CS-complex patients derived CS1USAU cells. Data in (c) were normalised to the EdU incorporation level measured in untreated cells. Data points and error bars represent average fluorescent intensities of quadruplicate wells and standard deviations, respectively.

Supplementary Figure 4 UDS and RRS complementation assays by an electroporation system (Neon, Life Technologies).

(a) The experimental scheme of complementation assay by electroporation. (b) UDS and (c) RRS assays for normal 48BR and XP-A-patient derived XP15BR cells. RRS was normalised to activity measurement in non-irradiated cells. Bars and error bars represent average fluorescent intensity of quadruplicate wells and standard deviations, respectively. (d, e) Transfection efficiency was confirmed by immunofluorescent staining with anti-V5 tag antibody. Cells were transfected with indicated plasmid by electroporation and then plated on a glass-bottomed 96-well plate. 40 hours after transfection, immunofluorescent staining was performed as described in Supplementary Method 5. Transfection efficiency was calculated as a number of Alexa 488-positive cells using the VTI system. (f) Total cell counts in the UDS assay. Scale bar, 100 μm; w/o, without transfection.

Supplementary Figure 5 Reproducibility and fluctuation of the assays.

Fluctuations and statistics data collected from past in-house experiments using the assay system. UDS and RRS data from normal 48BR, XP-patient-derived XP15BR and XP21BR, CS-patient-derived CS2AW, and UVSS-patient-derived Kps3 cells are summarised. Data are collected from independent experiments (n=3), and data points and S.D. value represent UDS/RRS level and standard deviations, respectively. Each data point in the UDS assay (a) and in the UDS virus complementation assay (c) represent a normalised UDS level of each experiment calculated as follows: normalised UDS = (Fluorescent intensity with UV-irradiation – Fluorescent intensity without UV-irradiation)/ Fluorescent intensity with UV-irradiation*100. Data in the UDS assay for RNA interference samples (e) were normalised to activity measurement in mock-transfection control cells. Data in the RRS assay (b), in the RRS virus complementation assay (d) and in the RRS assays for RNA interference samples (f) were normalised to activity measurement in non-irradiated cells. Error bars represent standard deviations. Statistical analysis was carried out by one-way ANOVA with post-hoc Scheffé method, p values <0.05 were considered as significant. NS, non-significant.

Supplementary information

Supplementary Figure 1

UDS and RRS assays for lymphoblastoid cell lines (LBLs). (PDF 389 kb)

Supplementary Figure 2

Cell sensitivity assay after γ-irradiation using the VTI system. (PDF 416 kb)

Supplementary Figure 3

Cell sensitivity assay after mitomycin C (MMC) treatment using the VTI system. (PDF 449 kb)

Supplementary Figure 4

UDS and RRS complementation assays by an electroporation system (Neon, Life Technologies). (PDF 845 kb)

Supplementary Figure 5

Reproducibility and fluctuation of the assays. (PDF 431 kb)

Supplementary Table 1

Data summary of cell sensitivity assay after UV-irradiation. (PDF 61 kb)

Supplementary Table 2

Data summary of cell sensitivity assay after γ-irradiation. (PDF 95 kb)

Supplementary Table 3

Data summary of cell sensitivity assay after Mitomycin C (MMC) treatment. (PDF 61 kb)

Supplementary Method 1

Instructions for image acquisition and data processing using the VTI system. (PDF 1770 kb)

Supplementary Method 2

Instructions for image acquisition and data processing for UDS assay using a standard fluorescence microscope and NIH ImageJ software. (PDF 4378 kb)

Supplementary Method 3

Instructions for UDS and RRS assays for lymphoblastoid cell lines. (PDF 189 kb)

Supplementary Method 4

Instructions for cell sensitivity assay after γ-irradiation and MMC treatment. (PDF 202 kb)

Supplementary Method 5

Instructions for complementation assay by electroporation (Neon, Life Technologies). (PDF 131 kb)

Supplementary Note 1

UDS assay parameters for the VTI system. (PDF 143 kb)

Supplementary Note 2

RRS assay parameters for the VTI system. (PDF 391 kb)

Supplementary Note 3

Cell sensitivity assay parameters for the VTI system. (PDF 144 kb)

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Jia, N., Nakazawa, Y., Guo, C. et al. A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents. Nat Protoc 10, 12–24 (2015). https://doi.org/10.1038/nprot.2014.194

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