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. 2010 Nov;17(11):1305-11.
doi: 10.1038/nsmb.1927. Epub 2010 Oct 10.

Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis

Yoshitami Hashimoto  1 , Arnab Ray Chaudhuri, Massimo Lopes, Vincenzo Costanzo
Affiliations

Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis

Yoshitami Hashimoto et al. Nat Struct Mol Biol. 2010 Nov.

Abstract

The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading- and lagging-strand DNA synthesis. In contrast, ssDNA gaps behind forks, which are prevalent on damaged templates, result from Mre11-dependent degradation of newly synthesized DNA strands and are suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct roles for Rad51 at replication forks, demonstrating that Rad51 protects newly synthesized DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.

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Figures

Figure 1
Figure 1
Rad51 binding to undamaged and damaged chromatin during DNA replication. (A) The time course of chromatin association of Rad51 and the indicated replication proteins. Immunoblotting was performed for the chromatin fractions that were incubated in 30 μl of egg extract for the indicated times in the presence or absence of aphidicolin (10 μg ml−1) or EcoRl (0.1 unit μl−1). Where indicated sperm nuclei were treated with 1,000 J m −2 UV and 1% (v/v) MMS, respectively. As a control, 0.5 μl egg extract was also immunoblotted (ext). To measure the relative amount of Rad51 per fork we calculated the ratio between Rad51 and Psf2 signal intensity at 40 min, which was 0.35 for untreated extracts, 0.57 for aphidicolin, 0.55 for UV and 0.51 for MMS treated extracts. (B) The effect of BRC4 on the chromatin association of Rad51 and replication fork proteins. Immunoblotting was performed for the chromatin fractions that were incubated in 25 μl of egg extract for 60 min in the presence of 0.5 mg ml−1 GST or 0.5 mg ml−1 GST-BRC4. Sperm nuclei were incubated in extracts that were unreated (−) or incubated with 50 μg ml−1 aphidicolin. Where indicated sperm nuclei were irradiated with UV at 1,000 J m−2 or treated with 1% (w/v) MMS before the incubation in egg extract. As a control, 1 μl egg extract was also immunoblotted (ext). (C) Quantification of Rad51 bound to damaged and undamaged chromatin in the presence (+ geminin) or in the absence (− geminin) of 160 nM geminin, and in the presence (+p27) or absence (−p27) of 40 μg ml−1 p27 recombinant protein. The graph shows the average relative values of several repeated experiments taking as reference the amount of Rad51 bound to undamaged chromatin in the presence of geminin or p27 (C; control). Error bars indicate standard deviations. Representative immunoblots are shown in Supplementary S1A and S1B.
Figure 2
Figure 2
Rad51 and PCNA modifications in DNA replication and ssDNA gap accumulation. (A) Rad51 and PCNA requirement for replication of untreated and MMS-treated DNA. Sperm nuclei were incubated in 10 μl egg extract with α32P-dATP for the indicated times in the presence or absence of 0.7 mg ml−1 GST or GST-BRC4 and MMS (− or +), and 0.2 mg ml−1 of recombinant wild type PCNA (WT) or mutated PCNA (K164R). Replication products were resolved on 1% (w/v) alkaline agarose gel and subjected to autoradiography. (B) The signal intensities obtained in (A) were quantified and reported on the graph. The experiments shown represent a typical result. (C) Gap labelling procedure using T4 DNA polymerase. Replicating genomic DNA was isolated and used as a template for gap-filling assay using T4 DNA polymerase. The labelled nascent molecules extended by T4 were then resolved on alkaline agarose gel. (D) Untreated (−MMS) and MMS treated (+MMS) sperm nuclei were incubated in 10 μl of egg extract in the presence of GST or GST-BRC4 for 60 min (1–4). Untreated sperm nuclei were incubated for 40, 60 or 80 min in the presence of PCNA-WT or PCNA-K164R (5–10). Genomic DNA was isolated and subjected to the gap labelling reaction followed by autoradiography. Exposure times are equivalent for the 2 gels although kinetic profile starts at 40 minutes in 5–10. The graph shows the relative fold increase in optical density measured for each lane taking as reference untreated chromatin recovered at 60 minutes. The experiment shows a typical result.
Figure 3
Figure 3
Rad51 is required to prevent replication fork uncoupling and ssDNA accumulation on damaged and undamaged templates. (A) and (C) Electron micrographs (and schematic drawings) of representative RIs isolated from sperm nuclei, incubated in GST-BRC4 treated extracts. Black arrows point to ssDNA regions at the replication fork. White arrows point to ssDNA gaps along the replicated duplexes (internal gaps). (B) Statistical distribution of internal gaps in the analyzed population of molecules. The total number of molecules analyzed is indicated in brackets. (D) Statistical distribution of ssDNA length at replication forks isolated in the indicated conditions. The total number of forks analyzed is indicated in brackets.
Figure 4
Figure 4
Accumulation of ssDNA gaps in the absence of Rad52 and Rad51 in S. cerevisiae. Upper panels: Electron micrographs of a representative RIs isolated from rad52 mutant S. cerevisiae growing cells. The black arrow points to an extended ssDNA regions at the replication fork. White arrows show ssDNA gap behind the fork. Lower panels: Statistical distribution of ssDNA gap length (left) and of the number of ssDNA gaps (right) observed on RIs isolated from wild type, Rad52 and Rad51 mutant S. cerevisiae growing cells.
Figure 5
Figure 5
Rad51 protects nascent strand DNA from Mre11-dependent degradation. (A) Statistical distribution of internal gaps in the analyzed population of molecules isolated from extracts that were supplemented with buffer (Control) or 100 μM Mirin and treated as indicated. The total number of molecules analyzed is indicated in brackets. (B) Statistical distribution of ssDNA length at replication forks isolated from extracts that were supplemented with buffer (Control) or 100 μM Mirin and treated as indicated. The total number of molecules analyzed is indicated in brackets. in the indicated conditions.
Figure 6
Figure 6
A model for possible roles of Rad51 during DNA replication. See text for explanation.
See this image and copyright information in PMC

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