BACH1 (BRCA1-associated C-terminal helicase 1), the product of the BRIP1 {BRCA1 [breast cancer 1, early onset]-interacting protein C-terminal helicase 1; also known as FANCJ [FA-J (Fanconi anaemia group J) protein]} gene mutated in Fanconi anaemia patients from complementation group J, has been implicated in DNA repair and damage signalling. BACH1 exerts DNA helicase activities and physically interacts with BRCA1 and MLH1 (mutL homologue 1), which differentially control DNA DSB (double-strand break) repair processes. The present study shows that BACH1 plays a role in both HR (homologous recombination) and MMEJ (microhomology-mediated non-homologous end-joining) and reveals discrete mechanisms underlying modulation of these pathways. Our results indicate that BACH1 stimulates HR, which depends on the integrity of the helicase domain. Disruption of the BRCA1–BACH1 complex through mutation of BACH1 compromised errorfree NHEJ (non-homologous end-joining) and accelerated error-prone MMEJ. Conversely, molecular changes in BACH1 abrogating MLH1 binding interfered neither with HR nor with MMEJ. Importantly, MMEJ is a mutagenic DSB repair pathway, which is derepressed in hereditary breast and ovarian carcinomas. Since BRCA1 and BACH1 mutations targeting the BRCA1–BACH1 interaction have been associated with breast cancer susceptibility, the results of the present study thus provide evidence for a novel role of BACH1 in tumour suppression.

INTRODUCTION

Biallelic mutations in BRIP1 {BRCA [breast cancer, early onset] 1-interacting protein C-terminal helicase 1; also know as FANCJ [FA-J (Fanconi anaemia group J) protein]}, were identified in FA-J patients; monoallelic carriers show increased susceptibility to breast cancer [1]. BRIP1, thus, together with BRCA2 [also known as FANCD (Fanconi anaemia, complementation group D) 1], PALB2 [partner and localizer of BRCA2, also know as FANCN (Fanconi anaemia, complementation group N)] and RAD51C (RAD51 homologue C), belongs to a group of genes which link diseases characterized by progressive bone marrow failure and increased risk for early-onset mammary and ovarian carcinomas [24]. Moreover, BACH1 (BRCA1-associated C-terminal helicase 1) was originally identified as a protein that C-terminally (amino acids 888–1063) binds the BRCT (BRCA1 C-terminal) repeat motifs in BRCA1, i.e. the product of a high-penetrance breast cancer susceptibility gene [5]. The BRCA1–BACH1 interaction is tightly controlled, as BACH1 is phosphorylated on Ser990 in the S/G2/M-phases of the cell cycle, which was shown to be a prerequisite for complex formation with BRCA1 [6]. The same study further reported that ionizing radiation treatment-induced checkpoint control during the transition from G2- to M-phase of the cell cycle depends on BRCA1, BACH1 and BRCA1–BACH1 complex formation.

BACH1 contains conserved ATPase/helicase motifs and Fe–S cluster domains, which are also found in BACH1-like helicases [1]. Missense mutations conferring increased breast cancer susceptibility were detected within the helicase, Fe–S domain and BRCA1-interacting domains of BRIP1. BACH1 exerts DNA-dependent ATPase and 5′–>3′ helicase activities preferentially targeting forked duplexes with a 5′-ssDNA (single-stranded DNA) tail and also D-loop structures, suggestive of a role during replication and/or HR (homologous recombination) [1,7]. Strikingly, the high- and moderate-penetrance breast cancer susceptibility gene products known today are directly or indirectly involved in DSB (double-strand break) repair, particularly in the HR pathway [24]. During replication stress, BACH1 was found to be dispensable for FANCD2 mono-ubiquitination, positioning BACH1 like BRCA1, BRCA2, PALB2 and RAD51C downstream of FANCD2 in the FA pathway [8]. HR was compromised in human cells after knockdown of BACH1, which could explain moderate sensitivity to ionizing irradiation and high sensitivity to cross-linking agents. However, results from chicken DT40 cells challenged the proposed role of BACH1 in homologous DSB repair and in DNA cross-link repair [9]. Of note, BACH1 in the chicken and lower organisms lacks the motif required for the interaction with BRCA1 in mammalian cells, so that BRCA1–BACH1 complexes may exhibit novel functions in mammalian cells [1]. Nevertheless, even in mammalian cells it remains unclear whether BACH1 is required for RAD51 filament formation as is the case with other breast cancer susceptibility gene products exerting HR functions. Thus RAD51 foci formation is reduced following radiomimetic treatment [10], but enhanced after replication fork stalling [8] in BACH1-depleted cells. Moreover, BACH1 was shown to inhibit rather than stimulate RAD51-mediated strand exchange in vitro [11]. One possible explanation, which was inspired by BACH1's capacity to resolve various secondary structures such as G-quadruplex DNA during replication and which may reconcile these at first sight contradictory data, is a controlling role of BACH1 in HR repair, i.e. termination of HR on aberrant DNA substrates and/or intermediates [1,11]. Another mechanism potentially underlying HR surveillance by BACH1 may involve another complex partner, namely the mismatch repair protein complex MLH1 (mutL homologue 1)–PMS2 (postmeiotic segregation increased 2) [12,13], as this complex has been implicated in the fidelity control of HR [14].

Aside from its scaffolding role in HR, the BACH1-binding partner BRCA1 was proposed to directly repress error-prone NHEJ (non-homologous end-joining) processes [15,16]. To better understand the roles of human BACH1 and its biochemical functions in distinct DSB repair processes, we have comparatively investigated the effect of BACH1 silencing and expression, BRIP1 mutations disrupting the helicase activity, BRCA1 or MLH1 interactions and expression of competitor peptides on HR and NHEJ pathways. From these experiments, we identified that coupling of BRCA1 to BACH1 is crucial to prevent error-prone and thus mutagenic NHEJ pathways.

EXPERIMENTAL

Cell culture and establishment of stable cell clones

Cells from the human cervix carcinoma cell line HeLa were cultivated in DMEM (Dulbecco's modified Eagle's medium; PAA Laboratories) supplemented with 10% FBS (fetal bovine serum; PAA Laboratories) and 2 mM L-glutamine (Biochrom). FA-J cells (EUFA 0030 skin fibroblasts) derived from a FA-J patient (AG656) were cultivated in DMEM supplemented with 15% FBS and 2 mM L-glutamine. For the analysis of NHEJ in the chromosomal context we stably transfected FA-J cells with EJ-EGFP (enhanced green fluorescent protein). Subsequently, puromycin-resistant (0.4 μg/ml; PAA Laboratories) colonies were screened for I-SceI-inducible cellular fluorescence. K562 (carrying recombinant HR-EGFP/3′EGFP) cells [17] were raised in RPMI 1640 medium (PAA Laboratories) supplemented with 10% FBS (PAA Laboratories) and 1% L-glutamine. The cell cultures used in the present study were free from Mycoplasma contamination.

