Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments

Abstract

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51^T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

Keywords: Brca2; DNA replication; Mre11; Rad51; Xenopus laevis; fork protection.

Graphical abstract

Figure 1

Brca2 and Rad51 Function in DNA Replication (A) The Xenopus Brca2 protein. See also Figures S1 and S2. (B) Brca2 and Rad51 immunoprecipitations (IPs) and western blots (WBs). (C) Brca2 and Rad51 depletion from egg extract. (D) Chromatin binding time course with or without recombinant geminin. NS, no sperm nuclei. (E) Brca2 and Rad51 chromatin binding normalized to histone H2B. Mean optical density ± SD of three experiments (n = 3) is shown. (F) Chromatin binding time course in mock and Brca2-depleted extracts. (G) Relative incorporation of α-³²P-dCTP over time in mock and Brca2-depleted extracts. Counts per minute for mock-treated extracts at 150 min were considered as 100%. (H) DNA replication in extracts depleted as indicated and reconstituted with 50 ng/μL recombinant Brca2c or Brca2d proteins. L-APH (3 μM) or L-APH plus S1 nuclease (0.1 U/μL) was added at the start where indicated. Nuclei were pre-treated with MMS (1% v/v) or UV (1,000 J/m²) where indicated. Counts per minute in mock-treated extracts at 150 min were considered as 100%. Mean values ± SD (n = 3) are shown. See also Figures S1–S3.

Figure 2

ssDNA Gaps and Nascent DNA Degradation in the Absence of Brca2 (A) EM micrographs showing RIs isolated from mock-treated (left) and Brca2- (center) and Rad51-depleted extracts (right). Empty arrows indicate newly replicated strands. Arrowheads indicate ssDNA gaps at fork junctions. (B) Graph showing the distribution of fork gaps with different lengths. Bars indicate the percentage of RIs with different gap lengths in mock-treated and Brca2- or Rad51-depleted extracts. Mean values ± SEM relative to 150 RIs counted in three experiments (n = 3) are shown. (C) EM micrograph showing RIs isolated from Brca2-depleted extract. The full black arrow indicates an internal ssDNA fork gap. (D) Distribution of RIs with the indicated number of internal gaps in mock-treated and Brca2- or Rad51-depleted extracts. Mean values ± SEM (n = 3) are shown. (E) EM micrograph showing an RI isolated from an undepleted extract incubated with H-APH (1.5 mM) as shown. (F) EM micrograph showing RIs isolated from mock-treated (top) or Brca2-depleted (bottom) extracts treated with H-APH as in (E). Insets show a higher magnification of replication bubbles. Empty arrows indicate double-stranded DNA. Arrowheads show ssDNA. See also Figure S4.

Figure 3

Brca2- and Rad51-Mediated Protection from Mre11 (A) Top: experimental scheme. Bottom: relative percentage of residual biotin-dUTP in sperm nuclei quantified using a fluorescence method. The fluorescence intensity of mock at 0 min was considered as 100%. Extracts were treated as indicated and supplemented with 100 μM mirin or recombinant Brca2c or Brca2d. Mean values ± SD (n = 3) are shown. (B and C) Gel showing the effect of Rad51^WT and Rad51^T131P (B) or RPA complex (C) pre-incubation with 5′ fluorescently labeled DNA substrate (20 nM), shown in the scheme containing phosphorothioate bonds (s), and subsequent incubation with Mre11 (30 nM). Reactions were resolved on denaturing 30% polyacrylamide gel. (D) Mre11-dependent DNA degradation rates in the presence of Rad51^WT, Rad51^T131P, or RPA relative to the amount of substrate shown in (B), lane (−), which was considered as 100%. Mean values ± SD (n = 3) are shown. (E) Electrophoretic mobility shift assay showing binding of Rad51^WT, Rad51^T131P, or RPA to the same fluorescently labeled DNA substrate (20 nM) resolved on 0.8% agarose gel. See also Figures S4 and S5.

