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Cell Cycle
2014 Jan 01;1313:2120-8. doi: 10.4161/cc.29157.
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Mta2 promotes Tipin-dependent maintenance of replication fork integrity.
Errico A
,
Aze A
,
Costanzo V
.
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Orderly progression of S phase requires the action of replisome-associated Tipin and Tim1 proteins, whose molecular function is poorly understood. Here, we show that Tipin deficiency leads to the accumulation of aberrant replication intermediates known as reversed forks. We identified Mta2, a subunit of the NuRD chromatin remodeler complex, as a novel Tipin binding partner and mediator of its function. Mta2 is required for Tipin-dependent Polymerase α binding to replicating chromatin, and this function is essential to prevent the accumulation of reversed forks. Given the role of the Mta2-NuRD complex in the maintenance of heterochromatin, which is usually associated with hard-to-replicate DNA sequences, we tested the role of Tipin in the replication of such regions. Using a novel assay we developed to monitor replication of specific genomic loci in Xenopus laevis egg extract we demonstrated that Tipin is directly required for efficient replication of vertebrate centromeric DNA. Overall these results suggest that Mta2 and Tipin cooperate to maintain replication fork integrity, especially on regions that are intrinsically difficult to duplicate.
Cancer Research UK, 206281 European Research Council, 13-0026 Worldwide Cancer Research, AICR_13-0026 Worldwide Cancer Research, ERC_206281 European Research Council
Figure 1. Tipin prevents the formation of reversed forks. (A) Immunoblot to assess Tipin depletion from egg extract. (B and C) Examples of reversed replication forks in Tipin-depleted extracts. Image magnification of the reversed fork shown in the corner box. (D) Quantification of reversed forks. Experiments were repeated at least 3 times. The graph shows the results of a typical experiment, where at least 100 forks were scored in mock or Tipin-depleted extracts.
Figure 2. Tipin directly interacts with Mta2. (A) Table indicating the Mta2 peptides identified via mass spectrometry analysis in Tipin immunoprecipitates. (B) Equal amounts of extract were immunoprecipitated with either anti-Tipin, anti-Mta2 antibodies or pre-immune serum. Purified proteins were immunoblotted with the indicated antibodies to detect associated proteins. (C) Pull down assay using His-Tipin and in vitro translated 35S-labeled Mta2. Mta2 was detected by autoradiograph.
Figure 3. Mta2 is required for DNA replication under minimum licensing conditions. (A) Immunoblot to assess Tipin or Mta2 depletion from egg extract. (B) Scheme of minimum licensing conditions experiments and replication assays. (C) DNA replication in âmaximumâ or âminimum licensedâ mock, Tipin or Mta2 depleted extract at 60 min. (D) The efficiency of DNA replication in âminimum licensedâ mock (blue), Tipin (red), Mta2 (yellow), and Tipin/Mta2 (green) co-depleted extracts at 60 min from nuclei addition to egg extract was tested. The defect in DNA replication observed in the Mta2 depleted extract (yellow) was rescued by the addition of 400 nM Mta2 recombinant protein (purple). Three independent experiments are averaged in the bar graphs. The error bars are standard deviation from the mean value.
Figure 4. Mta2 is required for Tipin loading on the chromatin. (A) Immunoblot analysis to detect the level of Mta2, Tipin, Orc1, and Histone H2B on the chromatin at different time points (30, 60, 90 min) in extract untreated or treated with geminin or aphidicolin. (B) Immunoblot to detect the levels of Mta2, Tipin, and Orc1 on the chromatin at 60 min from nuclei addition.
Figure 5. Mta2 is required for the efficient association Pol α to the chromatin in minimum licensing conditions. Sperm nuclei were added to mock or Mta2 depleted Xenopus egg extract. Geminin was supplemented 3 min post-nuclei addition to generate a minimum licensing system and chromatin was harvested at different times. Chromatin-bound proteins were analyzed by SDS-PAGE and immunoblot analysis was performed with the indicated antibodies.
Figure 6. Mta2 prevents the formation of reversed forks. (A) Example of reversed replication fork in Mta2 depleted extract. (B) Quantification of replication intermediates with reversed forks in mock or Mta2-depleted extract performed as in Figure 1D. Experiments were repeated at least 3 times. The graph shows the results of a typical experiment where at least 100 forks were scored in mock or Mta2-depleted extracts.
Figure 7. Tipin is required for replication of centromeric DNA regions. (A) BACs were incubated in egg extracts and analyzed for nuclei morphology (DAPI), membrane formation (DHCC staining), and replication (Cy3-dCTP incorporation). (B and C) Control and centromeric sequences containing BACs and were incubated in mock or Tipin-depleted extracts supplemented with α32P-dCTP to monitor DNA replication, which was measured following 3 h incubation. The graph in (B) shows the results of one representative experiment using 4 different centromeric BACs with the relative incorporation of α32P-dCTP. The graph in (C) shows the average efficiency of DNA replication obtained from 3 independents experiments using control and centromeric bacmids. Errors bars indicate standard deviation. (D and E) Chromatin isolated from mock or depleted extracts at the different times after centromeric BAC addition to egg extract and analyzed by western blotting with the indicated antibodies.
Figure 8. Model for TipinâMta2 function in maintaining fork integrity. (A) TipinâTim1 complex might create a bridge between replisome components such as Cdc45, GINS, Pol α, and the MCM complex necessary for the stable binding of Pol α to the replication fork. Mta2 might facilitate Tipin function at specific genome loci containing repetitive DNA sequences. The presence of Tipin might promote Pol α-mediated replication restart downstream lesions or structures that halt polymerases progression. (B) In the absence of Mta2 and/or Tipin inefficient re-priming and inability to resume fork progression leads to fork reversal.
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