DSB repair analysis

DSB repair via different pathways was investigated by the use of the EGFP-based test system as described in detail previously [17,18]. In the present study, we employed the DSB repair substrates HR-EGFP/5′EGFP and EJ-EGFP (Figure 1a). Briefly, HeLa cells were transfected with FuGENE HD reagent (Roche) and a mixture of 4 μg of plasmid DNA containing 2.5 μg of BACH1 expression vector for wt (wild-type) BACH1 (pcDNA3-wtBACH1), BACH1(K52R) [pcDNA3-BACH1(K52R)], BACH1(K141/142A) [(pcDNA3-BACH1(K141/142)], BACH1(S990A) [pcDNA3-BACH1(S990A)] or empty vector (pcDNA3.0), 0.5 μg of meganuclease expression plasmid pCMV-I-SceI, 0.5 μg of DSB repair substrate and 0.5 μg of pBS (pBlueScriptII KS, as a filler plasmid; Stratagene) or wtEGFP expression vector (for determination of transfection efficiency) [17,18]. For the DSB repair assays with FA-J cells, a plasmid mixture of 6 μg of DNA (3 μg of BACH1 expression vector/pcDNA3.0, 1 μg of DSB repair substrate, 1 μg of pCMV-I-SceI and 1 μg of pBS/wtEGFP expression vector) was introduced via nucleofection according to the Amaxa® protocol (Lonza). To measure recombination on chromosomally integrated substrates, FA-J (EJ-EGFP) cells were transfected with 5 μg of BACH1 expression vector/pcDNA3.0 together with 2.5 μg of pCMV-I-SceI and 2.5 μg of pBS/wtEGFP expression vector. Following cultivation for 24 h (HeLa and FA-J cell lines), 48 h (HeLa cells with pSUPER knockdown plasmid) or 72 h (FA-J cells with stably integrated EJ-EGFP), 105–106 cells were examined each to distinguish between EGFP-positive and -negative cells by the diagonal-gating method in the Fl1/Fl2 dot plot (FACSCalibur® FACScan, Becton Dickinson) [19]. Each quantification of green fluorescent cells in repair assays (with pBS) was normalized, with wtEGFP expression vector determined transfection efficiency (average transfection efficiency was 50%) to calculate the DSB repair frequency. The statistical significance of differences was determined using two-tailed Wilcoxon matched pairs test (P<0.05), using GraphPad Prism version 5.01. Reproducibility on at least two independent experimental days was assured for all data sets.

DSB repair in HeLa cells expressing differently mutated BACH1 proteins

Figure 1
DSB repair in HeLa cells expressing differently mutated BACH1 proteins

(a) DNA substrates for DSB repair analysis of HR and MMEJ pathways. DSB repair is monitored by flow cytometry from the fraction of green fluorescent cells after I-SceI-meganuclease-mediated cleavage of the mutated EGFP variants HR-EGFP and EJ-EGFP respectively. HR between internally mutated HR-EGFP and 3′ truncated 5′EGFP in substrate HR-EGFP/5′EGFP allows reconstitution of wtEGFP and CMV-driven expression. In the EJ-EGFP substrate, the I-SceI site is flanked by 5 bp microhomologies, enabling quantification of MMEJ. Black boxes, transcriptional promoter sequences; dark grey boxes, antibiotic resistance marker genes; and light grey boxes, microhomology sequence. (b) DSB repair as a function of BACH1. HeLa cells were co-transfected with meganuclease expression vector pCMV-I-SceI, substrates HR-EGFP/5′EGFP or EJ-EGFP, and an expression vector for wtBACH1, BACH1 (K52R), BACH1 (K141/142A) or BACH1 (S990A) compared with empty vector (control). After cultivation for 24 h, DSB repair frequencies were determined as detailed in Experimental section. Mean frequencies for cells expressing wtBACH1 were set to 100% (absolute values: 5.3×10−4 for HR-EGFP/5′EGFP, 7.5×10−4 for EJ-EGFP). Values are means±S.E.M. of six to nine measurements (statistical significance was calculated using wtBACH1 expressing samples as reference; *P<0.05; **P<0.01). (c) Immunoprecipitation of exogenous BACH1. HeLa cells were processed for DSB repair measurements as in (b). After transfection (48 h), cell extracts containing 27 mg of total proteins (Input) were prepared and Myc-tagged exogenous BACH1 variants immunoprecipitated (IP) with anti-(c-Myc) antibody followed by Western blotting specific for BRCA1, Myc-tagged BACH1 and MLH1. Immunoprecipitation with mouse IgG antiserum served as control for non-specific interactions. Adjacent images are derived from the same gel and autoradiography.

Figure 1
DSB repair in HeLa cells expressing differently mutated BACH1 proteins

(a) DNA substrates for DSB repair analysis of HR and MMEJ pathways. DSB repair is monitored by flow cytometry from the fraction of green fluorescent cells after I-SceI-meganuclease-mediated cleavage of the mutated EGFP variants HR-EGFP and EJ-EGFP respectively. HR between internally mutated HR-EGFP and 3′ truncated 5′EGFP in substrate HR-EGFP/5′EGFP allows reconstitution of wtEGFP and CMV-driven expression. In the EJ-EGFP substrate, the I-SceI site is flanked by 5 bp microhomologies, enabling quantification of MMEJ. Black boxes, transcriptional promoter sequences; dark grey boxes, antibiotic resistance marker genes; and light grey boxes, microhomology sequence. (b) DSB repair as a function of BACH1. HeLa cells were co-transfected with meganuclease expression vector pCMV-I-SceI, substrates HR-EGFP/5′EGFP or EJ-EGFP, and an expression vector for wtBACH1, BACH1 (K52R), BACH1 (K141/142A) or BACH1 (S990A) compared with empty vector (control). After cultivation for 24 h, DSB repair frequencies were determined as detailed in Experimental section. Mean frequencies for cells expressing wtBACH1 were set to 100% (absolute values: 5.3×10−4 for HR-EGFP/5′EGFP, 7.5×10−4 for EJ-EGFP). Values are means±S.E.M. of six to nine measurements (statistical significance was calculated using wtBACH1 expressing samples as reference; *P<0.05; **P<0.01). (c) Immunoprecipitation of exogenous BACH1. HeLa cells were processed for DSB repair measurements as in (b). After transfection (48 h), cell extracts containing 27 mg of total proteins (Input) were prepared and Myc-tagged exogenous BACH1 variants immunoprecipitated (IP) with anti-(c-Myc) antibody followed by Western blotting specific for BRCA1, Myc-tagged BACH1 and MLH1. Immunoprecipitation with mouse IgG antiserum served as control for non-specific interactions. Adjacent images are derived from the same gel and autoradiography.

K562 (HR-EGFP/3′EGFP) cells, carrying chromosomally integrated DSB repair substrate HR-EGFP/3′EGFP (see Figure 5a), were subjected to genomic PCR analysis as described by Volcic et al. [18]. Briefly, K562 (HR-EGFP/3′EGFP) cells were electroporated with 10 μg of pCMV-I-SceI, 10 μg of pBS/wtEGFP plasmid and 50 μg of expression plasmid for the BACH1 variant of interest (transfection efficiency was 60–80% for each construct). After a cultivation period of 48 h, cells were FACS sorted for green EGFP positivity and white EGFP negativity. Genomic DNA was isolated from white cells and PCR was performed with primers (Thermo Scientific) derived from sequences upstream of (5′-CCCGCAACCTCCCCTTCTAC-3′) and within (5′-CGGCTGAAGCATTACCCTGTTAT-3′) HR-EGFP, the latter partially covering the 3′-half of the I-SceI recognition sequence. As an internal control, we amplified a genomic RARα (retinoic acid receptor α) fragment as described in Volcic et al. [18]. PCR-Sce bands specific for the intact I-SceI site were quantified by use of a ChemImager 5500 with software (Alpha Imunotech Corporation) and normalized with the band specific for the RARα fragment each.