Figure 4

Smarcal1-Dependent Regulation of RVFs (A) EM micrograph showing RVF intermediate isolated from extracts treated with H-APH (1.5 mM). The arrow indicates a double-stranded reversed branch. The arrowhead indicates the single-stranded tail of the reversed branch. The inset shows a high magnification of the RVF junction. (B) Chromatin binding of the indicated factors at the indicated times following buffer or H-APH supplementation. Buffer and H-APH were added to egg extract 45 min after sperm nuclei. NS, absence of sperm nuclei. (C) RVF frequency in extracts treated as indicated. Mean values ± SEM (n = 3) are shown. ^∗,^∗∗,^∗∗∗p < 0.01, obtained by unpaired t test between the marked couples. (D) Scheme showing the assay to quantify RVFs with ssDNA tails (STAR Methods). (E) ELISA detection of BrdU in nascent ssDNA in nuclei incubated in extracts treated as shown. Where indicated, extracts were supplemented with 5 ng/μL recombinant human Smarcal1^WT or catalytically dead Smarcal1^HD. Mean intensity values ± SD (n = 3) are shown. See also Figure S6.

Figure 5

RVFs and Nascent DNA Degradation (A) Residual biotin-dUTP in nuclei replicated in extracts treated as shown. Where indicated, extracts were Smarcal1-depleted and supplemented with buffer or 5 ng/μL recombinant Smarcal1^WT or Smarcal1^HD proteins. The fluorescence intensity of mock at 0 min was considered as 100%. (B) ELISA detection of BrdU in nascent ssDNA in nuclei incubated in extracts treated as shown. Mean intensity values ± SD (n = 3) are shown. (C and D) Gel showing the effect of Rad51^WT and Rad51^T131P (C) or RPA (D) pre-incubation with 5′ fluorescently labeled RVF, shown in the scheme, and subsequent incubation with Mre11. Reactions were resolved on denaturing 30% polyacrylamide gel. (E) Mre11-dependent DNA degradation rates in the presence of Rad51^WT, Rad51^T131P, or RPA relative to the amount of substrate (20 nM) shown in (C), lane (−), which was considered as 100%. Mean values ± SD (n = 3) are shown. (F) DNA binding of Rad51 WT or RPA to the same fluorescently labeled RVF DNA substrate (20 nM) resolved on 0.8% agarose gel. See also Figure S6.

Figure 6

Rad51-Pol α Interaction at Stalled Forks (A) WB of egg extract IPs using the indicated antibodies. Flowthrough (FT) and extract input (XE) are also shown. (B) Top: Coomassie-stained gel showing pull-down experiments using Rad51 and hPol α complexes made of the Pol α catalytic subunit, B subunit, Pri-S, and Pri-L. The Pol α catalytic and B subunits were either full-length or missing the first 333 and 148 amino acids, respectively (ΔNTD). His-tagged Pri-S was used for the pull-down. Bottom: WB with anti-Rad51 antibodies of samples shown at the top. (C) Top: Coomassie-stained gel showing pull-down experiments using Rad51 and the first 109 amino acids of Pol α (MBP-Pol α NTD) bound to amylose resin. Bottom: WB with anti-Rad51 antibodies of the samples shown at the top. (D) Coomassie-stained gel showing pull-down experiments using Rad51 and the first 109 amino acids of Pol α (MBP-Pol α NTD) bound to amylose resin in the presence of increasing amounts of BRC4 peptide. (E) Chromatin binding time course following addition of recombinant human full-length (hPol α) and NTD truncated (ΔNTD) complexes shown in (B) in the presence of DMSO or M-APH. Anti-Xenopus Pol α p180 and anti-human Pol α p180 were used for the WB shown at the top and center, respectively. (F) IPOND showing proteins bound to chromatin containing nascent DNA following biotin pull- down with streptavidin beads. Extracts were supplemented with M-APH 45 min after nuclei addition and pulse-labeled for 10 min with biotin-dUTP as indicated. Pol α, δ, and ε inputs are also shown. (G) DNA replication in mock-treated or Rad51-depleted restarting extracts (Figure S7F). Values in mock-treated stalling extracts transferred to mock-treated restarting extracts were considered as 100%. Mean values ± SD (n = 3) are shown. See also Figure S7.

Figure 7

General Model See Discussion for an explanation.