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RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress
Short Article
RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress Graphical Abstract
Authors Rahul Bhowmick, Sheroy Minocherhomji, Ian D. Hickson
Correspondence [email protected]
In Brief Tumorigenesis is frequently driven by oncogene activation, which generates so-called DNA replication ‘‘stress’’ and instability at specific genomic loci, called common fragile sites. Bhowmick et al. show that DNA repair synthesis at fragile sites occurs in mitosis and define a role for the homologous recombination factor RAD52 in this process.
Highlights d
RAD52 promotes mitotic DNA synthesis (MiDAS) following replication stress
d
RAD52 promotes recruitment of MUS81 and POLD3 in mitosis
d
RAD52 deficiency increases mitotic chromosome missegregation
d
Mitotic DNA synthesis occurs independently of ATR, BRCA2, and RAD51
Bhowmick et al., 2016, Molecular Cell 64, 1117–1126 December 15, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2016.10.037
Molecular Cell
Short Article RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress Rahul Bhowmick,1,2 Sheroy Minocherhomji,1,2,3 and Ian D. Hickson1,4,* 1Center for Chromosome Stability and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen N, Denmark 2Co-first author 3Present address: Department of Discovery Toxicology, Amgen Inc., 1120 Veterans Boulevard, San Francisco, CA 94080, USA 4Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.10.037
SUMMARY
Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis. This mitotic DNA synthesis, termed MiDAS, requires the MUS81-EME1 endonuclease and a non-catalytic subunit of the Pol-delta complex, POLD3. Here, we examine the contribution of HR factors in promoting MiDAS in human cells. We report that RAD51 and BRCA2 are dispensable for MiDAS but are required to counteract replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective inhibition of MiDAS may comprise a potential therapeutic strategy to sensitize cancer cells undergoing replicative stress. INTRODUCTION The activation of oncogenes during tumorigenesis generates DNA replication stress (RS), which is a known driver of chromosomal instability (CIN) (Burrell et al., 2013; Macheret and Halazonetis, 2015). Certain regions of the human genome, such as common fragile sites (CFSs), are particularly sensitive to RS. These loci are difficult to replicate and are prone to generate copy number variations (CNVs) and chromosomal rearrangements in human cancers (Arlt et al., 2006; Burrow et al., 2009; Wilson et al., 2015). There are several features that the different CFS loci often have in common, including a propensity to form DNA secondary structures within AT-rich sequences, the presence of a long primary transcript that can potentially impede replication fork progression, and increased levels of R-loops (Helmrich et al., 2011; Le Tallec et al., 2013; Santos-Pereira
and Aguilera, 2015; Sarni and Kerem, 2016; Sollier and Cimprich, 2015; Zhang and Freudenreich, 2007). CFSs manifest as DAPInegative gaps on metaphase chromosomes (CFS expression), a phenomenon that is exacerbated by exposing cells in S-phase to a mild dose of aphidicolin (APH) (Glover et al., 1984). CFS expression is an active process that requires the MUS81EME1 DNA structure-selective endonuclease and its associated scaffold protein, SLX4 (Minocherhomji and Hickson, 2014; Minocherhomji et al., 2015; Naim et al., 2013; Ying et al., 2013). In response to RS, the FANCD2/FANCI protein complex forms ‘‘twin foci’’ (one on each sister chromatid) at CFS loci that can persist into mitosis (Chan et al., 2009; Howlett et al., 2005; Minocherhomji et al., 2015). These foci co-localize with SLX4 and serve as a useful surrogate marker of the location of CFSs in G2/M phase cells. Upon mitotic entry, CDK1-dependent phosphorylation of EME1 promotes the association of MUS81EME1 with SLX4 at under-replicated CFS loci (Minocherhomji et al., 2015; Wyatt et al., 2013; Ying et al., 2013). An important function of MUS81-EME1 at CFSs is to promote DNA repair synthesis that occurs in the prophase of mitosis (Minocherhomji et al., 2015). This mitotic DNA synthesis (henceforth termed ‘‘MiDAS’’) requires MUS81-EME1, SLX4, and a non-catalytic subunit of DNA polymerase d, POLD3 (Pol32 in yeast) (Bursomanno et al., 2015; Minocherhomji et al., 2015). Figure 1A summarizes the previously proposed roles of these factors. DNA synthesized via the MiDAS pathway is associated with the appearance of cytogenetically defined DAP1-negative gaps on metaphase chromosomes at CFS loci (Minocherhomji et al., 2015). In the absence of MiDAS, potentially lethal chromosome missegregation occurs, and daughter cells exhibit an increased frequency of prominent structures termed 53BP1 nuclear bodies (comprising OPT domains), which are known to associate with missegregated CFSs (Harrigan et al., 2011; Lukas et al., 2011; Minocherhomji et al., 2015; Naim et al., 2013; Ying et al., 2013). Homologous recombination (HR) has important roles in the repair of stalled or collapsed DNA replication forks, as well as of DNA double-strand breaks. Among the factors required for HR in human cells are RAD51, BRCA1, and BRCA2 (Davies et al., 2001; Sung and Klein, 2006; Xia et al., 2001). BRCA1 and BRCA2 are required for the efficient association of RAD51 with DNA, as well as stabilization of RAD51 filaments on singlestranded DNA (ssDNA). In yeast, Rad52 largely performs this so-called Rad51 mediator function (Jensen et al., 2010;
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Figure 1. RAD52 Persists at Sites of On-Going DNA Synthesis in Mitosis Following Replication Stress (A) Previously proposed model for MiDAS. (B and C) Representative images (B) and quantification (C) of EdU incorporation (red) on isolated metaphase chromosomes (DAPI, blue) following replication stress (RS) (+APH). (D and E) Representative western blots of soluble and insoluble fractions (D) and quantification (E) following pre-treatment with (+APH) or without (APH) APH. (F and G) Representative immunofluorescence (IF) images (F) and quantification (G) of the co-localization of RAD52 (green) with MUS81 or FANCD2 twin foci (red). DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 mm. See also Figure S1.
