Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences
2001; Springer Nature; Volume: 20; Issue: 17 Linguagem: Inglês
10.1093/emboj/20.17.4704
ISSN1460-2075
AutoresAndrew Tutt, David Bertwistle, J. S. Valentine, Anastasia Gabriel, Sally Swift, Gillian Ross, C.S. Griffin, John G. Thacker, Alan Ashworth,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle3 September 2001free access Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences Andrew Tutt Andrew Tutt The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author David Bertwistle David Bertwistle The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Janet Valentine Janet Valentine The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Anastasia Gabriel Anastasia Gabriel The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Sally Swift Sally Swift The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Gillian Ross Gillian Ross The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Carol Griffin Carol Griffin Medical Research Council Radiation and Genome Stability Unit, Harwell, OX11 0RD UK Search for more papers by this author John Thacker John Thacker Medical Research Council Radiation and Genome Stability Unit, Harwell, OX11 0RD UK Search for more papers by this author Alan Ashworth Corresponding Author Alan Ashworth The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Andrew Tutt Andrew Tutt The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author David Bertwistle David Bertwistle The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Janet Valentine Janet Valentine The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Anastasia Gabriel Anastasia Gabriel The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Sally Swift Sally Swift The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Gillian Ross Gillian Ross The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Carol Griffin Carol Griffin Medical Research Council Radiation and Genome Stability Unit, Harwell, OX11 0RD UK Search for more papers by this author John Thacker John Thacker Medical Research Council Radiation and Genome Stability Unit, Harwell, OX11 0RD UK Search for more papers by this author Alan Ashworth Corresponding Author Alan Ashworth The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Author Information Andrew Tutt1, David Bertwistle1, Janet Valentine1, Anastasia Gabriel1, Sally Swift1, Gillian Ross1, Carol Griffin2, John Thacker2 and Alan Ashworth 1 1The Breakthrough Toby Robins, Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK 2Medical Research Council Radiation and Genome Stability Unit, Harwell, OX11 0RD UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:4704-4716https://doi.org/10.1093/emboj/20.17.4704 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mutation of BRCA2 causes familial early onset breast and ovarian cancer. BRCA2 has been suggested to be important for the maintenance of genome integrity and to have a role in DNA repair by homology- directed double-strand break (DSB) repair. By studying the repair of a specific induced chromosomal DSB we show that loss of Brca2 leads to a substantial increase in error-prone repair by homology-directed single-strand annealing and a reduction in DSB repair by conservative gene conversion. These data demonstrate that loss of Brca2 causes misrepair of chromosomal DSBs occurring between repeated sequences by stimulating use of an error-prone homologous recombination pathway. Furthermore, loss of Brca2 causes a large increase in genome-wide error-prone repair of both spontaneous DNA damage and mitomycin C-induced DNA cross-links at the expense of error-free repair by sister chromatid recombination. This provides insight into the mechanisms that induce genome instability in tumour cells lacking BRCA2. Introduction Women who inherit loss-of-function mutations in either of the breast cancer susceptibility genes, BRCA1 and BRCA2, have a high risk of developing breast cancer (Rahman and Stratton, 1998). Since the wild-type allele is lost from tumours arising in heterozygous