BRCA2 Protects Nascent DNA Strands at Stalled Replication Forks
To obtain a better understanding of BRCA2’s role during DNA replication, DNA fiber analysis was utilized to monitor replication perturbation genome-wide at single-molecule resolution (). This procedure marks newly synthesized DNA strands just prior to (green fluorescent IdU) and after (red fluorescent CldU) exposure to HU, which transiently stalls replication by causing an imbalance in the deoxyribonucleoside triphosphate pool. Retention of the IdU label after HU treatment measures the stability of stalled forks (Σ IdU; ).
BRCA2 Protects Nascent DNA Strands at Stalled Replication Forks
In hamster cells expressing wild-type BRCA2 (V-C8+BRCA2), the median IdU tract length is maintained intact with or without HU treatment for 5 h (9.73 and 9.77 µm respectively, p
=0.924, two-tailed Mann-Whitney test; ), indicating that the integrity of stalled forks is not compromised during prolonged periods of replication stress. In contrast, nascent IdU tracts substantially shorten in cells deficient for BRCA2 (V-C8 (Wiegant et al., 2006
)) when replication forks are stalled compared to unperturbed replication (5.44 and 9.06 µm, respectively, p
<0.0001; ). As nascent IdU tracts are formed prior to treatment with HU, the disappearance of IdU label occurs during HU exposure. We confirmed that IdU shortening in BRCA2-deficient cells occurs irrespective of the choice of the replication poison by replacing HU with the chemotherapeutic gemcitabine, a nucleoside analogue that inhibits DNA elongation (Figure S1A
). Thus, BRCA2 functions in protecting nascent strands when replication forks are stalled.
To determine if the requirement for BRCA2 in protecting stalled replication forks is common to other cell types, we examined Brca2lex1/lex2
mouse embryonic stem (mES) cells, which express a C-terminal truncation of BRCA2 (Morimatsu et al., 1998
). As with V-C8 cells, IdU tracts shorten in Brca2lex1/lex2
cells exposed to HU compared with unimpeded replication (4.76 and 6.79 µm, respectively; p
=<0.0001), while the nascent tracts remain intact in wild-type mES cells (, Figure S1B
). In addition, human CAPAN-1 cells, which express a similar BRCA2
truncation as V-C8 cells (Goggins et al., 1996
), are defective in maintaining nascent tracts compared with unimpeded replication (5.41 µm and 9.53 µm, respectively; p
=<0.0001; , Figure S1C
). Reversion of the BRCA2 mutation in the CAPAN-1 cells by a nearby second-site mutation (C2-14 cells, (Sakai et al., 2008
); Figure S1C
) largely restores the protection of the stalled replication forks (). These results indicate that BRCA2 is required for the protection of stalled replication forks in multiple mammalian cell lines.
Recovery after Replication Stalling in BRCA2-Deficient Cells
Depletion of RAD51 has been reported to decrease replication restart after HU (Petermann et al., 2010
) and uncouple leading and lagging-strands (Hashimoto et al., 2010
). To address whether replication recovery is affected when BRCA2 is absent, CldU tract lengths formed after HU exposure were measured. Replication is substantially slowed after HU treatment, but similarly so in both control and BRCA2-deficient cells (5.93 and 5.67 µm, V-C8+BRCA2 and V-C8 after HU, respectively; ). In these experiments, the IdU and CldU labels are often quite separated, indicating that forks stall only transiently with HU in these hamster cells. Therefore, we assessed the frequency of replication restart (IdU→CldU) when CldU was present during HU. As with replication recovery after HU, we find no significant difference between BRCA2-proficient and deficient hamster cells (Figure S1D
). Further, the frequency of new replication tracts is also unaffected by BRCA2 status (Figure S1D
Given that replication restart may be more stringently controlled in human cells than rodent cells (Petermann et al., 2010
), we also assessed restart in CAPAN-1 and revertant cells. Consistent with a more efficient block in human cells, IdU and CdU tracts are more frequently joined when the CldU label is added after HU exposure than they are in hamster cells. This apparent replication restart (IdU→CldU) is not significantly impaired under this condition with or without BRCA2 (Figure S1E
), although we did observe a small increase in tracts labeled only with IdU in CAPAN-1 cells (13% to 19%, p
=0.279; Figure S1E
We noticed that most restart tracts have small gaps between the IdU and CldU labels (, right panel), indicating limited fork progression during exposure to HU. A larger portion of tracts from CAPAN-1 C2–C14 cells have gaps (77.5%) compared with CAPAN-1 cells (66.5%), implying that the block to restart is more efficient in the absence of BRCA2 (p
=0.0134, ). To examine transient replication restart during HU, we included CldU during HU in a control experiment in CAPAN-1 cells and found that ~97% of forks continue to incorporate CldU label even in the presence of HU (, left panel). Surprisingly, the continuous CldU tracts formed during HU are substantially longer than the gaps between the two labels in experiments where the label is omitted during HU (compare 9.73 µm CldU tract length during HU and 1.27 µm gap length during HU without label, ). We interpret this data to reflect asymmetric replication fork movement during HU, indicating leading and lagging strand uncoupling (Figure S1F
). We reasoned that gap tract lengths can be used to assess the ability of both strands to continue replication during HU. Gap lengths are smaller in CAPAN-1 compared to CAPAN-1 C2-14 cells (p
=<0.0001, ), providing evidence that BRCA2 can suppress uncoupling of the leading and lagging strands.