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Mortensen et al., 1996; San Filippo et al., 2006). Yeast cells also possess an additional Rad52-like protein called Rad59, which plays a more limited and specialized role in certain sub-pathways of HR (Bai and Symington, 1996; Pannunzio et al., 2008; Signon et al., 2001). One of these sub-pathways is break-induced replication (BIR), a form of recombination-dependent DNA replication that can repair one-ended DNA breaks that can arise, for example, when a replication fork collapses upon encountering a break in the template DNA. A subclass of BIR includes microhomology-mediated BIR (MMBIR), which can lead to the formation of CNVs without RAD51-mediated strand invasion (Hastings et al., 2009; Ottaviani et al., 2014; Sakofsky et al., 2015). The principal biochemical function of Rad52 family members is to promote the annealing of complementary ssDNA molecules, which probably explains how Rad52/59 can catalyze some HR processes in the absence of Rad51-mediated DNA strand invasion (Gasior et al., 1998; Kagawa et al., 2001, 2002; Lao et al., 2008; McIlwraith and West, 2008). It is not clear from analysis of sequence conservation whether human RAD52 is the functional homolog of yeast Rad52 or Rad59. Human RAD52 forms oligomers that have robust ssDNA annealing activity (Kagawa et al., 2001, 2002). Small molecule disruption of RAD52 oligomerization impairs some recombination activities of RAD52 (Chandramouly et al., 2015; Sullivan et al., 2016) and is synthetically lethal with BRCA2 deficiency (Chandramouly et al., 2015; Feng et al., 2011). Current models propose that RAD52 operates in a BRCA2-independent HR backup pathway to load RAD51 onto DNA (Lok and Powell, 2012). However, the precise role of RAD52 in human cells remains to be defined. The requirement for POLD3 in MiDAS suggests that the DNA synthesis might proceed via a BIR-like process (Costantino et al., 2014). We therefore investigated how HR impacts the mitotic DNA damage response in human cells following RS. We demonstrate that RAD52, but not RAD51 or BRCA2, is required for MiDAS. Consistent with RAD52 acting at an early step in MiDAS, RAD52 depletion disrupts the timely recruitment of MUS81-EME1 and POLD3 to CFSs in mitosis. RESULTS DNA Synthesis during MiDAS Frequently Occurs on a Single Sister Chromatid One possible mechanism for MiDAS would be for stalled replication forks at CFSs to simply re-start in early mitosis via canonical semi-conservative DNA replication. In this scenario, the newly replicated DNA would be evenly distributed on each sister chromatid of a metaphase chromosome. In contrast, BIR can occur by conservative DNA replication in yeast (Donnianni and Symington, 2013; Saini et al., 2013). To visualize the location of newly synthesized DNA in mitosis, we examined the pattern of EdU incorporation on isolated metaphase chromosomes. Interestingly, 50% of the newly synthesized DNA tracts in early mitosis were localized unequivocally to a single sister chromatid (Figures 1B and 1C). The remainder either was on both chromatids or gave variegated signals, which we collectively defined as ‘‘complex.’’ Most of the DNA synthesis leading to these complex patterns of EdU incorporation appeared to result from multiple DNA synthesis events occurring on a single sister, but the scoring of those
events was less reliable. These data indicate that EdU incorporation in mitosis most frequently exhibits a conservative pattern of DNA replication and that MiDAS, therefore, differs fundamentally from canonical semi-conservative DNA replication in S-phase. RAD52 Is Retained at CFSs on Mitotic Chromatin Following RS To assess the contribution of HR factors in promoting MiDAS, we examined whether HR factors were present in the soluble or the chromatin (insoluble) fraction from asynchronous (AS), G2-arrested (G2), or prometaphase (M) cells. We observed that BRCA2, RAD51, and RAD52 were all associated with chromatin from AS and G2-arrested cells irrespective of whether or not the cells were exposed to APH (Figures 1D and 1E; Figure S1A). However, we observed that RAD52, but not RAD51 or BRCA2, was retained on mitotic chromatin following APH treatment (Figures 1D and 1E; Figure S1A). Consistent with these data, RAD52 foci co-localized with FANCD2 twin foci in early mitosis alongside RPA and MUS81, whereas RAD51 and BRCA2 did not (Figures 1F and 1G; Figures S1B–S1D). MiDAS Is RAD52 Dependent To analyze whether MiDAS requires HR factors, we depleted BRCA2, RAD51, or RAD52 using small interfering RNA (siRNAs) and confirmed that the knockdowns did not elicit a cell cycle arrest in the presence of low-dose APH (Figures S2A–S2D). Depletion of either BRCA2 or RAD51 increased the number of MiDAS foci (FANCD2-associated EdU foci in prometaphase cells; Figures 2A and 2B) without altering the frequency of 53BP1 nuclear bodies in G1 cells (Figures 2C and 2D). In contrast, RAD52 depletion led to a failure to perform MiDAS and to a corresponding increase in the number of 53BP1 nuclear bodies (Figures 2A–2D; Figure S2E). Similar results were observed following administration of a RAD52 inhibitor (AICAR) (Chandramouly et al., 2015; Sullivan et al., 2016) in early mitosis (Figures 2E–2G). Because this inhibitor prevents RAD52 oligomerization, these data suggest that the creation of a higher-order structural form of RAD52 is required for MiDAS. Taken together, we propose that RAD51 or BRCA2 deficiency leads to increased CFS expression as a result of an increased reliance on the RAD52-dependent MiDAS pathway. RAD52 Promotes MUS81 Recruitment to CFSs MUS81-EME1 forms a complex with SLX4 in early mitosis following CDK1-dependent phosphorylation of the non-catalytic EME1 subunit (Castor et al., 2013; Garner et al., 2013; Minocherhomji et al., 2015; Wyatt et al., 2013). We investigated, therefore, whether RAD52 was required for the recruitment of either MUS81 or SLX4 to CFSs in early mitosis. We observed that recruitment of MUS81 to CFSs in early mitosis was impaired in RAD52-depleted cells (Figures 3A and 3B), whereas recruitment of SLX4 to CFSs was unaffected, albeit with increased numbers of FANCD2SLX4 foci per prometaphase cell (Figures 3C and 3D). Indeed, depletion of RAD52 generally inhibited recruitment of MUS81 to chromatin in mitosis (Figures 3E and 3F; Figure S3A). We also observed that SLX4-depleted cells failed to recruit RAD52 to chromatin in early mitosis (Figure S3B). In contrast, depletion of MUS81, or inhibition of PLK1 (with BI 2536; added in mitosis) (Le´na´rt et al., 2007), which block MiDAS to a similar extent as
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Figure 2. RAD52 Is Essential for MiDAS and MUS81 Recruitment in Mitosis (A and B) Representative IF images (A) and quantification (B) of EdU foci (red) that co-localize with FANCD2 twin foci (green) in prometaphase cells following the indicated siRNA depletions (top panel) and RS. (C and D) Representative IF images (C) and quantification (D) of 53BP1 nuclear bodies (green) in G1 daughter cells following the indicated siRNA depletions (top panel) and RS. (E–G) Experimental workflow (E), representative IF images (F), and quantification (G) of EdU foci (red) after RAD52i. DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 mm. See also Figure S2.
