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A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

Limsirichaikul S, Niimi A, Fawcett H, Lehmann A, Yamashita S, Ogi T - Nucleic Acids Res. (2009)

Bottom Line: We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU).We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads.Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 852-8523 Japan.

ABSTRACT
Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

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UV-induced EdU incorporation in non-S-phase cells is NER repair replication specific. (A–G) Levels of UV-UDS in NER-proficient and NER-deficient cells were examined. Primary fibroblasts were cultured on coverslips, UVC-irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Levels of EdU incorporation were analysed as in Figure 1A and presented as in Figure 1. (H) UDS assay performed by 3H-thymidine incorporation. Quiescent cells were UVC irradiated at the indicated dose and 3H-thymidine incorporation was measured for 3 h after UVC irradiation in the media containing 10 mM hydroxyurea. UDS levels were normalized and expressed as percentages of the UDS in the normal cells at 10 J/m2. Mean Normal represents the average UDS levels of the four different normal cell lines (1BR, 48BR, 142BR and 251BR). Asterisks indicate the mean values of nuclear fluorescent intensities, which correspond to the UDS levels. Δ represents UDS difference between irradiated and un-irradiated samples. (I) Typical photos of the EdU incorporation examined in Figure 2A–G are shown (2 h after 20 J/m2 UVC irradiation). Arrows indicate non-S-phase cells. 48BR, normal; 1BR, normal; XP15BR, XP-A; XP20BE, XP-G; XP13BR, XP-C; XP12BR, XP-D; CS10LO, CS-B.
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Figure 2: UV-induced EdU incorporation in non-S-phase cells is NER repair replication specific. (A–G) Levels of UV-UDS in NER-proficient and NER-deficient cells were examined. Primary fibroblasts were cultured on coverslips, UVC-irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Levels of EdU incorporation were analysed as in Figure 1A and presented as in Figure 1. (H) UDS assay performed by 3H-thymidine incorporation. Quiescent cells were UVC irradiated at the indicated dose and 3H-thymidine incorporation was measured for 3 h after UVC irradiation in the media containing 10 mM hydroxyurea. UDS levels were normalized and expressed as percentages of the UDS in the normal cells at 10 J/m2. Mean Normal represents the average UDS levels of the four different normal cell lines (1BR, 48BR, 142BR and 251BR). Asterisks indicate the mean values of nuclear fluorescent intensities, which correspond to the UDS levels. Δ represents UDS difference between irradiated and un-irradiated samples. (I) Typical photos of the EdU incorporation examined in Figure 2A–G are shown (2 h after 20 J/m2 UVC irradiation). Arrows indicate non-S-phase cells. 48BR, normal; 1BR, normal; XP15BR, XP-A; XP20BE, XP-G; XP13BR, XP-C; XP12BR, XP-D; CS10LO, CS-B.

Mentions: To apply the EdU technique to XP diagnosis, we estimated the levels of UV-induced EdU incorporation in several NER-deficient primary fibroblasts as well as normal controls (Figure 2A–G for histograms, and Figure 2I for typical photos). Since XP is a genetically heterogenous disorder, both the NER genes affected and the types of mutations determine the magnitude of the defect in UDS. We measured UDS in XP15BR (XP-A), XP20BE (XP-G/CS), XP13BR (XP-C), XP12BR (XP-D) and CS-patient-derived CS10LO (CS-B) cells, and compared the results with those obtained using 3H-thymidine incorporation. With 20 J/m2 UVC irradiation, we observed substantial UDS in normal fibroblasts, 48BR (Figure 2A) and 1BR (Figure 2B), as measured by EdU incorporation. We barely detected any UDS in severe XP-patient-derived XP15BR (XP-A, Figure 2C) and XP20BE (XP-G, Figure 2D) fibroblasts, while we detected marginal (∼20% of normal) UDS activity in XP-C patient-derived XP13BR (Figure 2E) fibroblasts.Figure 2.


A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).