Plasmid construction, BACH1 knockdown and BRCA1–BACH1 complex disruption

To down-regulate expression of wtBACH1 by RNA interference we cloned several independent shRNA (short hairpin RNA) vectors derived from pSUPER targeting various regions of the BRIP1 mRNA. The vector pSUPER-wtBACH1 resulted in reproducible BACH1 protein knockdown and was engineered by cloning oligonucleotides (Thermo Scientific): wtBACH1-1 (5′-GATCCCCCCAGATCCACAAGCCCAACTTCAAGAGAGTTGGGCTTGTGGATCTGGTTTTTGGAAA-3′), wtBACH1-2 (5′-AGCTTTTCCAAAAACCAGATCCACAAGCCCAACTCTCTTGAAGTTGGGCTTGTGGATCTGGGGG-3′) into the pSUPER vector following the procedure described by Brummelkamp et al. [20]. Knockdown of BACH1 during DSB repair measurements in HeLa cells was accomplished by adding 8 μg of shRNA expression vector pSUPER-wtBACH1 (in controls pSUPER was used) to the lipofection mixture.

To disrupt BRCA1–BACH1 interaction we used a vector expressing a 46-amino-acid BACH1 peptide that includes the region of BACH1 required for its interaction with BRCA1 [46BBD TRX (thioredoxin)]. As controls the 46S990ABBD TRX vector expressing the 46BBD peptide with a point mutation corresponding to S990A, the 56B2BD TRX vector expressing a peptide that disrupts the BRCA2–RAD51 interaction [21] (the same primer sets were used with the addition of BamHI and EcoRI as described below) or the empty vector TRX were used. For DSB repair measurements under conditions of BRCA1–BACH1 complex disruption, HeLa cells were co-transfected with one of each TRX vector (2.5 μg), 0.5 μg of pCMV-I-SceI, 0.5 μg of EJ-EGFP and 0.5 μg of pBS/wtEGFP expression plasmid using FuGENE HD (Roche). To generate a synthetic peptide antagonist expressed within the active site of TRX [22], the following primers were used: forward, 5′-TTGGATCCAGCATTATCTCCAGAAAGGAGA-3′, including a BamHI site, and reverse, 5′-AAGAATTCCAAAGAATTAAAGCTTGACCAG-3′ including an EcoRI site. PCR was performed using the wtBRIP1 or BRIP1(S990A) cDNA. The PCR product was digested with the noted restriction enzymes and cloned into the TRX vector. The final peptide antagonist was a 46-amino-acid peptide containing the BACH1 sequence required for BRCA1 binding (residues 963–1008: ISRKEKNDPVFLEEAGKAEKIVISRSTSPTFNKQTKRVSWSSFNSL).

Flow cytometric analysis of DNA content

For the generation of cell-cycle and apoptosis profiles, the cells were fixed in ice-cold 40% (v/v) ethanol/50% acetone solution for at least 1 h at −20°C, rehydrated and resuspended in a propidium iodide solution (50 μg/ml propidium iodide and 50 μg/ml RNase A, freshly prepared) and incubated for 30 min in the dark. Flow cytometric analysis of the cellular DNA content was performed on a FACSCalibur® using the CellQuest Pro software (Becton Dickinson).

Immunoblotting

For Western blot analysis cells were transfected and incubated for 24 h or 48 h as for DSB repair assays. Cellular lysates were prepared by incubation of the cells in freshly prepared lysis buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 25 mM NaF, 25 mM 2-glycerophosphate, 0.1 mM Na3VO4, 0.2% Triton X-100, 0.3% Nonidet P40 and proteinase inhibitors; Roche Diagnostics) for 30 min on ice. After clarification by centrifugation (20000 g for 15 min at 21°C), protein concentrations were determined with the BCA (bicinchoninic acid) protein assay kit (Pierce/Perbio Science) and 60 μg of total protein subjected to immunoblotting. Extracts were electrophoresed either by SDS/PAGE (4–8%) in Rotiphorese SDS/PAGE buffer (Roth) at room temperature (21°C) or in NuPAGE® Novex 4–12% gradient gels according to the manufacturer's instructions (Invitrogen) and transferred on to Hybond-C Extra (Amersham Biosciences) membranes on ice. Electrotransferred proteins were immunodetected by use of the following antibodies: anti-BACH1 rabbit B1310 (Sigma–Aldrich), anti-BACH1 rabbit Ab16608 (Biozol), anti-BRCA1 mouse monoclonal antibody Ab-1 (Calbiochem), anti-MLH1 mouse monoclonal antibody G168-15 (Becton Dickinson) and anti-tubulin mouse monoclonal antibody DM1A (Abcam). Primary antibodies were detected with HRP (horseradish peroxidase)-conjugated secondary antibodies (Pierce/Thermo Fisher Scientific). Western blot signals were visualized by SuperSignal West Pico Chemiluminescent Substrate or West Dura Extended Duration Substrate (Pierce). Densitometric quantification of band intensities was performed using a ChemImager 5500, analysing signals within the linear range and subtracting background signals. Protein expression levels were calculated by normalizing with tubulin signals individually.

Immunoprecipitation

For immunoprecipitation of protein complexes, cells were harvested and lysed in lysis buffer [20 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P40 and proteinase inhibitor; Roche) for 30 min on ice. Cell extracts were clarified by centrifugation (20000 g for 15 min at 4°C). Cell lysates were first incubated with Protein G beads (GE Healthcare) at 4°C for 1 h to eliminate unspecifically interacting molecules. After removal of the beads cell lysates were incubated with the respective antibody for 2–3 h or overnight {anti-(c-Myc) mouse monoclonal antibody 9E10, Abcam; anti-BACH1 E47 serum [13]; and mouse IgG antiserum, Santa Cruz Biotechnology}. Beads were added for 1 h, washed and subjected to immunoblotting.

RESULTS

BACH1 differentially regulates HR and MMEJ

BACH1 had been identified as a protein critically involved in HR [8]. Moreover, BACH1 interacts with MLH1, which modulates HR [14], and with BRCA1, which promotes HR and prevents DSB repair by error-prone repair pathways, MMEJ in particular [16,23,24]. These observations prompted us to examine possible changes in these DSB repair pathways upon alteration of the functional BACH1 status. For this purpose, we applied the fluorescence-based test system for distinct DSB repair pathways [17], which had successfully been used for the functional characterization of MLH1 and BRCA1 [14,24]. In this assay, targeted DSB formation by I-SceI-meganuclease triggers repair processes within differently mutated EGFP genes. According to the specific EGFP mutation and the assembly of differently mutated EGFP variants each substrate enables analysis of a particular DSB repair mechanism by quantification of EGFP gene reconstitution, i.e. the appearance of green fluorescent cells, as detected by FACS analysis.