SLX4 depletion, did not affect RAD52 recruitment during mitosis (Figures S3B–S3D). Therefore, RAD52 appears to act at an SLX4-dependent step in MiDAS prior to the involvement of MUS81 or POLD3.
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Interestingly, we observed that phosphorylated EME1 was still detectable on mitotic chromatin in RAD52-depleted cells (Figure S3E). This suggests that RAD52 and phosphorylated EME1 collectively promote the timely union of the active
Figure 3. RAD52 Is Required for the Recruitment of MUS81 and POLD3 to Chromatin in Mitosis (A and B) Representative IF images (A) and quantification (B) of co-localized MUS81 foci (red) and FANCD2 twin foci (green). (C and D) Representative images (C) and quantification (D) of co-localized SLX4 foci (red) and FANCD2 twin foci (green). (E and F) Representative western blots (E) and quantification (F) of soluble and insoluble fractions after the indicated siRNA treatments (top). DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 mm. See also Figure S3.
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Figure 4. Consequences of Defective MiDAS (A and B) Representative images (A) and quantification (B) of PICH-positive UFBs (red) with FANCD2 foci (green) at their bridge termini. (C–E) Experimental workflow (C), representative images (D), and quantification (E) of MiDAS (EdU foci, red) as indicated. DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 mm. (F and G) Clonogenic assays (F) and quantification (G) of U2OS cells following indicated treatments in S-phase or mitosis. (H) Model proposing that MiDAS can occur via an MMBIR-like process. Replication fork disruption, perhaps due to the presence of DNA secondary structures at AT-rich regions or R-loops, followed by limited 50 end resection of the generated DNA end, exposes a region of microhomology that can anneal with the partially (legend continued on next page)
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MUS81-EME1 endonuclease complex at CFSs. In contrast, we observed that depletion of RAD52 impaired the recruitment of POLD3 to chromatin during all stages of the cell cycle (Figures 3E and 3F; Figure S3A). Taken together, our data indicate that RAD52 is required for recruitment of MUS81 and POLD3 to CFSs in early mitosis and, consequently, for MiDAS. RAD52 Depletion Causes Chromosome Instability Next, we investigated the cellular consequences of RAD52 deficiency in human cells and whether this might lead to chromosomal instability and/or mitotic aberrations. We observed that RAD52 depletion resulted in increased frequencies of CFS-associated ultra-fine DNA bridges (UFBs) and chromatin bridges in anaphase, decreased CFS expression, and increased formation of micronuclei in G1 daughter cells (Figures 4A and 4B; Figures S4A–S4F). These phenotypes are highly similar to those observed in MUS81- and POLD3-depleted cells (Minocherhomji et al., 2015; Naim et al., 2013; Ying et al., 2013), suggesting that these mitotic defects are a common and direct consequence of impaired MiDAS. Combined Inhibition of ATR and RAD52 Leads to Increased Cancer Cell Death Increased RS is a hallmark of many cancer cells. Inhibition of ATR (ATRi) by pharmacological means further exacerbates RS in cancer cells to an extent that compromises cell viability. This strategy is currently in clinical trials as a potential anti-cancer therapy (Reaper et al., 2011; Toledo et al., 2011). As excessive RS causes an increased reliance on MiDAS for completion of DNA replication (Minocherhomji et al., 2015), and ATR inhibition increases CFS expression (Casper et al., 2002), we tested whether ATRi in S/G2 also increased the reliance on MiDAS. Consistent with a role for ATR in counteracting replication stress, low-dose APH in combination with ATRi led to an increase in the frequency of cells performing MiDAS (Figures 4C–4E). Moreover, combined inhibition of ATR and MiDAS (via RAD52i) caused a marked increase in cancer cell death following replication stress (Figures 4F and 4G). Taken together, our data suggest that exacerbating replication stress in cancer cells, combined with inhibition of MiDAS, could represent a novel anti-cancer therapeutic regimen. DISCUSSION We have identified a key role for the human RAD52 protein in promoting an early step in MiDAS. More specifically, RAD52 is required for the timely assembly of the active MUS81-EME1 endonuclease at unprocessed CFSs in mitosis. Because human RAD52 is poorly characterized and previously only thought to operate in a backup HR pathway to BRCA2, our data reveal a specific function of RAD52 that operates in BRCA2-proficient cells.
MiDAS does not require RAD51 or BRCA2. Nevertheless, RAD51 and BRCA2 function earlier in the cell cycle to reduce reliance on the MiDAS pathway. This is consistent with the known functions of RAD51 in the re-start of collapsed replication forks from a one-ended DNA break in late S/G2 phase cells (Sirbu et al., 2011; Zellweger et al., 2015). We propose that human RAD52 plays a more specialized role in promoting RAD51-independent DNA repair in mitosis at a time when BRCA2 and RAD51 are apparently excluded from chromatin. This most likely involves the ssDNA annealing activity of RAD52. Consistent with this, a RAD51 binding-defective derivative of RAD52 is still competent for DNA annealing (Kagawa et al., 2001). Our data are consistent with a role for RAD52 in BIR. Conventional BIR in yeast requires both Rad51 and Rad52 for homology-mediated template switching. In contrast, the microhomology-driven branch of BIR, MMBIR, can, under certain circumstances, be Rad51 independent (Anand et al., 2013; Hastings et al., 2009; Ottaviani et al., 2014; Sakofsky et al., 2015). Moreover, in yeast, some Rad52-dependent events involving annealing of regions of homology, such as would occur during BIR (Anand et al., 2013; Hastings et al., 2009; Mott and Symington, 2011), create DNA substrates that can be resolved efficiently by MUS81-EME1 (Gaillard et al., 2003; Osman et al., 2003). Both BIR and MMBIR require Pol32, whereas HR-mediated gene conversion does not (Donnianni and Symington, 2013; Hicks et al., 2010; Lydeard et al., 2007, 2010; Mayle et al., 2015; Sakofsky et al., 2015). Based on these considerations, therefore, we propose that MiDAS most likely occurs via a RAD51-independent MMBIR process in human cells. Given that RAD51-independent MMBIR requires much less homology (1–6 nucleotides) for template switching (Hastings et al., 2009; Ottaviani et al., 2014), this process would be optimal during the narrow time window in early mitosis when MiDAS occurs, as it negates the need for extensive DNA end resection and Rad51-driven homology searching. In the context of a collapsed replication fork, where the sister chromatid is in very close proximity, an extensive homology search is unnecessary and could even be undesirable. As illustrated in the model presented in Figure 4H, RAD52-mediated DNA annealing from a collapsed replication fork into regions of micro-homology could fulfill the requirement for a rapid completion of DNA synthesis to take place in mitosis, albeit at the potential cost of increased mutagenesis and CNVs at CFSs. Given the widespread occurrence of oncogene-induced RS and the increasing clinical interest in small molecule inhibitors that further exacerbate RS in human cancers (such as ATR inhibitors), our findings point to a protective role for RAD52 in the maintenance of cancer cell viability. As such, RAD52 could be a plausible target for therapies targeted at tumors with excessive RS and/or those exposed to agents that generate additional RS, such as ATRi. Furthermore, RAD52 inhibition might be
single-stranded template DNA. The annealing step might require a DNA helicase to facilitate template denaturation. Processing of the resulting replication intermediate by the activated SLX-MUS complex (SLX4 in complex with MUS81-EME1 and other nucleases) in early mitosis is then associated with POLD3dependent conservative DNA repair synthesis. This process would account for the high level of CNVs that arise at CFS loci in cancer cells. For clarity, the replication fork merging with the MiDAS bubble from the right is omitted. However, if this fork were also disrupted, DNA synthesis could arise on both sister chromatids via the same mechanism. See also Figure S4.
