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. 2012;7(5):e36970.
doi: 10.1371/journal.pone.0036970. Epub 2012 May 15.

The Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair

Ian R Kelsall  1 , Judith Langenick, Craig MacKay, Ketan J Patel, Arno F Alpi
Affiliations

The Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair

Ian R Kelsall et al. PLoS One. 2012.

Abstract

The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes--UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. UBE2T deficient cells are sensitive to a range of DNA damaging agents including UV light.
(A–F) Clonogenic survival assays to assess viability of indicated cell lines that have been exposed to MMS (methylmethanesulfonate) (A), HU (hydroxyurea) (B), IR (ionizing radiation) (C) and UV (UV-C, 254 nm) (D–F). Error bars represent one standard error of the mean from at least three independent experiments. t-tests have been performed for indicated survival curves. P≥0.05 not significant (NS), P≤0.05 statistically significant (*), P≤0.01 statistically significant (**);
Figure 2
Figure 2. UBE2T and FANCM cooperate to protect cells from UV-induced damage.
(A) Colony survival of indicated cell lines after exposure to increasing doses of UV light. Error bars represent one standard error of the mean from at least three independent experiments. t-tests have been performed for indicated survival curves. P≥0.05 not significant (NS); (B) Exponential growth curve of indicated cell lines. Cell density measurements were carried out over a 72 hr time period. Error bars represent one standard error of the mean from at least two independent experiments that were performed in duplicate. t-tests have been performed for indicated growth curves with calculated P values P≤0.05 (*); (C) Immunoblot analysis to monitor MMC-induced FANCD2 monoubiquitylation. Cells were mock treated (−) or exposed to 150 ng/ml MMC (+) for 18 hrs and whole cell lysates were probed for FANCD2 by immunoblotting. Monoubiquitylated FANCD2 resolves as a slower migrating band (indicated by D2-Ub). (D) Immunoblot analysis to monitor UV light-induced FANCD2 monoubiquitylation. Cells were irradiated with indicated doses of UV light and allowed to recover for 0, 0.5, 3 and 6 hrs. Whole cell lysates were probed for FANCD2 by immunoblotting.
Figure 3
Figure 3. Robust UV-induced checkpoint activation in ube2t
−/− cells. (A) Wild type and ube2t−/− cells were arrested in M phase with nocodazole. Cells were then released and synchronized cell populations in either G1 (black) or S (red) cell populations were treated with 0, 3 and 6 Jm−2 UV light. Cell survival was determined in a colony survival assay. Error bars represent one standard error of the mean from two independent experiments. In addition t-tests have been performed for indicated survival curves. P≤0.05 statistically significant (*) and P≤0.01 statistically significant (**). (B) Asynchronous populations of wild type and ube2t−/− cells were irradiated with 2 Jm−2, then fixed. Then cells were subjected to Propidium Iodide cell cycle analysis after indicated time points. The estimated percentages of cells in G1, S, G2/M phases are presented in a table. (C) Analysis for phospho-H2AX (γ-HA2X) in response to MMC (150 ng/ml) or UV (10 Jm−2). Soluble chromatin fractions were prepared from wild type and ube2t−/− cells and subjected to immunoblot analysis. Immunoblot analysis for Histone 3 (H3) and Ponceau S stained nitrocellulose membrane showing equal loading. (D) Analysis for phospho-CHK1 (pS345-CHK1) levels in response to UV (10 Jm−2). Whole cell lysates were prepared from wild type and ube2t−/− cells and subjected to immunoblot analysis using a phospho-CHK1 (pS345-CHK1) specific antibody. Actin immunoblot analysis was used as a loading control.
Figure 4
Figure 4. XPA is epistatic with UBE2T and FANCM in response to UV light.
(A) The two mechanistically different routes to resolve UV photolesions: Translesion synthesis (TLS) and Nucleotide Excision Repair (NER). (B, C, E, and F) Cellular viability after exposure to increasing UV doses was determined for the indicated cell lines by clonogenic survival assays. Error bars represent one standard error of the mean from at least three independent experiments. In addition t-tests have been carried out for indicated survival curves. P≥0.05 not significant (NS), P≤0.05 statistically significant (*). (D) Immunoblot showing PCNA monoubiquitylation in whole cell lysates of wild type and ube2t−/− cells after exposure to UV (60 Jm−2) and the indicated recovery time. Immunoblot for β-actin confirms equal protein loading.
Figure 5
Figure 5. UBE2T deficiency affects NER-mediated removal of cyclobutane pyrimidine dimers
(CPDs). Genomic DNA was isolated from UV irradiated (5 Jm−2) cell lines after indicated time points and analysed for the presence of photoproducts as described in Material and Methods. (A)(B) show the progression of (6–4)PP photoproduct removal and (D)(E) the removal CPDs. The amounts of both types of lesions are presented as percentages of those at time 0. (C) and (F) show representative immunoblots for (6–4)PPs and CPDs respectively. Error bars represent standard error of the mean from three (for (6–4)PP) and six (for CPDs) independent experiments. t-tests have been performed for indicated curves. P≥0.05 not significant (NS), P≤0.05 statistically significant (*).
Figure 6
Figure 6. UBE2T is not required for UV-induced XPC polyubiquitylation.
Immunoblot analysis for UV light induced XPC ubiquitylation in siUBE2T and non-target siCON transfected U2OS cells. Lysates of UV (20 Jm−2) irradiated cells were prepared after indicated chase times. Immunoblot for UBE2T and FANCD2 shows efficient knock down of UBE2T protein levels and defective UV-induced FANCD2 monoubiquitylation. Two time exposures are presented for anti-XPC.
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