Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11

Abstract

Breast cancer suppressor BRCA2 is critical for maintenance of genomic integrity and resistance to agents that damage DNA or collapse replication forks, presumably through homology-directed repair of double-strand breaks (HDR). Using single-molecule DNA fiber analysis, we show here that nascent replication tracts created before fork stalling with hydroxyurea are degraded in the absence of BRCA2 but are stable in wild-type cells. BRCA2 mutational analysis reveals that a conserved C-terminal site involved in stabilizing RAD51 filaments, but not in loading RAD51 onto DNA, is essential for this fork protection but dispensable for HDR. RAD51 filament disruption in wild-type cells phenocopies BRCA2 deficiency. BRCA2 prevents chromosomal aberrations on replication stalling, which are alleviated by inhibition of MRE11, the nuclease responsible for this form of fork instability. Thus, BRCA2 prevents rather than repairs nucleolytic lesions at stalled replication forks to maintain genomic integrity and hence likely suppresses tumorigenesis through this replication-specific function.

Figure 1. BRCA2 Protects Nascent DNA Strands at Stalled Replication Forks

(A) Schematic of single DNA fiber analysis. Green tracts, IdU; red tracts, CldU. Examples of various types of tracts are shown. (B) IdU tract length distributions from DNA fibers from BRCA2-deficient (V-C8) and proficient (V-C8+BRCA2) hamster cells in the presence (replication stalling) or absence (unperturbed replication) of HU. Sketch above delineates experimental design. Median tract lengths are given in parentheses here and in subsequent figures. Inset, cumulative distributions. (C) IdU tracts in Brca2^lex1/lex2 and wild-type mES, with and without HU. (D) IdU tracts in the BRCA2-deficient human CAPAN-1 and BRCA2 revertant of CAPAN-1 (C2-14), with and without HU. (E) CldU tracts of replication after exposure to HU or media in V-C8 and V-C8+BRCA2 cells. Inset, cumulative distributions; see also Figures S1D. (F) DNA fiber images from CAPAN-1 cells treated with HU and labeled with CldU either during HU (left panel) or after HU (right panel). (G) Gap lengths between IdU tracts before HU and CldU tracts after HU (yellow distribution) and CldU tract lengths during HU (red distribution) in CAPAN-1 cells. (H) Individual DNA fiber images from CAPAN-1 and C2-14 cells treated with HU and then labeled with CldU, marked to show the gap in label between the IdU and CldU labels. The % of fibers with gaps between the IdU and CldU labels is given for each cell line. Gap length frequency is shown for each cell line. See also Figure S1F. See also Table S1 for detailed information on data sets and statistical tests, including 95% confidence interval for cumulative distributions.

Figure 2. Inhibition of MRE11 Alleviates Nucleolytic Degradation of Stalled Forks

(A–B) Preformed IdU tract lengths in V-C8 (A) and V-C8+BRCA2 (B) cells during different exposure times to HU. Inset, the rate of IdU tract length change is 0.7 µm/h, estimated to be ~1.8 kb/h. (C) Sketch of design and expected outcome of nuclease directionality test. Tick marks delineate lagging strands. (D–E) Distribution curves of the ratio of CldU/IdU tract lengths with or without HU in V-C8 (D) and V-C8+BRCA2 (E) cells. (F) IdU tract lengths in V-C8 and V-C8+BRCA2 cells with or without HU in the presence of the MRE11 inhibitor mirin. (E) IdU tract lengths in Brca2^lex1/lex2 mES cells after shRNA treatment directed against MRE11 or control (shLuc) with or without HU. Western blot inset shows the MRE11 knockdown.

Figure 3. RAD54 and KU70 Deficiency Do Not Affect the Stability of Nascent Strands at Stalled Replication Forks

IdU tracts in Rad54^−/− (A) and Ku70^−/− (B) mES cells with or without HU. Inset, cumulative distributions.

Figure 4. BRCA2 Domain Analysis Reveals Differences in Fork Stability and HDR

(A) Graphical sketch of human BRCA2, PIR2, and BRC3-RPA. PALB2-interaction site (bright green), BRC repeats (dark green bars), DNA binding domain (DBD, black bar), C-terminal region (C-ter) with non-BRC RAD51 binding site (red bar). Arrows indicate truncations in V-C8 cells. (B–C) IdU tracts in V-C8 cells stably expressing the PIR2 (B) or BRC3-RPA (C) peptides with or without HU.

Figure 5. Highly Conserved BRCA2 C-ter is Essential in Maintaining Replication Fork Stability by Stabilizing RAD51 Filaments

(A) Blast alignments of BRCA2 C-terminal sequences; CDK phosphorylation target motif (SP, red letters); cyclin A recognition motif (RxL, blue; similar amino acids are in italics). See also Figure S4A. (B) IdU tracts in V-C8+BRCA2 S3291A cells with or without HU. (C) HDR assay for a DSB using the DR-GFP reporter. (D) HDR assay in V-C8 and BRCA2 BAC-complemented cells. GFP-positive cells indicate HDR events after I-SceI expression. Error bars indicate standard deviation; p-value derived from two-tailed Student’s t-test. (E) IdU tracts after BRC4 peptide expression in mES cells with or without HU. pCaggs, empty vector. Western blot inset shows BRC4 transient expression. (F) IdU tracts after RAD51 K133R or BRC4 expression in Brca2^lex1/lex2 mES cells with or without HU. Western blot inset shows RAD51 K133R transient expression.

Figure 6. Replication Fork Stalling Leads to Genomic Instability in BRCA2-Deficient Cells

(A–B) Chromosomal aberrations with or without HU treatment in the indicated V-C8 cell lines. Sketch above the graphs delineates experimental design. The % of metaphase spreads with the indicated aberrations (A) and the number of chromosome aberrations per metaphase (B) are plotted. p-value derived from two-tailed Student’s t-test. (C–D) Chromosomal aberrations with or without HU in V-C8 cells in the presence or absence of the MRE11-inhibitor mirin. Sketch above the graph delineates experimental design. The % of metaphase spreads with the indicated aberrations (C) and the number of chromosome aberrations per metaphase (D) are plotted. (G) Chromosomal aberrations in V-C8 cells exposed to HU include breaks (black arrowheads), gaps (red arrowhead), triradials, quadradials (green arrowhead) and other translocation (blue arrow). (F–G) Survival of indicated V-C8 cell lines upon continuous exposure to HU (F) and the Parp-inhibitor olaparib (G).

Figure 7. Model for HDR-independent Role of BRCA2 During DNA Replication

A replication fork can stall for various reasons, including insufficient pools of nucleotides as from HU. BRCA2 (pink circle) stabilizes RAD51 filaments on stalled replication forks (blue circles), thereby preventing fork reversal promoted by positive supercoils ahead of the fork (grey circles). Alternatively, stabilized filaments directly protect a reversed fork. Once the replication stall is removed, genome duplication can proceed until completed. BRCA2 is then no longer required for fork protection, such that CDK phosphorylates the BRCA2 C-ter, allowing residual RAD51 filaments to dissociate to promote progression into M-phase. Importantly, in the absence of BRCA2, nascent strands of the stalled fork are unprotected and degraded by MRE11 (yellow pacman), leading to chromosomal instability.