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selective, given the apparently limited role of RAD52 in genome maintenance in normal cells (Feng et al., 2011; Lok et al., 2013; Lok and Powell, 2012; Rijkers et al., 1998). Of possible relevance to this, abrogation of RAD52 function leads to increased cell death in lung tumors and in BRCA2-deficient cancer cells (Feng et al., 2011; Lok et al., 2013). We propose that this could be due to the abrogation of MiDAS, which is required to sustain viability in these cells. Additionally, amplification of the 12p13.33 locus comprising the RAD52 gene is associated with the development of squamous cell carcinomas of the lung (Lieberman et al., 2016). Hence, we propose that the treatment of patients with MiDAS inhibitors, in combination with agents such as ATR inhibitors, might synergistically and selectively target tumors exhibiting oncogene-activated RS. Sotiriou et al. (2016) report in this issue of Molecular Cell that RAD52 is required for the BIR-mediated repair of collapsed DNA replication forks in response to oncogene-induced replication stress. Their data are fully consistent with ours. EXPERIMENTAL PROCEDURES Cell Culture Osteosarcoma (U2OS) or cervical cancer (HeLa) cells were maintained in DMEM as described in Supplemental Experimental Procedures. Cell Synchronization Asynchronously growing cells were synchronized at G2 (+RO3306) for 16 hr and released into mitosis. Mitotic cells were shaken off at the times indicated. RNA Interference RNA interference was carried out using Lipofectamine RNAiMax reagent according to the manufacturer’s instructions. siRNAs used are described in the Supplemental Experimental Procedures. EdU Labeling and Detection EdU labeling and detection was performed as previously reported (Minocherhomji et al., 2015) and using Click chemistry according to the manufacturer’s instructions but with a final concentration of 13 of the Click-IT EdU buffer additive (Life Technologies). Metaphase Chromosome Spreads DAPI-stained metaphase chromosomes were visualized as described previously (Minocherhomji et al., 2015). Immunofluorescence Cells were fixed and permeabilized simultaneously at room temperature (RT). Following primary and secondary antibody detection, samples were air-dried and mounted using Vectashield with DAPI (Vector Laboratories). Images were captured using an Olympus BX63 microscope or a Zeiss LSM 700 confocal microscope. Image analysis utilized ImageJ or Zeiss software. Antibodies used are described in the Supplemental Experimental Procedures. Subcellular Fractionation Cells were harvested and subjected to subcellular fractionation using different centrifugation steps as described in Supplemental Experimental Procedures.
SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.molcel.2016.10.037. AUTHOR CONTRIBUTIONS R.B. and S.M. performed experiments. I.D.H., R.B., and S.M. designed experiments, interpreted results, and wrote/edited the manuscript. ACKNOWLEDGMENTS We thank Dr. Claudia Lukas, Dr. Jiri Lukas, and members of the I.D.H. laboratory for useful discussions and Hocine Mankouri and Ying Liu for critical reading of the manuscript. We also thank Thanos Halazonetis for sharing data prior to submission. Work in the authors’ laboratory is funded by the Danish National Research Foundation (DNRF115), The European Research Council (ERC Project Number 321717), and The Nordea Foundation. R.B. and S.M. were recipients of Danish Medical Research Council fellowships (DFF-4004-00155B, DFF-6110-00169B, and DFF-6110-00243B). Received: July 11, 2016 Revised: September 6, 2016 Accepted: October 28, 2016 Published: December 15, 2016 REFERENCES Anand, R.P., Lovett, S.T., and Haber, J.E. (2013). Break-induced DNA replication. Cold Spring Harb. Perspect. Biol. 5, a010397. Arlt, M.F., Durkin, S.G., Ragland, R.L., and Glover, T.W. (2006). Common fragile sites as targets for chromosome rearrangements. DNA Repair (Amst.) 5, 1126–1135. Bai, Y., and Symington, L.S. (1996). A Rad52 homolog is required for RAD51independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 10, 2025–2037. Burrell, R.A., McGranahan, N., Bartek, J., and Swanton, C. (2013). The causes and consequences of genetic heterogeneity in cancer evolution. Nature 501, 338–345. Burrow, A.A., Williams, L.E., Pierce, L.C., and Wang, Y.H. (2009). Over half of breakpoints in gene pairs involved in cancer-specific recurrent translocations are mapped to human chromosomal fragile sites. BMC Genomics 10, 59. Bursomanno, S., Beli, P., Khan, A.M., Minocherhomji, S., Wagner, S.A., Bekker-Jensen, S., Mailand, N., Choudhary, C., Hickson, I.D., and Liu, Y. (2015). Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells. DNA Repair (Amst.) 25, 84–96. Casper, A.M., Nghiem, P., Arlt, M.F., and Glover, T.W. (2002). ATR regulates fragile site stability. Cell 111, 779–789. Castor, D., Nair, N., De´clais, A.C., Lachaud, C., Toth, R., Macartney, T.J., Lilley, D.M., Arthur, J.S., and Rouse, J. (2013). Cooperative control of holliday junction resolution and DNA repair by the SLX1 and MUS81-EME1 nucleases. Mol. Cell 52, 221–233. Chan, K.L., Palmai-Pallag, T., Ying, S., and Hickson, I.D. (2009). Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat. Cell Biol. 11, 753–760.
Western Blotting Analysis Details are provided in Supplemental Experimental Procedures.
