BRCA2 associates with MCM10 to suppress PRIMPOL-mediated repriming and single-stranded gap formation after DNA damage

Abstract

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.

Fig. 1. BRCA2 restrains replication fork progression after DNA damage.

a Western blots showing levels of BRCA2 in U2OS cells treated with 2 different control and 3 different BRCA2 siRNAs. Similar results were obtained in n = 3 independent experiments. b Labeling scheme and a representative image of the DNA fiber assay. c Lengths of IdU-labeled replication tracts in U2OS cells treated with control and BRCA2 siRNA before and 6 h after 10 Gy of IR. d–f Replication tract lengths in U2OS cells treated with control (NSC1) and BRCA2 (1949) siRNAs before and 1, 3, and 6 h after treatment with 10 Gy of IR (d), 2 Gy of IR (e), or 10 µM of bleomycin (BLEO) (f). g Replication tract lengths in control and BRCA2-depleted U2OS cells after 6 h of continuous treatment with DMSO, BLEO (1 µM), MMS (50 µM), CPT (1 µM), and HU (50 µM). h Levels of BRCA2 and MCM10 in U2OS, VC8, and VC8 cells reconstituted with a human BRCA2 cDNA. i, j Replication tract lengths in VC8 and VC8 + BRCA2 cells before and 3 and 6 h after 10 Gy of IR (i) or 6 h after BLEO treatment (j). Data in (c–g) and (i, j) are presented as mean ± standard deviation (s.d.), with the number of dots shown below each column. Similar results were obtained from n = 2 biologically independent experiments. P values are calculated using two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Fig. 2. BRCA2 suppresses PRIMPOL-mediated repriming and ssDNA gap formation after DNA damage.

a Western blots showing siRNA-mediated depletion of HLTF, SMARCAL1, and ZRANB3 in U2OS cells. Two siRNAs for each gene were used. Similar results were obtained in n = 3 independent experiments. b, c Replication tract lengths in U2OS cells depleted of BRCA2, HLTF, SMARCAL1, or ZRANB3 either before (b) or 6 h after (c) 10 Gy of IR. d Replication tract lengths in U2OS cells co-depleted of BRCA2 with HLTF, SMARCAL1, or ZRANB3 at 6 h after 10 Gy of IR. e Western blots showing the levels of PRIMPOL depletion with two different siRNAs. Similar results were obtained in n = 3 independent experiments. f Replication tract lengths in U2OS cells depleted of PRIMPOL alone or co-depleted of PRIMPOL and BRCA2 before and 6 h after 10 Gy of IR. g Effect of S1 nuclease treatment on replication tract lengths in control and BRCA2-depleted U2OS cells at 6 h after 10 Gy of IR. h Replication tract lengths in U2OS cells depleted of BRCA2, HLTF, SMARCAL1, or ZRANB3 alone or in combination with PRIMPOL at 6 h after 10 Gy of IR. Data in (b–d) and (f–h) are presented as mean ± s.d., with the number of dots shown below each column. P values are calculated using two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Fig. 3. Impact of PRIMPOL loss on genome stability and cell viability after DNA damage in BRCA2-proficient and -deficient cells.

a Western blots showing levels of siRNA-mediated depletion of BRCA2, PRIMPOL, or BRCA2 + PRIMPOL in U2OS cells. Similar results were obtained in n = 3 independent experiments. b A representative image of a mitotic spread after Giemsa staining showing different forms of abnormal chromosomes. CTB, chromatid break; CSB, chromosomal break; Radial, radial chromosome. c, d Quantification of chromosomal aberrations in U2OS cells treated with control, BRCA2, PRIMPOL, or BRCA2 + PRIMPOL siRNAs without IR (c) or 6 h after 2 Gy of IR (d). Data are presented as mean ± s.d. from n = 3 independent experiments. e–h Sensitivity of the above siRNA-treated cells to IR (e), BLEO (f), MMS (g), and CPT (h). Cell viability was determined 72 h after treatment. Results are presented as mean ± s.d. from n = 3 independent experiments, each conducted in technical triplicates. Source data are provided as a Source Data file.