carriers, both BRCA1 and BRCA2 are thought to act as tumour suppressor genes. BRCA1 and BRCA2 encode unrelated nuclear proteins, which can both interact with Rad51 (Mizuta et al., 1997; Scully et al., 1997; Sharan et al., 1997; Chen et al., 1998), the eukaryotic equivalent of bacterial RecA. Rad51 catalyses strand exchange during homology-directed repair of DNA double-strand breaks (DSBs) by gene conversion. A direct interaction between BRCA2 and Rad51 has been demonstrated, and is mediated by a series of internal BRC repeats encoded by BRCA2 exon 11 (Chen et al., 1998), and an additional non-BRC domain located at the C-terminus of the protein (Mizuta et al., 1997; Sharan et al., 1997). These physical interactions, and the observation that BRCA1 and BRCA2 co-localize with Rad51 in ionizing radiation (IR)-induced nuclear foci (Chen et al., 1998), suggested a role for BRCA1 and BRCA2 in DNA repair by homologous recombination (HR). Subsequent studies, which have demonstrated that mouse and human cells deficient for wild-type BRCA1 or BRCA2 suffer from chromosome instability (Tirkkonen et al., 1997; Gretarsdottir et al., 1998; Patel et al., 1998; Tutt et al., 1999; Xu et al., 1999; Ban et al., 2001) and have a heightened sensitivity to DNA lesions that are repaired by HR (Patel et al., 1998; Shen et al., 1998; Scully et al., 1999; Yu et al., 2000; Wang et al., 2001a), have supported this contention. Mammalian cells can repair DNA DSBs by both HR and by non-homologous end-joining (NHEJ) (Karran, 2000; Khanna and Jackson, 2001). NHEJ of DSBs is non-conservative and is often associated with deletions, insertions and translocations. HR accounts for 30–50% of endonuclease-induced DSB repair events in dividing mammalian cells and can occur by two main pathways: gene conversion and single-strand annealing (SSA) (Liang et al., 1998). During gene conversion, the DSB is processed to produce 3′ single-stranded tails, which recruit Rad51 and thereby seek out a homologous template on the sister chromatid or homologous chromosome from which to accurately resynthesize the sequence surrounding the DSB (Baumann and West, 1998). Use of the identical sister chromatid in gene conversion, as opposed to homologous chromosomes, maintains genome integrity and is the preferred repair template (Johnson and Jasin, 2000). Gene conversion can occur in the absence (here referred to as GC) or presence (CO) of a crossover or exchange event. Sister chromatid crossover (CO) events can be equal (error-free events termed sister chromatid exchanges, SCE) or unequal depending on the template used for repair. Wild-type cells suppress unequal sister chromatid CO or CO events between chromosomes (Richardson et al., 1998; Johnson and Jasin, 2000) because these can cause duplications, deletions or translocations (Lupski, 1998; Jasin, 2000). An alternative, Rad51- independent, HR repair pathway is SSA. This competes with the GC pathway for the common 3′ single-stranded repair intermediate (Ivanov et al., 1996; Kang and Symington, 2000; Lambert and Lopez, 2000). SSA aligns and anneals regions of homology on either side of a DSB, repairing it but deleting the intervening sequence, causing deletions between repetitive elements or chromosome translocations when DSBs occur on more than one chromosome (Richardson and Jasin, 2000). Vertebrate cells with large repetitive genomes must, therefore, tightly regulate homologous DNA repair pathways in order to avoid genome instability (Jasin, 2000). Recently, embryonic stem (ES) cells with disruptions in Brca2 have been shown to be compromised for repair of restriction enzyme-induced DSBs by GC (Moynahan et al., 2001). It remains unknown whether the disruption of repair by sister chromatid GC is associated with repair of damage by error-prone recombination pathways such as gene conversion with unequal CO or SSA. Here we ask whether disruption of Brca2 in ES cells is associated with an increased frequency of DNA repair using these pathways. Common causes of spontaneous DSBs are arrested replication forks (Sasaki, 1980; Haber, 1999). Sister chromatid GC and equal sister chromatid CO events are thought to be an accurate mechanism responsible for their repair. SCE can be seen in untreated metaphase cells and following treatment with DNA-damaging agents, and are suggested to arise from the repair of arrested replication forks by equal sister chromatid CO (Sonoda et al., 1999). We therefore also examine the effect of disruption of Brca2 on the frequency of these events relative to other exchanges and aberrations that have arisen by error-prone repair. Our results suggest a mechanism for chromosome instability caused by loss of BRCA2. Results Strategy for assessment of the role of Brca2 in DSB repair in ES cells We wished to create a cell line carrying a conditionally mutable allele of the Brca2 gene to test its role in DNA repair and HR. It is thought that null mutations for Brca2 result in early embryonic lethality probably due to cell cycle arrest mediated by checkpoint activation (Bertwistle and Ashworth, 1998). The choice of DSB repair pathway may be cell cycle regulated; therefore, to avoid the confounding effect of significant cell cycle perturbation, we created a cell line with two hypomorphic Brca2 alleles. We used our previously described ES cell line carrying a hypomorphic allele Brca2Tr2014, which results in the truncation of the Brca2 open reading frame at amino acid 2014 (Connor et al., 1997a). We altered the other allele so that the final Brca2 exon (exon 27) was flanked by loxP sites, which could be conditionally deleted by transient expression of Cre recombinase. Deletion of exon 27 has also been shown to produce a hypomorphic allele, homozygosity for which causes ionizing radiation sensitivity in mouse ES cells (Morimatsu et al., 1998). An analogous truncating mutation in BRCA2 is associated with cancer predisposition in humans (Hakansson et al., 1997). Simultaneously with the modification of exon 27, we introduced a HR repair substrate, DR1Bsd. This allows the repair of an I-SceI-mediated DSB in DR1Bsd to be compared before and after Cre-mediated deletion of Brca2 exon 27 in the same (isogenic) cell line. An additional feature of the construct is that the modified exon 27 allele carries an in-frame myc (9E10) epitope tag, allowing monitoring of the endogenous Brca2 protein. The construct and the modified Brca2 allele are shown in Figure 1A. Figure 1.Brca2 exon 27 'knock in' strategy and analysis of clones. (A) Structure of the 3′ end of the mouse Brca2 locus and targeting vector containing HR repair substrate. The upper line represents the wild-type allele. The second and third lines represent the linearized targeting vector and the targeted allele, respectively. Exons 25, 26 and 27 are shown as grey boxes. The positions of relevant restriction enzyme sites are marked. 5′ and 3′ regions of homology between the targeting vector and the wild-type allele are enclosed between dashed lines flanking the 'knock in' region. This includes a mutated exon 27 with an in-frame 9E10 myc epitope tag shown as a black box. loxP sites flanking exon 27 myc are shown as small black squares. The lower line demonstrates the effect of Cre-mediated recombination between the labelled loxP sites to produce a Flox site. This deletes the intervening sequence including exon 27myc. Large black boxes represent the puromycin (Puro) selectable marker gene, the diphtheria toxin (DT) negative selection marker and the ampicillin (Amp) resistance gene. The HR repair substrate DR1Bsd is represented by a black speckled box. Regions of hybridization to the three probes (A, B and C) used in Southern blot analyses are indicated by large dark squares. The restriction enzymes and the probes used for Southern analysis of the targeted allele, and the positions and sizes of the fragments detected are shown at the bottom of the figure. (B) Southern blot analysis. The targeted allele was termed Brca2Ex27+ and the targeted cell line termed Brca2Tr/Ex27+. Southern blots of genomic DNA from parental cells Brca2Tr/Wt and a Brca2Tr/Ex27+-targeted