Nucleolytic Degradation of Stalled Forks Is Progressive and Has Distinct Directionalities
To better understand the mechanism of replication tract shortening during fork stalling, we monitored the integrity of the nascent strands by varying the exposure times to HU. Consistent with nucleolytic degradation of the stalled forks, preformed IdU tracts in V-C8 cells progressively shorten during HU ( and inset, Figure S2A
), with a rate of 0.7 µm/h, corresponding to ~1.8 kb/h. By contrast, preformed IdU tracts in cells with BRCA2 remain largely unchanged (, Figure S2B
Inhibition of MRE11 Alleviates Nucleolytic Degradation of Stalled Forks
To fine map the degradation, we consecutively labeled nascent tracts with IdU and CldU for equal periods of time before HU (). In unperturbed V-C8 cells, IdU and CldU tracts are similar in length (CldU/IdU=1.03; ). When challenged with HU, CldU tracts shorten while IdU tracts remain intact (CldU/IdU=0.65; , Figure S2C
), indicating that the more recently synthesized DNA is degraded first. Thus, leading strands are degraded 3’–5’, while lagging strands are degraded 5’-3’ (). In contrast, the CldU/IdU ratio in VC-8+BRCA2 cells is ~1 with or without HU (, Figure S2C
). These results imply that BRCA2 protects against degradation of stalled replication forks with opposite directionalities for the leading and lagging strands.
MRE11 Is Responsible for Nascent Strand Shortening at Stalled Forks
The slow kinetics of degradation (~1.8 kb/h) are reminiscent of another controlled degradative process, that of DNA end resection (~4 kb/h in yeast; (Fishman-Lobell et al., 1992
)). We considered that MRE11, which possesses 3’–5’ exonuclease activity and also promotes 5’-3’ end resection (Mimitou and Symington, 2009
; Williams et al., 2008
), could promote fork degradation in the absence of BRCA2. To test this, we used mirin, a chemical inhibitor of MRE11 nuclease activity (Dupre et al., 2008
). With mirin, IdU tracts are similar in length irrespective of replication stalling in both BRCA2-deficient and proficient cells (, Figure S2D
), suggesting that the MRE11 nuclease degrades stalled forks in the absence of BRCA2. To exclude off-target effects by the inhibitor, we expressed shRNA against MRE11 in Brca2lex1/lex2
cells (), which like mirin, substantially protects the nascent tracts during HU (6.18 and 4.05 µm with and without MRE11 knockdown, respectively; p
=<0.0001; ). These results further implicate the MRE11 complex, specifically its nuclease activity, in fork degradation in the absence of BRCA2.
Replication Fork Protection Is Independent of Canonical NHEJ and the HDR Protein RAD54
The KU heterodimer, a key component of canonical NHEJ, is important in the protection of DNA ends from nucleolytic digestion (Kass and Jasin, 2010
). We tested whether loss of KU would also lead to deprotection of DNA ends at stalled replication forks. Nascent IdU tracts are maintained intact in Ku70−/−
mES cells (), consistent with a specific role for KU at DSBs, but not at stalled forks (Pierce et al., 2001
RAD54 and KU70 Deficiency Do Not Affect the Stability of Nascent Strands at Stalled Replication Forks
We next examined whether protection of stalled forks is a property of all HDR proteins. RAD54 acts during late steps of HDR (Heyer et al., 2006
), downstream of BRCA2-mediated RAD51 nucleoprotein filament formation. Yet, RAD54 is not evidently involved in fork protection, as Rad54−/−
mES cells exhibit similar IdU tract lengths with or without HU (), thus indicating that not all HDR components are required to avoid fork degradation.
Domain Requirements for BRCA2 in Replication Fork Protection
BRCA2 contains both protein and DNA interaction domains, including several BRC repeats which bind RAD51, a DNA binding domain (DBD) consisting of several DNA binding modules, and a C-terminal site (C-ter) which also binds RAD51 (). Given the multidomain structure of BRCA2, we sought to characterize the domain requirements for replication fork stability.
