Tipin and Timeless form a mutually protective complex required for genotoxic stress resistance and checkpoint function

Abstract

Tipin is a mammalian protein that interacts with Timeless, which plays a role in DNA damage checkpoint responses. Here, we show that Tipin is a nuclear protein that associates with the replicative helicase and protects cells against genotoxic agents. Tipin is required for efficient cell cycle arrest in response to DNA damage, and depletion of Tipin renders cells sensitive to ionizing radiation as well as replication stress. Loss of Tipin results in spontaneous gamma-H2AX foci, a marker for DNA double-strand breaks. We find that Tipin and Timeless form a complex that maintains the level of both proteins in cells and that the loss of either one will lead to the loss of the interacting partner. This observation explains the similar checkpoint phenotypes observed in both Tipin- and Timeless-depleted cells.

Fig. 1.

Expression and localization of Tipin in proliferating cells. (A) Lysates from HeLa cells transfected with either an empty vector or a vector that expresses epitope-tagged Tipin were analyzed by Western blotting using the anti-Tipin antibody. (B) Lysates from HeLa cells transfected with control or Tipin siRNAs were analyzed by Western blotting. (C) HeLa cells were synchronized by sequential thymidine and nocodazole blocks, harvested at the indicated time points after nocodazole release, and analyzed by Western blotting using the indicated antibodies. (D) HeLa cells were grown overnight on coverslips and costained with the anti-Tipin antibody (green) and DAPI (blue). (E) HeLa or U2OS cells treated with either control or Tipin siRNAs were stained with propidium iodide (for DNA content) and analyzed by flow cytometry. Error bars indicate the standard deviation from three independent samples.

Fig. 2.

Endogenous Tipin associates with Timeless and Mcm proteins. Equal amounts of cell lysates from mock-treated or HU-treated (10 mM for 1 h) cells were immunoprecipitated with either anti-Tipin or anti-GAPDH (as control) antibodies. To rule out coprecipitation of proteins linked only by DNA, lysates were treated with Benzonase to digest cellular DNA. Precipitated proteins were immunoblotted with the indicated antibodies to detect coimmunoprecipitating proteins.

Fig. 3.

Tipin deficiency sensitizes cells to DNA damage and replication stress and compromises both the G₂/M and intra-S-phase DNA damage checkpoints. (A) HeLa cells were transfected with control, Tipin, or ATM siRNAs, plated at low density, and treated with the indicated doses of IR. Colonies were counted 2 weeks later. (B) HeLa cells were transfected with control, Tipin, or Timeless siRNAs, plated at low density, and treated with 2 mM HU for 16 h. Colonies were counted 2 weeks later. (C) U2OS cells were transfected with the indicated siRNAs and either mock-treated or exposed to 3 Gy of γ-irradiation 1 h before harvesting. Mitotic cells were detected by propidium iodide and phosphohistone H3 staining and analyzed by flow cytometry. Percentages of mitotic cells and their levels normalized to control (in parentheses) are shown. (D) Control, ATR, Tipin, or Timeless siRNA-treated U2OS cells were exposed to the indicated levels of γ-irradiation 1 h before harvesting. Mitotic cells were detected by propidium iodide and phosphohistone H3 staining and analyzed by flow cytometry. (E) U2OS cells transfected with control, ATR, Tipin, or Timeless siRNAs were exposed to 10 Gy of γ-irradiation and assayed for DNA synthesis 30 min later by [³H]thymidine incorporation. The amount of DNA synthesis after irradiation is expressed as a percentage of the level in untreated cells. Error bars indicate standard deviation from three independent experiments.

Fig. 4.

Loss of Tipin leads to spontaneous formation of γ-H2AX foci in the absence of exogenous damage. (A) Control or Tipin siRNA-treated HeLa cells were grown on coverslips, fixed with formaldehyde, and coimmunostained with anti-γ-H2AX and anti-Tipin antibodies. The merged picture shows cells stained with anti-γ-H2AX (green) and anti-Tipin (red) antibodies as well as DAPI (blue). (B) HeLa cells were treated with control or Tipin siRNAs and immunoblotted with anti-Tipin, anti-γ-H2AX, or anti-Ran antibodies.

Fig. 5.

Depletion of Tipin or Timeless leads to the loss of both proteins. (A) HeLa cells were transfected with control siRNA or one of the two different siRNAs targeting Tipin. Lysates were analyzed by Western blotting with antibodies against Tipin, Timeless, or GAPDH. (B) Lysates from cells treated with control siRNA or one of the two different siRNAs targeting Timeless were analyzed by Western blotting with antibodies against Timeless, Tipin, or GAPDH.