First, we comparatively examined HR and MMEJ as a function of the BACH1 status. HeLa cells were co-transfected with the corresponding DNA substrates HR-EGFP/5′EGFP or EJ-EGFP (Figure 1a) and I-SceI expression plasmid for targeted cleavage of the respective substrate. Concomitant introduction of expression plasmid for wtBACH1 caused a 4-fold stimulation of HR (P=0.0313), but no significant change in MMEJ (Figure 1b). When we expressed BACH1 carrying the amino acid exchange K52R, which abolishes BACH1 helicase activities [13], 2-fold reduced HR frequency (P=0.0313) and a 25% decrease in MMEJ (P=0.0244) compared with wtBACH1 was found. Interestingly, disrupting the MLH1-interaction in BACH1(K141/142A) did not significantly alter either HR or MMEJ. Similarly, the BACH1 (S990A) protein that can not bind BRCA1 did not cause a statistically significant decrease of HR. However, MMEJ frequencies increased by 35% (P=0.0034). To ensure that BACH1 bound MLH1 and BRCA1 as predicted after targeted DNA substrate cleavage in the DSB assay, we performed immunoprecipitations directed against Myc-tagged exogenous BACH1 proteins followed by immunoblotting for each complex partner. As shown in Figure 1(c), wtBACH1 and BACH1(K52R) both co-precipitated MLH1 and BRCA1, whereas MLH1 binding was lost with BACH1(K141/142A) and BRCA1 binding was lost with BACH1(S990A). Since BACH1, MLH1 and BRCA1 have been implicated in checkpoint control in response to genotoxic stress [2527], we examined potential changes in the cell-cycle distribution or apoptosis. However, under the conditions of the DSB repair assay, the percentages of cells in G0/G1-S- and G2/M-phases were equivalent in cells with or without expression of any of the BACH1 variants and the fraction of sub-G1 cells did not differ significantly (Supplementary Figure S1a at http://www.BiochemJ.org/bj/441/bj4410919add.htm). Therefore we conclude that the structural changes enforced on BACH1 protein directly influenced DNA repair rather than via checkpoint regulatory functions.

Abrogation of BRCA1–BACH1 interaction by BACH1 mutation or expression of competitor peptide up-regulate MMEJ

We wished to re-evaluate our previous finding on the role of the BRCA1–BACH1 interaction in preventing MMEJ under conditions with reduced interference by endogenously expressed wtBACH1 protein. Therefore we designed a pSUPER-derived plasmid [20] for the production of shRNA preferentially targeting wtBACH1- rather than BACH1(S990A)-encoding mRNA. Following transfection of HeLa cells with the corresponding knockdown plasmid, depletion of endogenous wtBACH1 was highly effective within 48 h, causing a decrease to 33% of the initial level according to densitometric quantification of band intensities and correction for tubulin signal intensities (Figure 2b). Concomitant expression of exogeneous wtBACH1 or BACH1(S990A) allowed re-accumulation of BACH1 proteins up to 86% and 129% respectively. Comparison of the BACH1 signals with and without knockdown suggested that the ratio of exogenous BACH1(S990A) to endogenous wtBACH1 was markedly elevated upon knockdown. When we compared MMEJ in these cells, we did not measure an effect of wtBACH1 knockdown in control cells transfected with empty vector or in cells expressing exogenous wtBACH1 (Figure 2a). However, expression of BACH1(S990A) enhanced MMEJ 7-fold (P=0.0313).

MMEJ in HeLa cells upon knockdown of endogenous wtBACH1 and expression of exogenous BACH1 defective for BRCA1 binding

Figure 2
MMEJ in HeLa cells upon knockdown of endogenous wtBACH1 and expression of exogenous BACH1 defective for BRCA1 binding

(a) Determination of MMEJ frequencies. HeLa cells were co-transfected with pCMV-I-SceI and EJ-EGFP together with wtBACH1 knockdown vector (pSUPER-wtBACH1) or the empty vector (pSUPER) plus expression vector for BACH1(S990A) in comparison with wtBACH1 expression or empty vector (control). DSB repair frequencies were determined 48 h after transfection. MMEJ frequencies of cells without knockdown (pSUPER) were set to 100%. Results are means±S.E.M. of six measurements (statistical significance was calculated for the comparison of pSUPER with pSUPER-wtBACH1 for each; *P<0.05). (b) Western blot analysis. Lysates of HeLa cells, cotransfected for DSB repair measurements and cultivated for 48 h, were immunoblotted using an antibody directed against BACH1. Tubulin immunostaining served as loading control and relative protein expression levels were calculated after normalization of band intensities with the corresponding tubulin signal.

Figure 2
MMEJ in HeLa cells upon knockdown of endogenous wtBACH1 and expression of exogenous BACH1 defective for BRCA1 binding

(a) Determination of MMEJ frequencies. HeLa cells were co-transfected with pCMV-I-SceI and EJ-EGFP together with wtBACH1 knockdown vector (pSUPER-wtBACH1) or the empty vector (pSUPER) plus expression vector for BACH1(S990A) in comparison with wtBACH1 expression or empty vector (control). DSB repair frequencies were determined 48 h after transfection. MMEJ frequencies of cells without knockdown (pSUPER) were set to 100%. Results are means±S.E.M. of six measurements (statistical significance was calculated for the comparison of pSUPER with pSUPER-wtBACH1 for each; *P<0.05). (b) Western blot analysis. Lysates of HeLa cells, cotransfected for DSB repair measurements and cultivated for 48 h, were immunoblotted using an antibody directed against BACH1. Tubulin immunostaining served as loading control and relative protein expression levels were calculated after normalization of band intensities with the corresponding tubulin signal.

We next employed competitor peptides to inhibit the formation of protein complexes between endogenously expressed BRCA1 and BACH1. For this purpose, we expressed a 46-amino-acid peptide derived from the BRCA1-interaction site within BACH1 encompassing Ser990 (46B). As specificity controls, we applied an expression plasmid for the same peptide with the amino acid change S990A as used for the mutant BACH1 experiments (46S). In addition, we expressed a 56-amino-acid peptide derived from BRCA2 disrupting the BRCA2–RAD51 interaction (56B) [21]. BACH1 co-immunoprecipitation analysis confirmed that BRCA1-specific bands were detectable in precipitates from controls (empty vector, 46S and 56B), but hardly from cells expressing the 46B peptide (Supplementary Figure S2a at http://www.BiochemJ.org/bj/441/bj4410919add.htm). Interference with BRCA1 interactions by the 46B peptide caused a 4.4-fold (P=0.0078) stimulation of MMEJ, whereas control peptides had no effect (Figure 3). Expression of peptide 46B, but not the 46S control peptide, suppressed HR 3.7-fold (P=0.0039), as was expected from the fact that 46B competitor peptide interferes with BRCA1 interactions via the BRCT motifs in general. From these observations we concluded that abrogation of BRCA1 interactions via the BRCT motif and BRCA1–BACH1 interaction in particular severely deregulate NHEJ.

Ablation of BRCA1 interactions via the BRCT motif by competitor peptide expression

Figure 3
Ablation of BRCA1 interactions via the BRCT motif by competitor peptide expression

MMEJ analysis. HeLa cells were co-transfected with pCMV-I-SceI and EJ-EGFP together with one of the following TRX vectors: a vector designed for expression of a 46-amino-acid BACH1 peptide derived from the interaction site with BRCA1-BRCT motifs (46B); expression of the same BACH1 peptide with S990A exchange (46S); expression of a 56-amino-acid peptide derived from BRCA2 (56B); or the empty vector (control). DSB repair was measured 24 h after transfection. MMEJ frequencies of controls were set to 100%. Results are means±S.E.M. of eight measurements (**P<0.01).

Figure 3
Ablation of BRCA1 interactions via the BRCT motif by competitor peptide expression

MMEJ analysis. HeLa cells were co-transfected with pCMV-I-SceI and EJ-EGFP together with one of the following TRX vectors: a vector designed for expression of a 46-amino-acid BACH1 peptide derived from the interaction site with BRCA1-BRCT motifs (46B); expression of the same BACH1 peptide with S990A exchange (46S); expression of a 56-amino-acid peptide derived from BRCA2 (56B); or the empty vector (control). DSB repair was measured 24 h after transfection. MMEJ frequencies of controls were set to 100%. Results are means±S.E.M. of eight measurements (**P<0.01).