clone subjected to restriction digestion and hybridization with the marked probe. Probe A is a flanking probe 5′ to the 5′ homology. Probe B is 3′ to the 3′ homology. Probe C is a fragment of Puro. In the left panel, BglI–SalI digestion shows the 7.2 kb wild-type fragment in Brca2Tr/Wt ES cells, and both the 7.2 kb wild-type fragment and the predicted 6.4 kb targeted fragment in Brca2Tr/Ex27+ cells. In the middle panel, ApaI digestion shows the 1.6 kb wild-type fragment and predicted 6.7 kb targeted fragment. These confirm correct integration into the Brca2 locus. The right panel shows ApaI digests probed with Puro, confirming the absence of additional random integrants in Brca2Tr/Ex27+ cells. Brca2Tr/Wt is a negative control. The two middle lanes are clones containing non-targeted random integrants. (C) Correct integration of the targeting construct into the wild-type allele of Brca2Tr/Wt ES cells was confirmed by confirmation of production of full-length myc-tagged Brca2 protein. Immunoblot analysis of whole-cell lysates of Brca2Tr/Wt and Brca2Tr/Ex27+ ES cells immunoprecipated (IP) with anti-myc and anti-Rad51 antibodies and immunoblotted (IB) with an anti-myc antibody. Download figure Download PowerPoint Targeted modification of Brca2 in ES cells We used targeted integration to obtain cell lines carrying a single copy of the DSB repair substrate at a defined chromosomal site. Following electroporation of Brca2Tr2014/Wt ES cells (Connor et al., 1997a) with the targeting construct, transformants were selected in puromycin and analysed by Southern blotting of genomic DNA (Figure 1B). In order to confirm integration into the wild-type allele, targeted clones and control parental Brca2Tr2014/Wt ES cells were lysed and the presence of a full-length myc-tagged Brca2 was confirmed by immunoprecipitation (IP) and immunoblotting (IB) using an anti-myc antibody (Figure 1C). The targeted allele was termed Brca2Ex27mycloxP (here Brca2Ex27+ for brevity) and the targeted cell line termed Brca2Tr2014/Ex27mycloxP (Brca2Tr/Ex27+). To confirm that addition of the six-amino-acid myc epitope to the C-terminus of Brca2 had not affected the ability of the protein to interact with Rad51, further reciprocal IP/IB experiments were performed using antibodies to Rad51 and to the myc epitope (Figure 1C and data not shown). Furthermore, co-localization of myc-tagged Brca2 and Rad51 in ionizing radiation-induced nuclear foci was confirmed by confocal immunofluorescent microscopy (data not shown). This established that the Brca2Ex27+ protein, in common with Brca2, was able to interact with Rad51. Transient expression of Cre recombinase in ES cells causes recombination between loxP sites and deletion of intervening sequence, resulting in deletion of Brca2 exon 27 and part of the intron between exons 26 and 27 (Figure 1A). Thus, the truncation of Brca2 will remove the C-terminal Rad51 binding domain and the myc epitope tag. The Brca2Tr/Ex27+ cell line was transiently transfected with the expression vector pCAGGS driving expression of an EGFP–Cre recombinase fusion protein or with enhanced green fluorescent protein (EGFP) alone as control. Cells were analysed by fluorescence-activated cell sorter (FACS) and the green fluorescent protein (GFP)-positive population sorted to >99% purity and returned to culture. Genomic DNA extraction and Southern blot analysis revealed the expected deletion of exon 27 in 60–70% of the alleles (data not shown). Following exon 27 deletion, the floxed allele is termed Brca2ΔEx27. Brca2Tr/ΔEx27 and Brca2Tr/Ex27+ control clonal cell lines were derived as described in Materials and methods. The presence of a slightly smaller C-terminally truncated Brca2 with loss of the myc epitope tag was confirmed in Brca2Tr/ΔEx27 cell lines by IP using antibodies to the N-terminal region of Brca2, and to the myc epitope and IB using the Brca2 antibody. It was apparent that the deletion of exon 27 was associated with a reduction in the abundance of the truncated form of Brca2, Brca2ΔEx27 (Figure 2A). Figure 