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RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress Graphical Abstract
Authors Rahul Bhowmick, Sheroy Minocherhomji, Ian D. Hickson
Correspondence [email protected]
In Brief Tumorigenesis is frequently driven by oncogene activation, which generates so-called DNA replication ‘‘stress’’ and instability at specific genomic loci, called common fragile sites. Bhowmick et al. show that DNA repair synthesis at fragile sites occurs in mitosis and define a role for the homologous recombination factor RAD52 in this process.
Highlights d
RAD52 promotes mitotic DNA synthesis (MiDAS) following replication stress
d
RAD52 promotes recruitment of MUS81 and POLD3 in mitosis
d
RAD52 deficiency increases mitotic chromosome missegregation
d
Mitotic DNA synthesis occurs independently of ATR, BRCA2, and RAD51
Bhowmick et al., 2016, Molecular Cell 64, 1117–1126 December 15, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2016.10.037
Molecular Cell
Short Article RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress Rahul Bhowmick,1,2 Sheroy Minocherhomji,1,2,3 and Ian D. Hickson1,4,* 1Center for Chromosome Stability and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen N, Denmark 2Co-first author 3Present address: Department of Discovery Toxicology, Amgen Inc., 1120 Veterans Boulevard, San Francisco, CA 94080, USA 4Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.10.037
SUMMARY
Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis. This mitotic DNA synthesis, termed MiDAS, requires the MUS81-EME1 endonuclease and a non-catalytic subunit of the Pol-delta complex, POLD3. Here, we examine the contribution of HR factors in promoting MiDAS in human cells. We report that RAD51 and BRCA2 are dispensable for MiDAS but are required to counteract replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective inhibition of MiDAS may comprise a potential therapeutic strategy to sensitize cancer cells undergoing replicative stress. INTRODUCTION The activation of oncogenes during tumorigenesis generates DNA replication stress (RS), which is a known driver of chromosomal instability (CIN) (Burrell et al., 2013; Macheret and Halazonetis, 2015). Certain regions of the human genome, such as common fragile sites (CFSs), are particularly sensitive to RS. These loci are difficult to replicate and are prone to generate copy number variations (CNVs) and chromosomal rearrangements in human cancers (Arlt et al., 2006; Burrow et al., 2009; Wilson et al., 2015). There are several features that the different CFS loci often have in common, including a propensity to form DNA secondary structures within AT-rich sequences, the presence of a long primary transcript that can potentially impede replication fork progression, and increased levels of R-loops (Helmrich et al., 2011; Le Tallec et al., 2013; Santos-Pereira
and Aguilera, 2015; Sarni and Kerem, 2016; Sollier and Cimprich, 2015; Zhang and Freudenreich, 2007). CFSs manifest as DAPInegative gaps on metaphase chromosomes (CFS expression), a phenomenon that is exacerbated by exposing cells in S-phase to a mild dose of aphidicolin (APH) (Glover et al., 1984). CFS expression is an active process that requires the MUS81EME1 DNA structure-selective endonuclease and its associated scaffold protein, SLX4 (Minocherhomji and Hickson, 2014; Minocherhomji et al., 2015; Naim et al., 2013; Ying et al., 2013). In response to RS, the FANCD2/FANCI protein complex forms ‘‘twin foci’’ (one on each sister chromatid) at CFS loci that can persist into mitosis (Chan et al., 2009; Howlett et al., 2005; Minocherhomji et al., 2015). These foci co-localize with SLX4 and serve as a useful surrogate marker of the location of CFSs in G2/M phase cells. Upon mitotic entry, CDK1-dependent phosphorylation of EME1 promotes the association of MUS81EME1 with SLX4 at under-replicated CFS loci (Minocherhomji et al., 2015; Wyatt et al., 2013; Ying et al., 2013). An important function of MUS81-EME1 at CFSs is to promote DNA repair synthesis that occurs in the prophase of mitosis (Minocherhomji et al., 2015). This mitotic DNA synthesis (henceforth termed ‘‘MiDAS’’) requires MUS81-EME1, SLX4, and a non-catalytic subunit of DNA polymerase d, POLD3 (Pol32 in yeast) (Bursomanno et al., 2015; Minocherhomji et al., 2015). Figure 1A summarizes the previously proposed roles of these factors. DNA synthesized via the MiDAS pathway is associated with the appearance of cytogenetically defined DAP1-negative gaps on metaphase chromosomes at CFS loci (Minocherhomji et al., 2015). In the absence of MiDAS, potentially lethal chromosome missegregation occurs, and daughter cells exhibit an increased frequency of prominent structures termed 53BP1 nuclear bodies (comprising OPT domains), which are known to associate with missegregated CFSs (Harrigan et al., 2011; Lukas et al., 2011; Minocherhomji et al., 2015; Naim et al., 2013; Ying et al., 2013). Homologous recombination (HR) has important roles in the repair of stalled or collapsed DNA replication forks, as well as of DNA double-strand breaks. Among the factors required for HR in human cells are RAD51, BRCA1, and BRCA2 (Davies et al., 2001; Sung and Klein, 2006; Xia et al., 2001). BRCA1 and BRCA2 are required for the efficient association of RAD51 with DNA, as well as stabilization of RAD51 filaments on singlestranded DNA (ssDNA). In yeast, Rad52 largely performs this so-called Rad51 mediator function (Jensen et al., 2010;
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Figure 1. RAD52 Persists at Sites of On-Going DNA Synthesis in Mitosis Following Replication Stress (A) Previously proposed model for MiDAS. (B and C) Representative images (B) and quantification (C) of EdU incorporation (red) on isolated metaphase chromosomes (DAPI, blue) following replication stress (RS) (+APH). (D and E) Representative western blots of soluble and insoluble fractions (D) and quantification (E) following pre-treatment with (+APH) or without (APH) APH. (F and G) Representative immunofluorescence (IF) images (F) and quantification (G) of the co-localization of RAD52 (green) with MUS81 or FANCD2 twin foci (red). DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 mm. See also Figure S1.