BACH1 modulates extra- and intra-chromosomal MMEJ

To study the influences of functionally distinct BACH1 variants in cells devoid of endogenous BACH1, we transiently expressed wtBACH1 and the mutants BACH1(K52R), BACH1(K141/142A) and BACH1(S990A) in FA-J cells, i.e. immortalized fibroblasts from a patient with FANCJ (Figure 4b). When we analysed MMEJ repair after co-transfection with MMEJ substrate, the results strikingly resembled those obtained in HeLa cells (Figures 1b and 2a). Thus expression of neither wtBACH1 nor BACH1(K141/142A) significantly altered MMEJ in FA-J cells (Figure 4a). The amino acid exchange K52R in BACH1 associated with helicase defectiveness caused a reduction in MMEJ of 35% (P=0.0117). Abrogation of BRCA1-binding in BACH1 (S990A) up-regulated MMEJ 3.1-fold (P=0.0391). Importantly, for the different BACH1 variants we found cell-cycle profiles identical with those of cells transfected with empty vector (Supplementary Figure S1b), thus demonstrating that the DSB repair changes observed were not indirectly caused by altered checkpoint control functions.

DSB repair by MMEJ in FA-J cells expressing differently mutated BACH1 proteins

Figure 4
DSB repair by MMEJ in FA-J cells expressing differently mutated BACH1 proteins

(a) Extrachromosomal repair. FA-J cells were transfected with pCMV-I-SceI, substrate EJ-EGFP and expression plasmids for different BACH1 variants (see Figure 1b), and DSB repair was measured after cultivation for 24 h. DSB repair frequencies for cells expressing wtBACH1 were defined as 100% (absolute value=1.5×10−3). Results are means±S.E.M. of nine to twelve measurements (statistical significance was calculated using wtBACH1 expression as reference; *P<0.05). (b) BACH1 expression analysis. Lysates of FA-J cells, processed as for DSB repair measurements, were immunoblotted with an antibody against BACH1. Tubulin immunostaining served as loading control. (c) Intrachromosomal repair. Cells from two independent FA-J (EJ-EGFP) clones, i.e. FA-J cells with chromosomally integrated EJ-EGFP substrate, were co-transfected with pCMV-I-SceI and expression plasmids for different BACH1 variants. After cultivation for 24 h DSB repair frequencies were determined and mean frequencies for cells expressing wtBACH1 defined as 100% (absolute value=1.2×10−4). Results are means±S.E.M. of 11–12 measurements (statistical significance was calculated using wtBACH1-expressing samples as reference; *P<0.05; **P<0.01).

Figure 4
DSB repair by MMEJ in FA-J cells expressing differently mutated BACH1 proteins

(a) Extrachromosomal repair. FA-J cells were transfected with pCMV-I-SceI, substrate EJ-EGFP and expression plasmids for different BACH1 variants (see Figure 1b), and DSB repair was measured after cultivation for 24 h. DSB repair frequencies for cells expressing wtBACH1 were defined as 100% (absolute value=1.5×10−3). Results are means±S.E.M. of nine to twelve measurements (statistical significance was calculated using wtBACH1 expression as reference; *P<0.05). (b) BACH1 expression analysis. Lysates of FA-J cells, processed as for DSB repair measurements, were immunoblotted with an antibody against BACH1. Tubulin immunostaining served as loading control. (c) Intrachromosomal repair. Cells from two independent FA-J (EJ-EGFP) clones, i.e. FA-J cells with chromosomally integrated EJ-EGFP substrate, were co-transfected with pCMV-I-SceI and expression plasmids for different BACH1 variants. After cultivation for 24 h DSB repair frequencies were determined and mean frequencies for cells expressing wtBACH1 defined as 100% (absolute value=1.2×10−4). Results are means±S.E.M. of 11–12 measurements (statistical significance was calculated using wtBACH1-expressing samples as reference; *P<0.05; **P<0.01).

To assess the significance of these findings for DNA repair in the chromatin context we stably transfected FA-J cells with the MMEJ substrate EJ-EGFP (Figure 1a). We successfully isolated two independent FA-J (EJ-EGFP) clones that showed marked I-SceI meganuclease-triggered DSB repair activities, thus enabling the comparison of chromosomal MMEJ activities as a function of distinct BRIP1 mutations. In comparison with the results obtained with extrachromosomal MMEJ substrates, expression of wtBACH1 in these BACH1-negative cells caused a 2.2-fold stimulation of MMEJ (P=0.0020) (Figure 4c). As was expected from the analyses with transiently transfected repair substrate, expression of BACH1(S990A) was followed by a further 1.7-fold (P=0.0122) increase in MMEJ, whereas BACH1(K52R) expression did not induce a statistically significant change.

To further characterize chromosomal NHEJ, we made use of K562 (HR-EGFP/3′EGFP) cells with chromosomally integrated substrate HR-EGFP/3′EGFP (Figure 5a). First, we expressed I-SceI and allowed repair of I-SceI-mediated cleavage for 48 h. Secondly, we separated green fluorescent and white non-fluorescent cells flow cytometrically [17,18]. Thirdly, we performed genomic PCR on the white cells. Green cells had undergone successful homologous DSB repair at frequencies corresponding to the ones displayed in Supplementary Figure S3 (at http://www.BiochemJ.org/bj/441/bj4410919add.htm), indicating successful cleavage of the genomic DNA. Analysis of genomic DNA from the white cells enabled analysis of NHEJ processes exclusively. Reduced amplification of a PCR fragment specific for the intact I-SceI recognition sequence in pCMV-I-SceI-transfected cells compared with untransfected cells suggested a lack of repair or repair by error-prone NHEJ mechanisms (Figure 5b). Interestingly, upon additional expression of wtBACH1 the I-SceI-specific PCR signal was intensified 4.5-fold indicating repair by error-free NHEJ. However, BACH1(S990A) expression resulted in only a 1.5-fold increase, BACH1(K52R) expression in intermediate PCR amplification (2.6-fold increase). These observations substantiated that disruption of BRCA1–BACH1 complex formation promotes both extra- and intra-chromosomal MMEJ and compromises error-free NHEJ.

Genomic PCR analysis of NHEJ

Figure 5
Genomic PCR analysis of NHEJ

(a) Schematic representation of the primer positions for PCR-Sce within the DSB repair substrate HR-EGFP/3′EGFP, which is chromosomally integrated in K562 (HR-EGFP/3′EGFP) cells. Note that the 3′-positioned primer is specific for the intact I-SceI recognition sequence within the mutated EGFP variant HR-EGFP. The arrow indicates the PCR primer. (b) Genomic PCR. K562 (HR-EGFP/3′EGFP) cells were transfected with 10 μg of pCMV-I-SceI, 10 μg of pBS/wtEGFP plasmid and 50 μg of pcDNA3-wtBACH1, pcDNA3-BACH1(S990A), pcDNA3-BACH1(K52R) or pcDNA3.0 (control), followed by cultivation for 48 h, when EGFP-negative white cells were isolated by FACS sorting. Genomic PCR-Sce was performed to assess the fraction of error-free NHEJ after I-SceI cleavage. As an internal control, we amplified a genomic RARα fragment. Uncleaved genomic DNA from untransfected cells served as positive control. A representative image from two separate experiments with equivalent results is shown. Mean transfection efficiencies from duplicate measurements with these DNA mixtures were 74% for pcDNA3.0, 71% for pcDNA3-wtBACH1, 73% for pcDNA3-BACH1(S990A) and 65% for pcDNA3-BACH1(K52R).