2.Cre-mediated deletion of the C-terminus of Brca2 in ES cells. (A) Immunoblotting analysis of whole-cell extract of Brca2Tr/Ex27+ and Brca2Tr/ΔEx27 clonal cell lines immunoprecipated (IP) with anti-myc, anti-Rad51 and N-terminal anti-Brca2 antibodies, and immunoblotted (IB) with the anti-Brca2 antibody. The positions of full-length Brca2, the exon 27-deleted (Brca2ΔEx27) and the exon 11-truncated (Brca2Tr2014) proteins are indicated. (B) Failure of normal induction of Rad51 foci in Brca2Tr/ΔEx27 ES cells. Brca2Tr/Ex27+ and Brca2Tr/ΔEx27 ES cells mock irradiated (left upper and lower panels) or irradiated with 10 Gy (right upper and lower panels) were fixed and analysed by immunofluorescent microscopy. DNA is labelled with DAPI and appears blue. Rad51 was detected with anti-Rad51 antibody and a secondary FITC-conjugated antibody. The right upper panel shows induction of Rad51-containing nuclear foci in Brca2Tr/Ex27+ cells. The right lower panel shows failure of induction of Rad51 foci in Brca2Tr/ΔEx27 ES cells. Download figure Download PowerPoint Brca2ΔEx27 and Brca2Tr2014 associate with Rad51 but inhibit X-ray-induced Rad51 nuclear focus formation Despite deletion of the C-terminal Rad51 binding domain, Brca2ΔEx27 still contains the eight BRC repeats encoded by exon 11 of Brca2. Brca2Tr2014 is predicted to retain seven BRC repeats (Connor et al., 1997a). We wished to establish whether either Brca2Tr2014 or Brca2ΔEx27 associates with Rad51. IP with anti-Rad51 antibody and IB with antibody to the N-terminal region of Brca2 revealed that both Brca2Tr2014 and Brca2ΔEx27 associate with Rad51 (Figure 2A). The greater intensity of the Brca2ΔEx27 band when immunoprecipitated with anti-Rad51 antibody rather than anti-Brca2 may be due to a greater affinity of Brca2ΔEx27 for Rad51 than for the anti-Brca2 antibody. To ascertain whether Brca2ΔEx27 affected Rad51 nuclear focus formation, we irradiated Brca2Tr/Ex27+ and Brca2Tr/ΔEx27 ES cells with 10 Gy of X-rays and analysed the formation of Rad51 nuclear foci 5 h later. We found that Brca2Tr/Ex27+ cells formed Rad51 foci after irradiation with 10 Gy, but this failed to induce Rad51 foci in Brca2Tr/ΔEx27 ES cells (Figure 2B). This shows that although Brca2ΔEx27 interacts with Rad51, the C-terminus of Brca2 is required for the formation or stabilization of IR-induced Rad51 foci. The DR1Bsd recombination test substrate To investigate the effect of these hypomorphic Brca2 mutations on the repair of DNA DSBs by homology-directed repair we have used a chromosomal DSB repair substrate that allows reporting of both gene conversion (GC and CO) and SSA homology-directed repair events at a defined chromosomal locus within our cell lines. This can be achieved both by antibiotic selection of colonies and by analysis of genomic DNA repair products. The DR1Bsd substrate contains a central zeocin selectable marker gene (Zeo) flanked by two differentially mutated blasticidin antibiotic resistance (Bsd) genes (Figure 3). S1Bsd is a full-length 693 bp Bsd gene that contains an in-frame insertion of the 18 bp recognition sequence of the restriction endonuclease I-SceI at a unique SalI site 279 bp into the coding sequence of Bsd. This insertion encodes two in-frame stop codons and renders Bsd non-functional. The 3′ repeat (5′ΔBsd) is a 659 bp promoterless fragment of Bsd inactivated by truncation of the 5′ 34 bp. Both repeats are in the same orientation and are therefore termed direct repeats. Figure 3.HR repair substrate DR1Bsd. (A) The repair substrate is represented in a 5′ to 3′ orientation. Relevant restriction endonuclease recognition sites are marked. The 5′ mutated Bsd repeat is shown as a green line and is labelled S1Bsd. The upstream TK promoter sequence from pMCINeo is marked with an arrow. The site of mutation of the wild-type SalI site by insertion of the 18 bp recognition sequence of the I-SceI endonuclease is shown as a red bar. The central Zeo antibiotic selection marker is shown as a blue line with its upstream PGK promoter marked with an arrow. The downstream