BRCA2 Domain Analysis Reveals Differences in Fork Stability and HDR
V-C8 cells have two Brca2
alleles encoding proteins truncated for the C-ter and DBD domain () (Wiegant et al., 2006
) and are defective in both HDR (Saeki et al., 2006
) and maintenance of fork stability (). As BRCA2 could directly inhibit degradation via protein-DNA interactions requiring the DBD domain, we generated a V-C8 cell line stably expressing a BRCA2 peptide missing the entire DBD domain (PIR2, ), but which is proficient at HDR (Figure S3A
; (Edwards et al., 2008
)). No substantial difference in IdU tract lengths was observed with or without HU (9.69 and 8.74 µm, respectively; , Figure S3B
). Thus, the BRCA2 DBD is not required for the protection of stalled replication forks, indicating that it is unlikely that BRCA2 directly inhibits nucleolytic degradation by binding to nascent DNA.
V-C8 cells stably expressing a peptide consisting of one BRC repeat fused to the large subunit of the ssDNA-binding protein RPA (BRC3-RPA, ) are proficient in HDR (Saeki et al., 2006
), suggesting that the main function of BRCA2 in HDR is to deliver RAD51 to ssDNA. These cells, however, exhibit shorter nascent IdU tract lengths when replication is stalled with HU (6.35 and 8.81 µm with and without HU, respectively, p
<0.0001; , Figure S3C
). Although the degradation is not as extensive as with V-C8 cells (), these results suggest that the delivery of RAD51 to ssDNA is not sufficient protect stalled forks.
Highly Conserved BRCA2 C-ter RAD51 Interaction Site Is Essential for Replication Fork Stability but Dispensable for HDR
BRCA2 interacts with RAD51 through both the BRC repeats and the C-ter. While RAD51 loading through the BRC repeats does not seem to be sufficient for the protection of stalled forks, the conserved C-ter appears to provide an essential function, given our results with the Brca2lex1/lex2
cells (). The C-ter binds RAD51 differently than the BRC repeats in that it interacts with RAD51 oligomers and stabilizes RAD51 filaments (Davies and Pellegrini, 2007
; Esashi et al., 2007
). Moreover, the RAD51 binding site at the C-ter contains a cyclin dependent kinase (CDK) phosphorylation consensus sequence that is conserved throughout vertebrates (Esashi et al., 2005
) as well as some invertebrates (, Figure S4A
); phosphorylation by CDK at this site (S3291) abrogates the C-ter-RAD51 interaction (Davies and Pellegrini, 2007
; Esashi et al., 2005
), thereby promoting RAD51 filament disassembly which in turn promotes entry into mitosis (Ayoub et al., 2009
Highly Conserved BRCA2 C-ter is Essential in Maintaining Replication Fork Stability by Stabilizing RAD51 Filaments
To further investigate the function of the C-ter, we utilized V-C8 cells stably expressing full-length BRCA2 containing the S3291A mutation which, like phosphorylation, disrupts RAD51 binding at this site (Davies and Pellegrini, 2007
; Esashi et al., 2005
). As with the Brca2lex1/lex2
cells, IdU tracts shorten with HU (5.59 and 10.03 µm with and without HU, respectively, p
=<0.0001; , Figure S4B
), implicating RAD51 interaction at the C-ter in the protection of stalled replication forks, perhaps through the stabilization of RAD51 filaments.
To determine the relationship between the protection of stalled forks and HDR, we quantified HDR in V-C8+BRCA2 S3291A cells using the DR-GFP reporter, where a DSB introduced by the I-SceI endonuclease followed by HDR leads to GFP-positive cells (). V-C8 cells are highly defective in HDR compared with V-C8+BRCA2 cells (~25-fold; ;(Saeki et al., 2006
)). Importantly, V-C8+BRCA2 S3291A cells are as efficient for HDR as those expressing wild-type BRCA2. Thus, while the BRCA2 C-ter RAD51 interaction site is essential for the protection of stalled replication forks, it is dispensable for HDR, providing a clear separation of function mutation for the two processes.
BRCA2 Maintains Nascent Replication Tracts by Stabilizing RAD51 Filaments
To directly test the involvement of RAD51 in the protection of stalled forks, we expressed the BRC4 peptide (Saeki et al., 2006
), which suppresses DNA binding of RAD51 and thus perturbs RAD51 filaments (Davies et al., 2001
; Hashimoto et al., 2010
). As with defective BRCA2, expression of the BRC4 peptide in wild-type mES cells leads to substantially shorter IdU tracts upon HU (3.67 and 6.65 µm with and without HU, respectively, p
=<0.0001; , Figure S4D
). These data indicate that disruption of RAD51 filaments leads to nascent strand degradation.