Figure 5
Genomic PCR analysis of NHEJ

(a) Schematic representation of the primer positions for PCR-Sce within the DSB repair substrate HR-EGFP/3′EGFP, which is chromosomally integrated in K562 (HR-EGFP/3′EGFP) cells. Note that the 3′-positioned primer is specific for the intact I-SceI recognition sequence within the mutated EGFP variant HR-EGFP. The arrow indicates the PCR primer. (b) Genomic PCR. K562 (HR-EGFP/3′EGFP) cells were transfected with 10 μg of pCMV-I-SceI, 10 μg of pBS/wtEGFP plasmid and 50 μg of pcDNA3-wtBACH1, pcDNA3-BACH1(S990A), pcDNA3-BACH1(K52R) or pcDNA3.0 (control), followed by cultivation for 48 h, when EGFP-negative white cells were isolated by FACS sorting. Genomic PCR-Sce was performed to assess the fraction of error-free NHEJ after I-SceI cleavage. As an internal control, we amplified a genomic RARα fragment. Uncleaved genomic DNA from untransfected cells served as positive control. A representative image from two separate experiments with equivalent results is shown. Mean transfection efficiencies from duplicate measurements with these DNA mixtures were 74% for pcDNA3.0, 71% for pcDNA3-wtBACH1, 73% for pcDNA3-BACH1(S990A) and 65% for pcDNA3-BACH1(K52R).

DISCUSSION

Stimulation of HR by BACH1 depends on the integrity of the helicase domain

The results of the present study show that BACH1 plays a critical role in both of the DSB repair pathways, HR and MMEJ. Analysis of cells expressing BACH1 mutated in distinct functional domains revealed discrete mechanisms underlying modulation of either HR or MMEJ. Thus the mutation K52R, which disrupts the integrity of the helicase domain, caused HR reduction, which is in agreement with the results obtained by Xie et al. [10]. The BACH1 helicase has been proposed to resolve structural obstacles, particularly when associated with stalled replication forks and in a synergistic fashion with the helicase BLM [1,31,32]. In this way, the BACH1 helicase may promote HR, and enable recombinative replication fork bypass and thus timely progression through S-phase [33]. Concomitantly, BACH1 may prevent deletions at replication forks arrested at aberrant DNA structures and thus inhibit genomic instability.

Abrogation of neither MLH1 or of BRCA1 binding caused the same detrimental effect on BACH1-mediated HR as was observed after helicase inactivation. Taken together with our previous MLH1-knockdown experiments in FA-J cells [14], these observations show that BACH1 is dispensable for MLH1-mediated HR regulation and vice versa. The results obtained with BACH1(S990A) are consistent with studies in DT40 and human cells indicating that the ATPase, but not the BRCA1-interaction domain, is required for DNA cross-link repair, which involves HR after adduct removal [13,31]. However, it has also been reported that disruption of BRCA1–BACH1 interaction channels cross-link repair from HR to bypass by translesion synthesis in a manner dependent on MLH1, and that BACH1 mutations inactivating either the helicase or the BRCA1 interaction are equally detrimental to HR [10]. In the present study, we did not observe a statistically significant difference between HR frequencies after wtBACH1 compared with BACH1(S990A) expression in HeLa, FA-J (results not shown) or K562 (HR-EGFP/3′EGFP) cells, even though HR values for BACH1(S990A) were intermediate when compared with the values for wtBACH1 and BACH1(K52R).

BRCA1–BACH1 complex formation is required to prevent MMEJ processes

Interestingly, upon expression of BACH1(S990A) in HeLa cells we observed MMEJ enhancement to the extent seen after knockdown of BRCA1 [34]. Previous reports have shown that the fidelity of NHEJ is reduced in BRCA1-deficient mouse embryo fibroblasts and in BRCA1+/− patient cell lines [16,24,35]. The results of the present study show that binding of BACH1 to BRCA1 could be important to ensure MMEJ repression by BRCA1. Aside from BACH1 CtIP [CTBP (C-terminal binding protein)-interacting protein], Abraxas and Rap80 (receptor associated protein 80) also form a complex with the BRCA1–BRCT domain [36,37]. Recruitment of CtIP by BRCA1 is necessary for homologous DSB repair [38]. Consequently, a simple shift from BRCA1–BACH1 to BRCA1–CtIP complexes upon BACH1(S990A) expression would be expected to increase HR rather than decrease MMEJ, the latter of which was shown in the present study. Moreover, our experiments employing the 46B peptide, which blocks BRCA1–BRCT domain interactions, make compensatory binding by other BRCA1–BRCT interaction partners an unlikely explanation for this finding, because MMEJ was stimulated also upon complete blockage of these interactions. The significant HR decrease with competitor peptide, but not with mutant BACH1(S990A), could mean that BRCA1–CtIP (or other) interactions via the BRCT motifs are more important for HR activation than the BRCA1–BACH1 interaction.

Upon disruption of the integrity of the BACH1 helicase domain by the K52R mutation, we observed a modest decrease in extrachromosomal, but not intrachromosomal, MMEJ. In analogy to the yeast SRS2 (suppressor of rad six) DNA helicase, BACH1 may counteract improper RAD51 filament formation on extrachromosomal DSB repair substrates [11,39,40]. In contrast with the BACH1(K52R) mutant, the BRCA1-interaction mutant consistently displayed the same MMEJ-specific effect in the extra- and intra-chromosomal context. We therefore consider this interaction critical for MMEJ regulation. MMEJ enhancement after loss of BRCA1–BACH1 complex formation cannot simply be explained by a shift of DSB repair from HR to MMEJ, because MMEJ was still elevated in BRCA2-silenced cells (results not shown). From this, disruption of BRCA1–BACH1 complexes appears to more directly derepress MMEJ.

We further noticed that wtBACH1 expression had a stimulatory effect on both error-prone MMEJ (Figure 4c) and error-free NHEJ (Figure 5b) in the chromatin context. Disruption of BRCA1–BACH1 complex formation further increased MMEJ and diminished error-free NHEJ. From this, we propose that BACH1 modulates MMEJ in chromosomes by at least two mechanisms. First, BACH1 promotes MMEJ as part of a general NHEJ stimulatory effect, possibly by the recruitment/stabilization of repair components/complexes to/at sites of DNA damage. Secondly, BACH1 activates BRCA1-dependent MMEJ repression by BRCA1 complex formation. These two opposite effects on MMEJ could neutralize each other in the scenario with extrachromosomal DSB repair substrate (no effect of wtBACH1 knockdown/expression). In the complex chromatin, BACH1 functions in recruitment/stabilization might be more important, thus resulting in a net MMEJ enhancement in wtBACH1 expression in FA-J (EJ-EGFP) cells. BACH1 was described to physically interact with, and promote loading of, the human ssDNA-binding protein RPA (replication protein A) following DNA damage [25,41]. RPA represents the initial signal/sensor for DNA damage that facilitates recruitment of MRE11 (meiotic recombination 11) complexes and ATM (ataxia telangiectasia mutated)/ATR (ATM- and RAD3-related) to DSBs [42], so that BACH1–RPA interactions offer one possible explanation for a general NHEJ stimulatory effect of BACH1.