promoterless direct repeat 5′ΔBsd is marked as a green line. The position of the wild-type SalI site is marked. A black bar indicates the position of a TK promoter probe that can hybridize in all repair products (B, lower panels) equally. The effect of transient expression of I-SceI from the pCAGGS expression vector is illustrated. The upper line represents the undamaged repair substrate. The I-SceI expression vector is shown as a circle. The lower line demonstrates the site of induction of a DNA DSB at the I-SceI recognition sequence in S1Bsd. (B) Mechanisms by which wild-type Bsd may be created by HR repair of the I-SceI DSB in DR1Bsd. Repair by use of the SSA pathway is depicted in the left panels. This involves 5′–3′ resection of one strand on either side of the DSB, leaving a 3′ tail. When complementary Bsd sequences from S1Bsd and 5′ΔBsd on either side of the DSB are exposed, they can anneal. This is indicated by thin vertical lines. The single-stranded tails are resected by a nuclease, gaps are filled in and nicks ligated. This process deletes all sequence between S1Bsd and 5′ΔBsd, and thus results in the creation of wild-type Bsd and the deletion of Zeo. The repair product and the size of predicted restriction fragments are marked in the left lower panel. Repair of S1Bsd by use of the GC pathway (right panels) involves similar 5′–3′ resection to leave 3′ single-stranded tails. These invade and pair with homologous 5′ΔBsd sequence on either the same chromatid (central panel) or sister chromatid (right panel). The break may thus be repaired using wild-type sequence as the template. Regions of pairing are indicated with a cross and may be resolved either with or without a crossover (CO) event. If the substrate is repaired without CO, the repair product contains wild-type Bsd, Zeo and 5′ΔBsd. The repair product and the size of predicted restriction fragments are marked in the right lower panel. If an unequal CO event takes place, the central Zeo is removed. This product, referred to as the 'Pop out' repair product, is identical whether repair is by SSA or CO (left lower panel). Equal CO events recreate S1Bsd and are, therefore, not recovered. Download figure Download PowerPoint DSB induction and repair events in DR1Bsd Transient expression of the rare cutting endonuclease I-SceI linked to a triplicated nuclear localization signal (3 × nls I-SceI) is non-toxic in mouse ES cells and induces a DSB at a chromosomally integrated I-SceI site (Rouet et al., 1994; Moynahan et al., 2001), as in S1Bsd (Figure 3). Following repair by HR between S1Bsd and 5′ΔBsd, the disrupted SalI site of S1Bsd can be restored, recreating wild-type Bsd and consequently resulting in the resistance of ES cells to blasticidin (Figure 3). Repair of DR1Bsd by SSA leads to blasticidin resistance, but consequent deletion of Zeo renders cells sensitive to zeocin. HR by GC may occur using either 5′ΔBsd on the same chromatid as a donor (intra-chromatid GC) or 5′ΔBsd on the sister chromatid following DNA replication (sister chromatid GC). Clones derived by HR repair using GC will be resistant to both blasticidin and zeocin. Gene conversion may be associated with an unequal crossing over event, in which case the central Zeo gene is removed as an excised circle (intra-chromatid CO), or in sister chromatid CO Zeo is transferred to the donor sister chromatid. Therefore, I-SceI DSB repair of DR1Bsd by any of the above homology-directed mechanisms will induce blasticidin resistance in daughter cells. Whereas clones repaired by GC will be resistant to both blasticidin and zeocin, clones repaired by SSA or unequal CO will be resistant to blasticidin, but sensitive to zeocin. The successful repair of DR1Bsd by HR mechanisms may also be detected and further characterized by Southern analysis of pooled blasticidin-resistant clones (Figure 3). Digestion of the HR repaired construct in blasticidin-resistant clones with BglII and SalI reveals a change in size of the fragment from 2.8 kb in the parental construct to 0.84 kb. Excision of the