We next asked whether stabilization of filaments could suppress this degradation. ATP hydrolysis by RAD51 is required for efficient dissociation from DNA while association with DNA is unaffected (Benson et al., 1994
; van Mameren et al., 2009
). RAD51 K133R, which is devoid of ATPase activity, forms stable filaments and promotes strand exchange in vitro
(Morrison et al., 1999
) while suppressing turnover or completion of successful HDR in vivo
(Stark et al., 2002
). Over-expression of RAD51 K133R in Brca2lex1/lex2
cells exposed to HU renders IdU tracts resistant to degradation (7.22 and 6.81 µm with and without HU, respectively, p
=0.077; , Figure S4E
), indicating that stabilized RAD51 filaments rescue the protection of stalled forks and consistent with the requirement for the C-ter in this process. Expression of BRC4 has no effect in these cells (, Figure S4E
), suggesting that BRCA2 and RAD51 are epistatic for fork protection.
Replication Fork Stalling Leads to MRE11-Dependent Genomic Instability in BRCA2-Deficient Cells
We next sought to determine the physiological consequences of replication perturbation in cells that cannot maintain the integrity of stalled replication forks. Both V-C8 and V-C8+BRCA2 S3291A cells exhibit elevated spontaneous chromosomal abnormalities compared to cells with wild-type BRCA2, revealing intrinsic genomic instability in cells expressing BRCA2 S3291A (). Exposure to HU substantially increases the average number of aberrations per metaphase spread in both cell lines, from 0.51 to 1.77 for V-C8 cells and from 0.12 to 0.37 for V-C8+BRCA2 S3291A cells (). Thus, replication stalling induces chromosomal aberrations in cells which are defective in protecting forks from degradation.
Replication Fork Stalling Leads to Genomic Instability in BRCA2-Deficient Cells
Treatment of cells with HU has been reported to lead to DSB formation after prolonged exposure times, while treatment with HU for a few hours, as performed here, does not cause such lesions (Hanada et al., 2007
; Petermann et al., 2010
). Given that the degradation of forks is not correlated to HDR (), we sought to further distinguish genomic instability arising from DSBs from genomic instability arising from degraded forks by treating cells with colcemid immediately after HU exposure. As with delayed colcemid treatment, breaks/gaps increase in V-C8 cells with HU exposure, and triradial/quadriradial chromosomes increase even further (), suggesting that unprotected replication forks expose potential sites for aberrant interchromosomal interactions. Overall, the average number of chromosomal aberrations per metaphase in V-C8 cells increases from 0.56 to 2.5 with HU (). Similarly, aberrations increase with HU in V-C8+BRCA2 S3291A cells, while the number of aberrations in cells expressing wild-type BRCA2 is substantially lower (Figures S5A–C
). Thus, cells in which stalled replication forks degrade exhibit greater genomic instability.
As the MRE11-inhibitor mirin alleviates degradation of stalled forks in BRCA2-deficient cells (), we asked whether inhibition of MRE11 could also alleviate HU-induced chromosome aberrations. Both breaks/gaps and radial chromosomes were reduced in V-C8 cells treated with HU and mirin compared to HU alone (). Overall, the average number of chromosomal aberrations per metaphase decreased more than 2-fold when MRE11 nuclease activity was inhibited during fork stalling (1.07 and 2.54 with and without mirin, respectively, p=0.015; ). As inhibiting the degradation of stalled replication forks in BRCA2-deficient cells is associated with reduced numbers of chromosome aberrations, these data support a relationship between degradation of stalled replication forks by MRE11 and genomic instability.
Replication Stalling In BRCA2-Deficient Cells Does Not Reduce Cellular Survival
To investigate long-term effects of stalled replication, cell survival was examined following exposure to agents that have differential effects on replication. After continuous exposure to HU, V-C8 cells and V-C8+BRCA2 S3291A show only modest sensitivity to HU relative to cells expressing wild-type BRCA2 (), suggesting that fork degradation has little effect on cell survival. We also pulsed cells under the conditions used in the DNA fiber experiments, and again saw no significant difference in survival (data not shown). Thus, although chromosomal aberrations increase, cell survival is not compromised, indicating that BRCA2 deficiency will be associated with increased mutagenesis when replication is perturbed.
V-C8 cells are exquisitely sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors like olaparib (; (Farmer et al., 2005
)). By contrast, cells expressing either wild-type BRCA2 or the S3291A mutant are similarly resistant to this drug (). Given that PARP inhibition leads to ssDNA breaks which are converted to DSBs during S phase, efficient repair of these DSBs is consistent with the BRCA2 S3291A mutant being proficient at DSB repair by HDR. Supporting this, similar results were obtained with other agents that require HDR for their repair, i.e., 6-thioguanine and mitomycin C (Figures S5D and S5E