Fig. 4. Identification of MCM10 as a BRCA2-ineracting protein.

a Numbers of tryptic peptides obtained from two independent PALB2 tandem affinity purification (TAP)-LC-MS/MS experiments. b Co-IP of endogenous PALB2 and BRCA2 with ectopic MCM10. FLAG-HA-tagged MCM10 was transiently overexpressed in 293T cells and IPed with anti-FLAG. c Co-IP of endogenous MCM10 and endogenous PALB2 and BRCA2 in unperturbed and HU-treated U2OS cells. d Co-IP of GFP-tagged BRCA2 variants and FLAG-HA-tagged MCM10 overexpressed in 293T cells. e Association of endogenous BRCA2 with ectopic FLAG-HA-tagged MCM10. The procedure was performed as in (b) except that the final IP material was treated with DNase I on the beads and then washed before being subjected to western blotting. f Co-IP of BRCA2 and MCM10 in S-phase-synchronized U2OS cells at 3 h after treatment with DMSO, 10 Gy of IR, 2 Gy of IR, BLEO (1 µM), CPT (1 µM), or cisplatin (CDDP, 1 µM). Cells were synchronized in the S phase by a single thymidine block for 24 h and released for 2 h prior to the treatments. For (b–f), similar results were obtained in n = 3 independent experiments. Source data are provided as a Source Data file.

Fig. 5. The BRCA2–MCM10 association restrains replication fork progression after DNA damage.

a Schematic diagram of MCM10 domain structure and amino acid sequence alignment of the MCM10 N-terminal coiled-coil (CC) motif. b Co-IP of endogenous BRCA2 with FLAG-HA-tagged MCM10 proteins transiently overexpressed in 293T cells. c Expression levels and association between exogenous MCM10 proteins and endogenous BRCA2 in U2OS cell lines stably expressing FLAG-HA-tagged WT and CC mutant MCM10 proteins. For (b) and (c), similar results were obtained in n = 3 independent experiments. d–f Replication tract lengths in U2OS cells selectively expressing exogenous WT or mutant MCM10 proteins before or 6 h after 10 Gy of IR (d), 3 h after 2 Gy of IR (e), or after 6 h of continuous treatment with DMSO (vehicle) or 1 µM BLEO (f). g Replication tract lengths in the above cells before and after IR as determined by the DNA combing assay. A representative combing image is shown on the left. h Stability of stalled replication forks in the above cells. Assay scheme is shown on the top, and IdU/CldU tract length ratio after HU treatment is shown below. Data in (d–h) are presented as mean ± s.d., with the number of dots shown below each column. P values are determined two-tailed unpaired Student’s t test. Similar results were obtained from n = 2 independent experiments. Source data are provided as a Source Data file.

Fig. 6. The BRCA2–MCM10 association suppresses PRIMPOL-mediated repriming and ssDNA gap formation after DNA damage.

a Effect of PRIMPOL depletion on replication fork progression in stable U2OS cells selectively expressing exogenous WT vs ΔCC mutant MCM10 proteins before and after IR. The endogenous MCM10 was depleted together with PRIMPOL with siRNAs targeting the 3′-UTR of its mRNA. b Effect of S1 nuclease treatment on detected replication tract length in the above cells after IR. Data in (a) and (b) are presented as mean ± s.d., with the number of dots shown below each column. P values are calculated using two-tailed unpaired Student’s t test. Similar results were obtained in n = 2 independent experiments. c A proposed model for the role of BRCA2 and the BRCA2–MCM10 association in replication fork progression after DNA damage. BRCA2 is recruited to the replication fork via its association with MCM10. When the fork encounters a lesion, polymerase ε stalls, whereas the MCM2-7 helicase complex continues to unwind downstream DNA, leading to excessive ssDNA formation. RPA binds to the ssDNA and may recruit PRIMPOL; however, PRIMPOL recruitment is prevented by BRCA2, which can displace RPA from ssDNA. When BRCA2 is lost or the BRCA2–MCM10 association is compromised, increased and/or persistent presence of RPA on ssDNA leads to PRIMPOL recruitment, hence repriming of DNA synthesis downstream of the lesion and formation of ssDNA gaps. See text for further details.