MRN, CtIP, and BRCA1 mediate repair of topoisomerase II-DNA adducts

Abstract

Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein-DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)-DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2-DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication.

Figure 1.

Replication of etoposide-treated chromosomal DNA requires CtIP. (A) CtIP immunodepletion from LSS extracts. Control (ΔMock) and CtIP-depleted extracts (ΔCtIP) were probed with the indicated antibodies. (B) Mock and CtIP-depleted extracts were incubated with sperm nuclei. DNA replication was monitored by agarose gel electrophoresis after incorporation of α-[³²P]dCTP into genomic DNA at the indicated time points. (C) Quantification of three independent experiments as shown in B. The mean is plotted, and error bars indicate the standard deviation. (D) Control (mock) and CtIP-depleted extracts were supplemented with sperm nuclei, and DNA replication of Xenopus sperm nuclei was monitored by alkaline gel electrophoresis after incorporation of α-[³²P]dCTP into genomic DNA at the indicated time points. (E) DNA replication was monitored as in B in the presence of a low dose (2 µM) of etoposide in mock- or CtIP-depleted extracts, below quantification of three independent experiments (**, P = 0.003, two-tailed unpaired t test; n = 3). (F) Effect of ATM and ATR inhibitors in the sensitivity to low-dose etoposide (2 µM). Bar graph shows quantification of three independent experiments (*, P = 0.014, two-tailed unpaired t test; n = 3).

Figure 2.

MRN and CtIP remove Top2 adducts during S phase. (A) Mock or CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and incubated in the presence of 100 µM etoposide for 40 min. Reactions were stopped and diluted in denaturing buffer, and genomic DNA was fractionated via CsCl gradients. The fractions were transferred and probed with anti-Xenopus Top2 antibodies. (B) Relative quantification of three independent experiments as shown in A. Error bars indicate standard deviations (*, P = 0.021, two-tailed unpaired t test). (C) CtIP, Mre11, or CtIP/Mre11 immunodepletion from LSS extracts. Control (ΔMock) and depleted extracts were blotted with the indicated antibodies. (D) Quantification of Top2–DNA adducts in Mre11-depleted, CtIP-depleted, or MRE11- and CtIP-depleted extracts as described in A. (*, P < 0.05; **, P < 0.01, two-tailed unpaired t test; n = 3). (E) Quantification of Top2–DNA adducts in CtIP-depleted extracts and CtIP-depleted extracts supplemented with recombinant CtIP protein (100 nM in extract; *, P = 0.048, two-tailed unpaired t test; n = 3). (F) Quantification of Top2–DNA adducts in Mre11-depleted extracts and Mre11-depleted extracts supplemented with recombinant MRN protein complex (500 nM in extract; *, P = 0.018, two-tailed unpaired t test; n = 3).

Figure 3.

CtIP regulates resection from Top2 adducts. (A) Control (ΔMock) and CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and incubated with 0.05 U/µl PflMI or 100 µM etoposide (Etop.). Chromatin was purified at the indicated times and processed for Western blot with Top2, CtIP, RPA70, Ku70, and H3 (loading control) antibodies. NS, no sperm control. (B) Plot of RPA binding as shown in A for control and CtIP-depleted extracts treated with PflMI or etoposide. Shown are mean RPA intensities. Error bars represent SD; n = 3. (C) Chromatin-bound RPA70 was monitored in control (ΔMock) and CtIP-depleted extracts treated with etoposide or ICRF-193. a.u., arbitrary units.

Figure 4.

Exo1 cannot process Top2–DNA adducts. (A) Mock, Exo1, or CtIP immunodepletions from LSS extracts. Control (ΔMock) and depleted extracts were blotted with the indicated antibodies. (B) Quantification of Top2–DNA adducts in CtIP-depleted or Exo1-depleted extracts treated with etoposide as described (*, P = 0.02, two-tailed unpaired t test; NS, not statistically significant). (C) Control (ΔMock) or CtIP-depleted replication-incompetent, membrane-free extracts (HSS) were supplemented with sperm nuclei (5,000/µl) and treated with etoposide. (D) Chromatin fractions from extracts in C were blotted with the indicated antibodies.

Figure 5.

BRCA1 is required to process Top2–DNA adducts. (A) Mock and BRCA1 immunodepletions from LSS extracts; extracts were blotted with the indicated antibodies. (B) DNA replication in BRCA1-depleted extracts is sensitive to low-dose etoposide. Top: control (ΔMock) and BRCA1-depleted extracts were incubated with sperm nuclei. DNA replication was monitored by agarose gel electrophoresis after incorporation of α-[³²P]dCTP into genomic DNA; bottom: quantification of three independent experiments. The mean is plotted, and error bars indicate the standard deviation (*, P < 0.026, unpaired two-tailed t test; n = 3). (C) Relative quantification of Top2–DNA adducts in BRCA1-depleted extracts (*, P = 0.02, two-tailed unpaired t test; n = 3). (D) Mock or BRCA1-depleted extracts were supplemented with sperm nuclei (5,000/µl), treated with PflMI or etoposide as indicated, and incubated for 40 min. Chromatin was isolated and immunoblotted with the indicated antibodies.

Figure 6.

CtIP–BRCA1 interactions are required for CtIP-dependent processing of etoposide-induced DSBs. (A) Control extract (no damage), extracts treated with etoposide (Etop.), or extracts treated with PflM1 were incubated with the CDK inhibitor roscovitine. Chromatin was isolated at 0 or 40 min and processed for Western blot with the indicated antibodies. Roscovitine inhibits genomic DNA replication (bottom). (B) Control (ΔMock) and CtIP-depleted extracts (ΔCtIP) with or without etoposide were supplemented with sperm nuclei, and replication was monitored by agarose gel electrophoresis after incorporation of α-[³²P]dCTP into genomic DNA. CtIP-depleted extracts were supplemented with recombinant xCtIP WT or CtIP-S328A mutant (xCtIP S328A). Bar graph shows quantification of three independent experiments (*, P = 0.026, two-tailed unpaired t test). (C) Relative quantification of Top2–DNA adducts in CtIP-depleted extracts supplemented with buffer, xCtIP WT, or xCtIP S328A (*, P = 0.035, two-tailed unpaired t test; NS, not statistically significant [P = 0.08]; n = 3). (D) Control (ΔMock) and CtIP-depleted extracts were supplemented with sperm nuclei (5,000/µl) and treated with either PflM1 endonuclease or etoposide. CtIP-depleted extracts were supplemented with recombinant xCtIP WT or xCtIP S328A. Chromatin was isolated and processed for Western blotting with the indicated antibodies. NS, no sperm control; Ext., extract.