Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts - PubMed (original) (raw)

Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts

Alessia Errico et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Tipin and its interacting partner Tim1 (Timeless) form a complex at replication forks that plays an important role in the DNA damage checkpoint response. Here we identify Xenopus laevis Tipin as a substrate for cyclin E/cyclin-dependent kinases 2 that is phosphorylated in interphase and undergoes further phosphorylation upon entry into mitosis. During unperturbed DNA replication, the Tipin/Tim1 complex is bound to chromatin, and we were able to detect interactions between Tipin and the MCM helicase. Depletion of Tipin from Xenopus extracts did not significantly impair normal replication but substantially blocked the ability of stalled replication forks to recover after removal of a block imposed by aphidicolin. Tipin-depleted extracts also showed defects in the activation of Chk1 in response to aphidicolin, probably because of a failure to load the checkpoint mediator protein Claspin onto chromatin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Tipin is a substrate for cyclin E/CDK2. (A) _In vitro_-translated Tipin was supplemented or not with recombinant cyclin E/CDK2 and ATP. Alternatively, 35S-labeled protein was added to a Xenopus extract in the presence or absence of roscovitine. (B) Cyclin E- or cyclin A-CDK2 and ATP were added to _in vitro_-translated Tipin (wild type or S326A mutant). (C) Recombinant GST-Tipin (wild type or S326A) was incubated with increasing concentrations of cyclin E- or cyclin A-CDK2 and [γ-32P]ATP. (D) 35S-labeled Tipin was added to a Xenopus cycling extract, and samples were collected from 0 to 40 min. (E) Wild-type 35S-labeled Tipin was added to an interphase or metaphase arrested extract (CSF, cytostatic factor-mediated metaphase II arrested extract). Samples were analyzed by SDS/PAGE and autoradiography.

Fig. 2.

Endogenous Tipin is a chromatin-associated phosphoprotein that interacts with Tim1 and MCM7. (A) Xenopus extract was treated (lane 2) or not (lane 1) with λ-phosphatase in the presence (lane 3) or absence of sodium vanadate. Endogenous Tipin was detected by immunoblotting. (B) Equal amounts of extract were immunoprecipitated with anti-Tipin and anti-Tim1 antibodies or preimmune serum. Purified proteins were immunoblotted as indicated. (C) Sperm nuclei (3,000 nuclei per microliter) were added to Xenopus extract, and chromatin was harvested at different times. Chromatin-bound proteins were analyzed by immunoblotting. (D) Sperm nuclei were added to Xenopus extract, and chromatin was harvested in the presence or absence of geminin (5 ng/μl). Samples were probed with anti-Tipin antibody. (E) Xenopus extract was immunoprecipitated with either anti-Tipin or preimmune serum. Samples were probed with anti-MCM7 antibody.

Fig. 3.

Tipin is not essential for replication. (A) Mock- or Tipin-depleted extracts were supplemented with sperm nuclei (3,000 nuclei per microliter) and cy3-dATP (Amersham). After 30 min, 3 μl was spotted on a microscope slide and mixed with a fixative containing Hoechst 33258 (blue fluorescence). Red fluorescence due to cy3-dATP incorporation indicates a replicating nucleus. (B) Sperm nuclei (1,000 nuclei per microliter) were added to an interphase Xenopus extract (mock- or Tipin-depleted). Replication was labeled with [α-32P]dATP from 0 to 30 min, from 30 to 60 min, from 60 to 90 min, or from 90 to 120 min from nuclei addition. Replicated DNA was purified, loaded on agarose gel, and analyzed by autoradiography.

Fig. 4.

Tipin is required for Chk1 phosphorylation and Claspin loading on chromatin. (A) Sperm nuclei were added to mock-depleted (lanes 1 and 2) or Tipin-depleted (lanes 3 and 4) extract in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of aphidicolin and incubated at 23°C for 70 min. Nuclei were purified, and proteins were analyzed by immunoblot with anti-phospho-Chk1 antibody. Recombinant Tipin (wild type, lane 5; S326A, lane 6) or 1 μl of mock-depleted extract (LSS, lane 7) were added to the depleted extract, and the experiment was performed as described above. (B) Mock- or Tipin-depleted extract (1 μl) was analyzed by immunoblotting as indicated. (C) Chromatin was purified from mock-depleted (lane1) or Tipin-depleted (lanes 2–5) extract supplemented with aphidicolin and recombinant wild-type Tipin (lane 3) or S326A Tipin (lane 4) or 1 μl of extract (LSS, lane 5). Samples were probed with anti-Claspin antibody.

Fig. 5.

Tipin depletion induces accumulation of ssDNA and impairs recovery from stalled replication forks. (A) Mock-depleted (lanes 1–3) or Tipin-depleted (lanes 4–6) extract was incubated with 20 μM aphidicolin. Chromatin was harvested at 10, 20, and 40 min and analyzed by immunoblotting with anti-RPA and anti-ORC1 antibodies. The amount of RPA loaded on the chromatin was determined with respect to ORC1. Three independent experiments are averaged in the bar graphs; mock-depleted extract is represented by blue bars, and Tipin-depleted extracts are represented by red bars. (B) Chromatin replication was initiated in an interphase extract (damaging extract). Sperm nuclei were added to the extract and incubated at 23°C to allow origins to fire and replication to start. After 15 min, fork progression was blocked by adding 40 μM aphidicolin and 0.5 mM roscovitine. Damaged forks were isolated at 60 min and added to a second extract (restarting extract) made incompetent for origin firing and origin assembly by addition of 0.5 mM roscovitine and 5 ng/μl geminin. Different damaging and restarting (mock- or Tipin-depleted) extracts were used as stated in the different lanes. Lanes 1 and 2, Xenopus extract either with (lane 2) or without (lane 1) aphidicolin in the first incubation; lanes 3–7, all had aphidicolin in the first incubation; lane 3, both damaging and restarting extract were mock-depleted; lanes 4 and 5, the damaging extract was Tipin-depleted, and forks were restarted in a mock-depleted extract; lane 5, caffeine was added to the second incubation; lane 6, both damaging and restarting extract were Tipin-depleted; lane 7, the same as lane 6 except that recombinant Tipin was added in the second incubation. In all cases, [α-32P]dCTP was added in the second incubation to monitor replication. Incorporation was analyzed by alkaline agarose gel electrophoresis and autoradiography. Three independent experiments are averaged in the bar graphs, taking the amount of replication of the positive control (e.g., lane 2) as 100%. The error bars represent standard deviations from the mean values.

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Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts - PubMed (original) (raw)