entire substrate with KpnI gives a 3.3 kb fragment if repair is by HR by GC or a 1.4 kb deletion product following repair by HR by SSA or CO. The outcomes of SSA and CO are identical at the DNA level in blasticidin-resistant cells. This repair product will be referred to here as the 'Pop out' recombination product. Repair of the DSB by the NHEJ mechanism either involves precise re-ligation of the I-SceI overhangs, or more commonly, endonuclease processing of the broken ends. This is associated with small deletions and insertions. Under these circumstances, S1Bsd remains mutant and the clone is sensitive to blasticidin. Precise deletion of the 18 bp I-SceI site and accurate microannealing of the duplicated flanking SalI overhangs by NHEJ repair mechanisms might theoretically lead to restoration of wild-type Bsd. This event is reported to occur two orders of magnitude less frequently than HR between the repeats in similar constructs (Moynahan and Jasin, 1997; Lin et al., 1999). To test the requirement for a homologous repeat for recreation of wild-type Bsd in ES cells, we compared the frequency of blasticidin-resistant colonies induced by transient I-SceI expression in cell lines expressing full-length Brca2 containing either only S1Bsd or the entire repair substrate DR1Bsd. We found that the frequency of blasticidin resistance is 9 × 10−4 for S1Bsd alone compared with 5 × 10−2 for DR1Bsd (Figure 4A). Blasticidin resistance induced by NHEJ repair of S1Bsd is, therefore, a very rare event. Figure 4.Repair of DR1Bsd leads to blasticidin resistance by homology-directed repair. (A) Bar graph showing the number of blasticidin-resistant colonies per thousand cells plated (corrected for transfection and cloning efficiencies). The left column represents Brca2Tr/Wt cells containing S1Bsd alone and the right column Brca2Tr/Ex27+ cells that contain both the S1Bsd and the homologous donor repeat 5′ΔBsd in DR1Bsd. There is very little induction of blasticidin resistance in S1Bsd; therefore, the frequency of repair of a DSB in S1Bsd relative to wild-type Bsd by NHEJ is extremely low. Error bars represent ± 1 SEM. (B) A representative Southern blot of BglII–SalI-digested genomic DNA from Brca2Tr/Ex27+ or Brca2Tr/ΔEx27 ES cells before (marked No Tx) and after I-SceI-induced DSB repair and subsequent blasticidin selection (marked Bsdr). A TK promoter fragment (indicated in Figure 3) was used as a probe. A dominant 2.8 kb restriction fragment is seen in both cell lines before transfection of pCAGGS 3 × nls I-SceI and is from the unbroken DR1Bsd substrate. After DSB induction, repair and selection of blasticidin-resistant colonies, the predicted 0.84 kb HR fragment is dominant in all cell lines. This arises due to HR with 5′ΔBsd and transfer of the wild-type SalI-containing sequence from 5′ΔBsd to S1Bsd, to create Bsd. Download figure Download PowerPoint Brca2Tr/Ex27+ and Brca2Tr/ΔEx27 ES cells were transfected with pCAGGS 3 × nls I-SceI or with pCAGGS EGFP as control, replated and selected with blasticidin (see Materials and methods). DNA from pooled resistant colonies was digested with BglII and SalI, followed by Southern blotting. This confirmed that blasticidin-resistant colonies arise in both cell lines by HR with 5′ΔBsd (Figure 4B) rather than by NHEJ or another novel mechanism. The effect of Brca2 exon 27 deletion on HR repair by gene conversion As Rad51 binds the C-terminus of Brca2 and is known to have a key role in DSB repair by homologous strand invasion and gene conversion, we wished to test the effect of our Brca2ΔEx27 mutation on this process. Brca2Tr/Ex27+ and Brca2Tr/ΔEx27 ES cells were transfected with pCAGGS 3 × nls I-SceI and selected with blasticidin as described in Materials and methods. In each of three experiments, using three Brca2Tr/Ex27+ and three independently derived Brca2Tr/ΔEx27 ES cell clones, resulting blasticidin-resistant clones were isolated, expanded and then double selected with blasticidin and zeocin. Parental Brca2Tr/Ex27
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