Conclusion

In summary, we provide evidence for two independent DSB repair regulatory functions of BACH1. It enhances, first, highfidelity HR requiring the helicase activity and, secondly, high-fidelity NHEJ, which depends on binding to BRCA1. This dual controlling role of BACH1 is reminiscent of the tumoursuppressor functions of BRCA1. BRCA1 mutations in patient cells cause a shift from error-free HR to mutagenic pathways such as MMEJ [24,34,43] causing genomic instability, a mutator phenotype and accelerated carcinogenesis. BRCA1 germline mutations within the BRCT domain have been documented in individuals with a history of breast and ovarian cancer [44]. BACH1 recognizes the BRCT domain of BRCA1 [36] and mutation or deletion disrupts the BRCA1–BACH1 interaction [5,6]. The results of the present study therefore provide a new mechanism underlying susceptibility to mammary carcinogenesis in individuals with mutations in BRIP1 or the BRCT-encoding region of the BRCA1 gene. In the light of ongoing clinical cancer trials involving PARP [poly(ADP-ribose) polymerase] inhibitor treatment, it is of interest that deregulated MMEJ may provide a new marker for BACH1 dysfunction, which could be useful for the prediction of therapeutic responsiveness.

Abbreviations

     
  • ATM

    ataxia telangiectasia mutated

  •  
  • BRCA1-associated

    C-terminal helicase 1

  •  
  • BRCA

    breast cancer, early onset

  •  
  • BACH1

    BRCA1-associated C-terminal helicase 1

  •  
  • BRCT

    BRCA1 C-terminal

  •  
  • BRIP1

    BRCA1-interacting protein C-terminal helicase 1

  •  
  • CtIP

    CTBP-interacting protein

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • DSB

    double-strand break

  •  
  • EGFP

    enhanced green fluorescent protein

  •  
  • FA

    Fanconi anaemia

  •  
  • FA-J

    FA group J

  •  
  • FA-J cell

    EUFA 0030 skin fibroblast

  •  
  • FANCJ

    FA-J protein

  •  
  • FANCD

    FA complementation group D

  •  
  • FBS

    fetal bovine serum

  •  
  • HR

    homologous recombination

  •  
  • MLH1

    mutL homologue 1

  •  
  • MMEJ

    microhomology-mediated non-homologous end-joining

  •  
  • NHEJ

    non-homologous end-joining

  •  
  • PALB2

    partner and localizer of BRCA2

  •  
  • pBS

    pBlueScriptII KS

  •  
  • RAD51C

    RAD51 homologue C

  •  
  • RPA

    replication protein A

  •  
  • shRNA

    short hairpin RNA

  •  
  • ssDNA

    single-stranded DNA

  •  
  • TRX

    thioredoxin

  •  
  • wt

    wild-type

AUTHOR CONTRIBUTION

Lisa Wiesmüller designed and co-ordinated the study and wrote the paper. Lisa Dohrn and Daniela Salles performed most, and Simone Siehler and Julie Kaufmann performed part of the experiments. Lisa Dohrn, Daniela Salles and Simone Siehler evaluated the data.

We thank Professor Hans Joenje, VU Medical Center, Amsterdam, The Netherlands, for the gift of FA-J cells and Dr Sharon Cantor, University of Massachusetts, Worcester, MA, U.S.A., for the BACH1 and peptide expression plasmids.

FUNDING

This work was supported by the Deutsche Forschungsgesellschaft [grant number Wi 3099/7-2] and by the Federal Ministry of Education and Research (BMBF) grant ‘BRENDA’ [grant number 012P0505].

References

References
1
Wu
Y.
Suhasini
A. N.
Brosh
R. M.
Jr
Welcome the family of FANCJ-like helicases to the block of genome stability maintenance proteins
Cell. Mol. Life Sci.
2009
, vol. 
66
 (pg. 
1209
-
1222
)
2
Walsh
T.
King
M. C.
Ten genes for inherited breast cancer
Cancer Cell
2007
, vol. 
11
 (pg. 
103
-
105
)
3
Vaz
F.
Hanenberg
H.
Schuster
B.
Barker
K.
Wiek
C.
Erven
V.
Neveling
K.
Endt
D.
Kesterson
I.
Autore
F.
, et al. 
Mutation of the RAD51C gene in a Fanconi anemia-like disorder
Nat. Genet.
2010
, vol. 
42
 (pg. 
406
-
409
)
4
Meindl
A.
Hellebrand
H.
Wiek
C.
Erven
V.
Wappenschmidt
B.
Niederacher
D.
Freund
M.
Lichtner
P.
Hartmann
L.
Schaal
H.
, et al. 
Germline mutations in breast and ovarian cancer pedigrees establish RAD51C as a human cancer susceptibility gene
Nat. Genet.
2010
, vol. 
42
 (pg. 
410
-
414
)
5
Cantor
S. B.
Bell
D. W.
Ganesan
S.
Kass
E. M.
Drapkin
R.
Grossman
S.
Wahrer
D. C.
Sgroi
D. C.
Lane
W. S.
Haber
D. A.
Livingston
D. M.
BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function
Cell
2001
, vol. 
105
 (pg. 
149
-
160
)
6
Yu
X.
Chini
C. C.
He
M.
Mer
G.
Chen
J.
The BRCT domain is a phospho-protein binding domain
Science
2003
, vol. 
302
 (pg. 
639
-
642
)
7
Cantor
S.
Drapkin
R.
Zhang
F.
Lin
Y.
Han
J.
Pamidi
S.
Livingston
D.
The BRCA1-associated protein BACH1 is a DNA helicase targeted by clinically relevant inactivating mutations
Proc. Natl. Acad. Sci. U.S.A.
2004
, vol. 
101
 (pg. 
2357
-
2362
)
8
Litman
R.
Peng
M.
Jin
Z.
Zhang
F.
Zhang
J.
Powell
S.
Andreassen
P. R.
Cantor
S.B.
BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ
Cancer Cell
2005
, vol. 
8
 (pg. 
255
-
265
)
9
Bridge
W. L.
Vandenberg
C. J.
Franklin
R. J.
Hiom
K.
The BRIP1 helicase functions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslink repair
Nat. Genet.
2005
, vol. 
37
 (pg. 
953
-
957
)
10
Xie
J.
Litman
R.
Wang
S.
Peng
M.
Guillemette
S.
Rooney
T.
Cantor
S. B.
Targeting the FANCJ–BRCA1 interaction promotes a switch from recombination to poleta-dependent bypass
Oncogene
2010
, vol. 
29
 (pg. 
2499
-
2508
)
11
Sommers
J. A.
Rawtani
N.
Gupta
R.
Bugreev
D. V.
Mazin
A. V.
Cantor
S. B.
Brosh
R. M.
Jr
FANCJ uses its motor ATPase to destabilize protein–DNA complexes, unwind triplexes, and inhibit RAD51 strand exchange
J. Biol. Chem.
2009
, vol. 
284
 (pg. 
7505
-
7517
)
12
Cannavo
E.
Gerrits
B.
Marra
G.
Schlapbach
R.
Jiricny
J.
Characterization of the interactome of the human MutL homologues MLH1, PMS1, and PMS2
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
2976
-
2986
)
13
Peng
M.
Litman
R.
Xie
J.
Sharma
S.
Brosh
R. M.
Jr
Cantor
S. B.
The FANCJ/MutLa interaction is required for correction of the cross-link response in FA-J cells
EMBO J.
2007
, vol. 
26
 (pg. 
3238
-
3249
)
14
Siehler
S. Y.
Schrauder
M.
Gerischer
U.
Cantor
S.
Marra
G.
Wiesmüller
L.
Human MutL-complexes monitor homologous recombination independently of mismatch repair
DNA Repair
2009
, vol. 
8
 (pg. 
242
-
252
)
15
Paull
T. T.
Cortez
D.
Bowers
B.
Elledge
S. J.
Gellert
M.
Direct DNA binding by Brca1
Proc. Natl. Acad. Sci. U.S.A.
2001
, vol. 
98
 (pg. 
6086
-
6091
)
16
Baldeyron
C.
Jacquemin
E.
Smith
J.
Jacquemont
C.
De Oliveira
I.
Gad
S.
Feunteun
J.
Stoppa-Lyonnet
D.
Papadopoulo
D.
A single mutated BRCA1 allele leads to impaired fidelity of double strand break end-joining
Oncogene
2002
, vol. 
21
 (pg. 
1401
-
1410
)
17
Akyüz
N.
Boehden
G. S.
Süsse
S.
Rimek
A.
Preuss
U.
Scheidtmann
K. H.
Wiesmüller
L.
DNA substrate dependence of p53-mediated regulation of double-strand break repair
Mol. Cell. Biol.
2002
, vol. 
22
 (pg. 
6306
-
6317
)
18
Volcic
M.
Karl
S.
Baumann
B.
Salles
D.
Daniel
P.
Fulda
S.
Wiesmüller
L.
NF-κB regulates DNA double-strand break repair in conjunction with BRCA1–CtIP complexes
Nucleic Acids Res.
2011
 