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Mortensen et al., 1996; San Filippo et al., 2006). Yeast cells also possess an additional Rad52-like protein called Rad59, which plays a more limited and specialized role in certain sub-pathways of HR (Bai and Symington, 1996; Pannunzio et al., 2008; Signon et al., 2001). One of these sub-pathways is break-induced replication (BIR), a form of recombination-dependent DNA replication that can repair one-ended DNA breaks that can arise, for example, when a replication fork collapses upon encountering a break in the template DNA. A subclass of BIR includes microhomology-mediated BIR (MMBIR), which can lead to the formation of CNVs without RAD51-mediated strand invasion (Hastings et al., 2009; Ottaviani et al., 2014; Sakofsky et al., 2015). The principal biochemical function of Rad52 family members is to promote the annealing of complementary ssDNA molecules, which probably explains how Rad52/59 can catalyze some HR processes in the absence of Rad51-mediated DNA strand invasion (Gasior et al., 1998; Kagawa et al., 2001, 2002; Lao et al., 2008; McIlwraith and West, 2008). It is not clear from analysis of sequence conservation whether human RAD52 is the functional homolog of yeast Rad52 or Rad59. Human RAD52 forms oligomers that have robust ssDNA annealing activity (Kagawa et al., 2001, 2002). Small molecule disruption of RAD52 oligomerization impairs some recombination activities of RAD52 (Chandramouly et al., 2015; Sullivan et al., 2016) and is synthetically lethal with BRCA2 deficiency (Chandramouly et al., 2015; Feng et al., 2011). Current models propose that RAD52 operates in a BRCA2-independent HR backup pathway to load RAD51 onto DNA (Lok and Powell, 2012). However, the precise role of RAD52 in human cells remains to be defined. The requirement for POLD3 in MiDAS suggests that the DNA synthesis might proceed via a BIR-like process (Costantino et al., 2014). We therefore investigated how HR impacts the mitotic DNA damage response in human cells following RS. We demonstrate that RAD52, but not RAD51 or BRCA2, is required for MiDAS. Consistent with RAD52 acting at an early step in MiDAS, RAD52 depletion disrupts the timely recruitment of MUS81-EME1 and POLD3 to CFSs in mitosis. RESULTS DNA Synthesis during MiDAS Frequently Occurs on a Single Sister Chromatid One possible mechanism for MiDAS would be for stalled replication forks at CFSs to simply re-start in early mitosis via canonical semi-conservative DNA replication. In this scenario, the newly replicated DNA would be evenly distributed on each sister chromatid of a metaphase chromosome. In contrast, BIR can occur by conservative DNA replication in yeast (Donnianni and Symington, 2013; Saini et al., 2013). To visualize the location of newly synthesized DNA in mitosis, we examined the pattern of EdU incorporation on isolated metaphase chromosomes. Interestingly, 50% of the newly synthesized DNA tracts in early mitosis were localized unequivocally to a single sister chromatid (Figures 1B and 1C). The remainder either was on both chromatids or gave variegated signals, which we collectively defined as ‘‘complex.’’ Most of the DNA synthesis leading to these complex patterns of EdU incorporation appeared to result from multiple DNA synthesis events occurring on a single sister, but the scoring of those
events was less reliable. These data indicate that EdU incorporation in mitosis most frequently exhibits a conservative pattern of DNA replication and that MiDAS, therefore, differs fundamentally from canonical semi-conservative DNA replication in S-phase. RAD52 Is Retained at CFSs on Mitotic Chromatin Following RS To assess the contribution of HR factors in promoting MiDAS, we examined whether HR factors were present in the soluble or the chromatin (insoluble) fraction from asynchronous (AS), G2-arrested (G2), or prometaphase (M) cells. We observed that BRCA2, RAD51, and RAD52 were all associated with chromatin from AS and G2-arrested cells irrespective of whether or not the cells were exposed to APH (Figures 1D and 1E; Figure S1A). However, we observed that RAD52, but not RAD51 or BRCA2, was retained on mitotic chromatin following APH treatment (Figures 1D and 1E; Figure S1A). Consistent with these data, RAD52 foci co-localized with FANCD2 twin foci in early mitosis alongside RPA and MUS81, whereas RAD51 and BRCA2 did not (Figures 1F and 1G; Figures S1B–S1D). MiDAS Is RAD52 Dependent To analyze whether MiDAS requires HR factors, we depleted BRCA2, RAD51, or RAD52 using small interfering RNA (siRNAs) and confirmed that the knockdowns did not elicit a cell cycle arrest in the presence of low-dose APH (Figures S2A–S2D). Depletion of either BRCA2 or RAD51 increased the number of MiDAS foci (FANCD2-associated EdU foci in prometaphase cells; Figures 2A and 2B) without altering the frequency of 53BP1 nuclear bodies in G1 cells (Figures 2C and 2D). In contrast, RAD52 depletion led to a failure to perform MiDAS and to a corresponding increase in the number of 53BP1 nuclear bodies (Figures 2A–2D; Figure S2E). Similar results were observed following administration of a RAD52 inhibitor (AICAR) (Chandramouly et al., 2015; Sullivan et al., 2016) in early mitosis (Figures 2E–2G). Because this inhibitor prevents RAD52 oligomerization, these data suggest that the creation of a higher-order structural form of RAD52 is required for MiDAS. Taken together, we propose that RAD51 or BRCA2 deficiency leads to increased CFS expression as a result of an increased reliance on the RAD52-dependent MiDAS pathway. RAD52 Promotes MUS81 Recruitment to CFSs MUS81-EME1 forms a complex with SLX4 in early mitosis following CDK1-dependent phosphorylation of the non-catalytic EME1 subunit (Castor et al., 2013; Garner et al., 2013; Minocherhomji et al., 2015; Wyatt et al., 2013). We investigated, therefore, whether RAD52 was required for the recruitment of either MUS81 or SLX4 to CFSs in early mitosis. We observed that recruitment of MUS81 to CFSs in early mitosis was impaired in RAD52-depleted cells (Figures 3A and 3B), whereas recruitment of SLX4 to CFSs was unaffected, albeit with increased numbers of FANCD2SLX4 foci per prometaphase cell (Figures 3C and 3D). Indeed, depletion of RAD52 generally inhibited recruitment of MUS81 to chromatin in mitosis (Figures 3E and 3F; Figure S3A). We also observed that SLX4-depleted cells failed to recruit RAD52 to chromatin in early mitosis (Figure S3B). In contrast, depletion of MUS81, or inhibition of PLK1 (with BI 2536; added in mitosis) (Le´na´rt et al., 2007), which block MiDAS to a similar extent as
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Figure 2. RAD52 Is Essential for MiDAS and MUS81 Recruitment in Mitosis (A and B) Representative IF images (A) and quantification (B) of EdU foci (red) that co-localize with FANCD2 twin foci (green) in prometaphase cells following the indicated siRNA depletions (top panel) and RS. (C and D) Representative IF images (C) and quantification (D) of 53BP1 nuclear bodies (green) in G1 daughter cells following the indicated siRNA depletions (top panel) and RS. (E–G) Experimental workflow (E), representative IF images (F), and quantification (G) of EdU foci (red) after RAD52i. DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 mm. See also Figure S2.