Limsirichaikul S, Niimi A, Fawcett H, Lehmann A, Yamashita S, Ogi T - Nucleic Acids Res. (2009)

UV-induced EdU incorporation in non-S-phase cells is NER repair replication specific. (A–G) Levels of UV-UDS in NER-proficient and NER-deficient cells were examined. Primary fibroblasts were cultured on coverslips, UVC-irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Levels of EdU incorporation were analysed as in Figure 1A and presented as in Figure 1. (H) UDS assay performed by 3H-thymidine incorporation. Quiescent cells were UVC irradiated at the indicated dose and 3H-thymidine incorporation was measured for 3 h after UVC irradiation in the media containing 10 mM hydroxyurea. UDS levels were normalized and expressed as percentages of the UDS in the normal cells at 10 J/m2. Mean Normal represents the average UDS levels of the four different normal cell lines (1BR, 48BR, 142BR and 251BR). Asterisks indicate the mean values of nuclear fluorescent intensities, which correspond to the UDS levels. Δ represents UDS difference between irradiated and un-irradiated samples. (I) Typical photos of the EdU incorporation examined in Figure 2A–G are shown (2 h after 20 J/m2 UVC irradiation). Arrows indicate non-S-phase cells. 48BR, normal; 1BR, normal; XP15BR, XP-A; XP20BE, XP-G; XP13BR, XP-C; XP12BR, XP-D; CS10LO, CS-B.
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Figure 2: UV-induced EdU incorporation in non-S-phase cells is NER repair replication specific. (A–G) Levels of UV-UDS in NER-proficient and NER-deficient cells were examined. Primary fibroblasts were cultured on coverslips, UVC-irradiated (20 J/m2), followed by 2-h incubation with 10 μM EdU. Levels of EdU incorporation were analysed as in Figure 1A and presented as in Figure 1. (H) UDS assay performed by 3H-thymidine incorporation. Quiescent cells were UVC irradiated at the indicated dose and 3H-thymidine incorporation was measured for 3 h after UVC irradiation in the media containing 10 mM hydroxyurea. UDS levels were normalized and expressed as percentages of the UDS in the normal cells at 10 J/m2. Mean Normal represents the average UDS levels of the four different normal cell lines (1BR, 48BR, 142BR and 251BR). Asterisks indicate the mean values of nuclear fluorescent intensities, which correspond to the UDS levels. Δ represents UDS difference between irradiated and un-irradiated samples. (I) Typical photos of the EdU incorporation examined in Figure 2A–G are shown (2 h after 20 J/m2 UVC irradiation). Arrows indicate non-S-phase cells. 48BR, normal; 1BR, normal; XP15BR, XP-A; XP20BE, XP-G; XP13BR, XP-C; XP12BR, XP-D; CS10LO, CS-B.
Mentions: To apply the EdU technique to XP diagnosis, we estimated the levels of UV-induced EdU incorporation in several NER-deficient primary fibroblasts as well as normal controls (Figure 2A–G for histograms, and Figure 2I for typical photos). Since XP is a genetically heterogenous disorder, both the NER genes affected and the types of mutations determine the magnitude of the defect in UDS. We measured UDS in XP15BR (XP-A), XP20BE (XP-G/CS), XP13BR (XP-C), XP12BR (XP-D) and CS-patient-derived CS10LO (CS-B) cells, and compared the results with those obtained using 3H-thymidine incorporation. With 20 J/m2 UVC irradiation, we observed substantial UDS in normal fibroblasts, 48BR (Figure 2A) and 1BR (Figure 2B), as measured by EdU incorporation. We barely detected any UDS in severe XP-patient-derived XP15BR (XP-A, Figure 2C) and XP20BE (XP-G, Figure 2D) fibroblasts, while we detected marginal (∼20% of normal) UDS activity in XP-C patient-derived XP13BR (Figure 2E) fibroblasts.Figure 2.

Bottom Line: We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU).We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads.Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 852-8523 Japan.

ABSTRACT
Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed 'unscheduled DNA synthesis (UDS)', is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

Show MeSH
Related in: MedlinePlus