doi:10.1093/nar/gkr687
19
Baumann
C.
Boehden
G. S.
Bürkle
A.
Wiesmüller
L.
Poly(ADP-RIBOSE) polymerase-1 (Parp-1) antagonizes topoisomerase I-dependent recombination stimulation by p53
Nucleic Acids Res.
2006
, vol. 
34
 (pg. 
1036
-
1049
)
20
Brummelkamp
T. R.
Bernards
R.
Agami
R.
A system for stable expression of short interfering RNAs in mammalian cells
Science
2002
, vol. 
296
 (pg. 
550
-
553
)
21
Esashi
F.
Christ
N.
Gannon
J.
Liu
Y.
Hunt
T.
Jasin
M.
West
S. C.
CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair
Nature
2005
, vol. 
434
 (pg. 
598
-
604
)
22
Colas
P.
Cohen
B.
Jessen
T.
Grishina
I.
McCoy
J.
Brent
R.
Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2
Nature
1996
, vol. 
380
 (pg. 
548
-
550
)
23
Moynahan
M. E.
Chiu
J. W.
Koller
B. H.
Jasin
M.
Brca1 controls homology-directed DNA repair
Mol. Cell
1999
, vol. 
4
 (pg. 
511
-
518
)
24
Keimling
M.
Kaur
J.
Bagadi
S. A.R.
Kreienberg
R.
Wiesmüller
L.
Ralhan
R.
A sensitive test for the detection of specific DSB repair defects in primary cells from breast cancer specimens
Int. J. Cancer
2008
, vol. 
123
 (pg. 
730
-
736
)
25
Gong
Z.
Kim
J. E.
Leung
C. C.
Glover
J. N.
Chen
J.
BACH1/FANCJ acts with TopBP1 and participates early in DNA replication checkpoint control
Mol. Cell
2010
, vol. 
37
 (pg. 
438
-
446
)
26
O'Brien
V.
Brown
R.
Signalling cell cycle arrest and cell death through the MMR system
Carcinogenesis
2006
, vol. 
27
 (pg. 
682
-
692
)
27
Reference deleted
28
Reference deleted
29
Reference deleted
30
Zhuang
J.
Jiang
G.
Willers
H.
Xia
F.
Exonuclease function of human Mre11 promotes deletional nonhomologous end joining
J. Biol. Chem.
2009
, vol. 
284
 (pg. 
30565
-
30573
)
31
Wang
W.
Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins
Nat. Rev. Genet.
2007
, vol. 
8
 (pg. 
735
-
748
)
32
Suhasini
A. N.
Rawtani
N. A.
Wu
Y.
Sommers
J. A.
Sharma
S.
Mosedale
G.
North
P. S.
Cantor
S. B.
Hickson
I. D.
Brosh
R. M.
Jr
Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome
EMBO J.
2011
, vol. 
30
 (pg. 
692
-
705
)
33
Kumaraswamy
E.
Shiekhattar
R.
Activation of BRCA1/BRCA2-associated helicase BACH1 is required for timely progression through S phase
Mol. Cell. Biol.
2007
, vol. 
27
 (pg. 
6733
-
6741
)
34
Keimling
M.
Volcic
M.
Csernok
A.
Wieland
B.
Dörk
T.
Wiesmüller
L.
Functional characterization connects individual patients' mutations in ataxia telangiectasia mutated (ATM) with dysfunction of specific DNA double-strand break repair signalling pathways
FASEB J.
2011
, vol. 
25
 (pg. 
3849
-
3860
)
35
Bau
D. T.
Fu
Y. P.
Chen
S. T.
Cheng
T. C.
Yu
J. C.
Wu
P. E.
Shen
C. Y.
Breast cancer risk and the DNA double-strand break end-joining capacity of nonhomologous end-joining genes are affected by BRCA1
Cancer Res.
2004
, vol. 
64
 (pg. 
5013
-
5019
)
36
Yu
X.
Wu
L. C.
Bowcock
A. M.
Aronheim
A.
Baer
R.
The C-terminal (BRCT) domains of BRCA1 interact in vivo with CtIP, a protein implicated in the CtBP pathway of transcriptional repression
J. Biol. Chem.
1998
, vol. 
273
 (pg. 
25388
-
25392
)
37
Wang
B.
Matsuoka
S.
Ballif
B. A.
Zhang
D.
Smogorzewska
A.
Gygi
S. P.
Elledge
S. E.
Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response
Science
2007
, vol. 
316
 (pg. 
1194
-
1198
)
38
Yun
M. H.
Hiom
K.
CtIP–BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle
Nature
2009
, vol. 
459
 (pg. 
460
-
464
)
39
Hegde
V.
Klein
H.
Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast
Nucleic Acids Res.
2000
, vol. 
28
 (pg. 
2779
-
2783
)
40
Carter
S. D.
Vigasová
D.
Chen
J.
Chovanec
M.
Aström
S. U.
Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism
Proc. Natl. Acad. Sci. U.S.A.
2009
, vol. 
106
 (pg. 
12037
-
12042
)
41
Gupta
R.
Sharma
S.
Sommers
J. A.
Kenny
M. K.
Cantor
S. B.
Brosh
J, R. M.
FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein
Blood
2007
, vol. 
110
 (pg. 
2390
-
2398
)
42
Robison
J. G.
Bissler
J. J.
Dixon
K.
Replication protein A is required for etoposide-induced assembly of MRE11/RAD50/NBS1 complex repair foci
Cell Cycle
2007
, vol. 
6
 (pg. 
2408
-
2416
)
43
Gudmundsdottir
K.
Ashworth
A.
The roles of BRCA1 and BRCA2 and associated proteins in the maintenance of genomic stability
Oncogene
2006
, vol. 
25
 (pg. 
5864
-
5874
)
44
Lee
M. S.
Green
R.
Marsillac
S. M.
Coquelle
N.
Williams
R. S.
Yeung
T.
Foo
D.
Hau
D. D.
Hui
B.
Monteiro
A. N.
Glover
J. N.
Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays
Cancer Res.
2010
, vol. 
70
 (pg. 
4880
-
4890
)

Author notes

1

These authors contributed equally to this work.

Supplementary data