SLX4 depletion, did not affect RAD52 recruitment during mitosis (Figures S3B–S3D). Therefore, RAD52 appears to act at an SLX4-dependent step in MiDAS prior to the involvement of MUS81 or POLD3.
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Interestingly, we observed that phosphorylated EME1 was still detectable on mitotic chromatin in RAD52-depleted cells (Figure S3E). This suggests that RAD52 and phosphorylated EME1 collectively promote the timely union of the active
Figure 3. RAD52 Is Required for the Recruitment of MUS81 and POLD3 to Chromatin in Mitosis (A and B) Representative IF images (A) and quantification (B) of co-localized MUS81 foci (red) and FANCD2 twin foci (green). (C and D) Representative images (C) and quantification (D) of co-localized SLX4 foci (red) and FANCD2 twin foci (green). (E and F) Representative western blots (E) and quantification (F) of soluble and insoluble fractions after the indicated siRNA treatments (top). DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 mm. See also Figure S3.
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Figure 4. Consequences of Defective MiDAS (A and B) Representative images (A) and quantification (B) of PICH-positive UFBs (red) with FANCD2 foci (green) at their bridge termini. (C–E) Experimental workflow (C), representative images (D), and quantification (E) of MiDAS (EdU foci, red) as indicated. DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 mm. (F and G) Clonogenic assays (F) and quantification (G) of U2OS cells following indicated treatments in S-phase or mitosis. (H) Model proposing that MiDAS can occur via an MMBIR-like process. Replication fork disruption, perhaps due to the presence of DNA secondary structures at AT-rich regions or R-loops, followed by limited 50 end resection of the generated DNA end, exposes a region of microhomology that can anneal with the partially (legend continued on next page)
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MUS81-EME1 endonuclease complex at CFSs. In contrast, we observed that depletion of RAD52 impaired the recruitment of POLD3 to chromatin during all stages of the cell cycle (Figures 3E and 3F; Figure S3A). Taken together, our data indicate that RAD52 is required for recruitment of MUS81 and POLD3 to CFSs in early mitosis and, consequently, for MiDAS. RAD52 Depletion Causes Chromosome Instability Next, we investigated the cellular consequences of RAD52 deficiency in human cells and whether this might lead to chromosomal instability and/or mitotic aberrations. We observed that RAD52 depletion resulted in increased frequencies of CFS-associated ultra-fine DNA bridges (UFBs) and chromatin bridges in anaphase, decreased CFS expression, and increased formation of micronuclei in G1 daughter cells (Figures 4A and 4B; Figures S4A–S4F). These phenotypes are highly similar to those observed in MUS81- and POLD3-depleted cells (Minocherhomji et al., 2015; Naim et al., 2013; Ying et al., 2013), suggesting that these mitotic defects are a common and direct consequence of impaired MiDAS. Combined Inhibition of ATR and RAD52 Leads to Increased Cancer Cell Death Increased RS is a hallmark of many cancer cells. Inhibition of ATR (ATRi) by pharmacological means further exacerbates RS in cancer cells to an extent that compromises cell viability. This strategy is currently in clinical trials as a potential anti-cancer therapy (Reaper et al., 2011; Toledo et al., 2011). As excessive RS causes an increased reliance on MiDAS for completion of DNA replication (Minocherhomji et al., 2015), and ATR inhibition increases CFS expression (Casper et al., 2002), we tested whether ATRi in S/G2 also increased the reliance on MiDAS. Consistent with a role for ATR in counteracting replication stress, low-dose APH in combination with ATRi led to an increase in the frequency of cells performing MiDAS (Figures 4C–4E). Moreover, combined inhibition of ATR and MiDAS (via RAD52i) caused a marked increase in cancer cell death following replication stress (Figures 4F and 4G). Taken together, our data suggest that exacerbating replication stress in cancer cells, combined with inhibition of MiDAS, could represent a novel anti-cancer therapeutic regimen. DISCUSSION We have identified a key role for the human RAD52 protein in promoting an early step in MiDAS. More specifically, RAD52 is required for the timely assembly of the active MUS81-EME1 endonuclease at unprocessed CFSs in mitosis. Because human RAD52 is poorly characterized and previously only thought to operate in a backup HR pathway to BRCA2, our data reveal a specific function of RAD52 that operates in BRCA2-proficient cells.
MiDAS does not require RAD51 or BRCA2. Nevertheless, RAD51 and BRCA2 function earlier in the cell cycle to reduce reliance on the MiDAS pathway. This is consistent with the known functions of RAD51 in the re-start of collapsed replication forks from a one-ended DNA break in late S/G2 phase cells (Sirbu et al., 2011; Zellweger et al., 2015). We propose that human RAD52 plays a more specialized role in promoting RAD51-independent DNA repair in mitosis at a time when BRCA2 and RAD51 are apparently excluded from chromatin. This most likely involves the ssDNA annealing activity of RAD52. Consistent with this, a RAD51 binding-defective derivative of RAD52 is still competent for DNA annealing (Kagawa et al., 2001). Our data are consistent with a role for RAD52 in BIR. Conventional BIR in yeast requires both Rad51 and Rad52 for homology-mediated template switching. In contrast, the microhomology-driven branch of BIR, MMBIR, can, under certain circumstances, be Rad51 independent (Anand et al., 2013; Hastings et al., 2009; Ottaviani et al., 2014; Sakofsky et al., 2015). Moreover, in yeast, some Rad52-dependent events involving annealing of regions of homology, such as would occur during BIR (Anand et al., 2013; Hastings et al., 2009; Mott and Symington, 2011), create DNA substrates that can be resolved efficiently by MUS81-EME1 (Gaillard et al., 2003; Osman et al., 2003). Both BIR and MMBIR require Pol32, whereas HR-mediated gene conversion does not (Donnianni and Symington, 2013; Hicks et al., 2010; Lydeard et al., 2007, 2010; Mayle et al., 2015; Sakofsky et al., 2015). Based on these considerations, therefore, we propose that MiDAS most likely occurs via a RAD51-independent MMBIR process in human cells. Given that RAD51-independent MMBIR requires much less homology (1–6 nucleotides) for template switching (Hastings et al., 2009; Ottaviani et al., 2014), this process would be optimal during the narrow time window in early mitosis when MiDAS occurs, as it negates the need for extensive DNA end resection and Rad51-driven homology searching. In the context of a collapsed replication fork, where the sister chromatid is in very close proximity, an extensive homology search is unnecessary and could even be undesirable. As illustrated in the model presented in Figure 4H, RAD52-mediated DNA annealing from a collapsed replication fork into regions of micro-homology could fulfill the requirement for a rapid completion of DNA synthesis to take place in mitosis, albeit at the potential cost of increased mutagenesis and CNVs at CFSs. Given the widespread occurrence of oncogene-induced RS and the increasing clinical interest in small molecule inhibitors that further exacerbate RS in human cancers (such as ATR inhibitors), our findings point to a protective role for RAD52 in the maintenance of cancer cell viability. As such, RAD52 could be a plausible target for therapies targeted at tumors with excessive RS and/or those exposed to agents that generate additional RS, such as ATRi. Furthermore, RAD52 inhibition might be
single-stranded template DNA. The annealing step might require a DNA helicase to facilitate template denaturation. Processing of the resulting replication intermediate by the activated SLX-MUS complex (SLX4 in complex with MUS81-EME1 and other nucleases) in early mitosis is then associated with POLD3dependent conservative DNA repair synthesis. This process would account for the high level of CNVs that arise at CFS loci in cancer cells. For clarity, the replication fork merging with the MiDAS bubble from the right is omitted. However, if this fork were also disrupted, DNA synthesis could arise on both sister chromatids via the same mechanism. See also Figure S4.
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selective, given the apparently limited role of RAD52 in genome maintenance in normal cells (Feng et al., 2011; Lok et al., 2013; Lok and Powell, 2012; Rijkers et al., 1998). Of possible relevance to this, abrogation of RAD52 function leads to increased cell death in lung tumors and in BRCA2-deficient cancer cells (Feng et al., 2011; Lok et al., 2013). We propose that this could be due to the abrogation of MiDAS, which is required to sustain viability in these cells. Additionally, amplification of the 12p13.33 locus comprising the RAD52 gene is associated with the development of squamous cell carcinomas of the lung (Lieberman et al., 2016). Hence, we propose that the treatment of patients with MiDAS inhibitors, in combination with agents such as ATR inhibitors, might synergistically and selectively target tumors exhibiting oncogene-activated RS. Sotiriou et al. (2016) report in this issue of Molecular Cell that RAD52 is required for the BIR-mediated repair of collapsed DNA replication forks in response to oncogene-induced replication stress. Their data are fully consistent with ours. EXPERIMENTAL PROCEDURES Cell Culture Osteosarcoma (U2OS) or cervical cancer (HeLa) cells were maintained in DMEM as described in Supplemental Experimental Procedures. Cell Synchronization Asynchronously growing cells were synchronized at G2 (+RO3306) for 16 hr and released into mitosis. Mitotic cells were shaken off at the times indicated. RNA Interference RNA interference was carried out using Lipofectamine RNAiMax reagent according to the manufacturer’s instructions. siRNAs used are described in the Supplemental Experimental Procedures. EdU Labeling and Detection EdU labeling and detection was performed as previously reported (Minocherhomji et al., 2015) and using Click chemistry according to the manufacturer’s instructions but with a final concentration of 13 of the Click-IT EdU buffer additive (Life Technologies). Metaphase Chromosome Spreads DAPI-stained metaphase chromosomes were visualized as described previously (Minocherhomji et al., 2015). Immunofluorescence Cells were fixed and permeabilized simultaneously at room temperature (RT). Following primary and secondary antibody detection, samples were air-dried and mounted using Vectashield with DAPI (Vector Laboratories). Images were captured using an Olympus BX63 microscope or a Zeiss LSM 700 confocal microscope. Image analysis utilized ImageJ or Zeiss software. Antibodies used are described in the Supplemental Experimental Procedures. Subcellular Fractionation Cells were harvested and subjected to subcellular fractionation using different centrifugation steps as described in Supplemental Experimental Procedures.
SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.molcel.2016.10.037. AUTHOR CONTRIBUTIONS R.B. and S.M. performed experiments. I.D.H., R.B., and S.M. designed experiments, interpreted results, and wrote/edited the manuscript. ACKNOWLEDGMENTS We thank Dr. Claudia Lukas, Dr. Jiri Lukas, and members of the I.D.H. laboratory for useful discussions and Hocine Mankouri and Ying Liu for critical reading of the manuscript. We also thank Thanos Halazonetis for sharing data prior to submission. Work in the authors’ laboratory is funded by the Danish National Research Foundation (DNRF115), The European Research Council (ERC Project Number 321717), and The Nordea Foundation. R.B. and S.M. were recipients of Danish Medical Research Council fellowships (DFF-4004-00155B, DFF-6110-00169B, and DFF-6110-00243B). Received: July 11, 2016 Revised: September 6, 2016 Accepted: October 28, 2016 Published: December 15, 2016 REFERENCES Anand, R.P., Lovett, S.T., and Haber, J.E. (2013). Break-induced DNA replication. Cold Spring Harb. Perspect. Biol. 5, a010397. Arlt, M.F., Durkin, S.G., Ragland, R.L., and Glover, T.W. (2006). Common fragile sites as targets for chromosome rearrangements. DNA Repair (Amst.) 5, 1126–1135. Bai, Y., and Symington, L.S. (1996). A Rad52 homolog is required for RAD51independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 10, 2025–2037. Burrell, R.A., McGranahan, N., Bartek, J., and Swanton, C. (2013). The causes and consequences of genetic heterogeneity in cancer evolution. Nature 501, 338–345. Burrow, A.A., Williams, L.E., Pierce, L.C., and Wang, Y.H. (2009). Over half of breakpoints in gene pairs involved in cancer-specific recurrent translocations are mapped to human chromosomal fragile sites. BMC Genomics 10, 59. Bursomanno, S., Beli, P., Khan, A.M., Minocherhomji, S., Wagner, S.A., Bekker-Jensen, S., Mailand, N., Choudhary, C., Hickson, I.D., and Liu, Y. (2015). Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells. DNA Repair (Amst.) 25, 84–96. Casper, A.M., Nghiem, P., Arlt, M.F., and Glover, T.W. (2002). ATR regulates fragile site stability. Cell 111, 779–789. Castor, D., Nair, N., De´clais, A.C., Lachaud, C., Toth, R., Macartney, T.J., Lilley, D.M., Arthur, J.S., and Rouse, J. (2013). Cooperative control of holliday junction resolution and DNA repair by the SLX1 and MUS81-EME1 nucleases. Mol. Cell 52, 221–233. Chan, K.L., Palmai-Pallag, T., Ying, S., and Hickson, I.D. (2009). Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat. Cell Biol. 11, 753–760.
Western Blotting Analysis Details are provided in Supplemental Experimental Procedures.
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