Double-strand Break-induced Homologous Recombination Research Articles

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells.

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions.

RAD52 inactivation is synthetically lethal with deficiencies in BRCA1 and PALB2 in addition to BRCA2 through RAD51-mediated homologous recombination

Synthetic lethality is an approach to study selective cell killing based on genotype. Previous work in our laboratory has shown that loss of RAD52 is synthetically lethal with BRCA2 deficiency, while exhibiting no impact on cell growth and viability in BRCA2-proficient cells. We now show that this same synthetically lethal relationship is evident in cells with deficiencies in BRCA1 or PALB2, which implicates BRCA1, PALB2 and BRCA2 in an epistatic relationship with one another. When RAD52 was depleted in BRCA1- or PALB2-deficient cells, a severe reduction in plating efficiency was observed, with many abortive attempts at cell division apparent in the double-depleted background. In contrast, when RAD52 was depleted in a BRCA1- or PALB2-wildtype background, a negligible decrease in colony survival was observed. The frequency of ionizing radiation-induced RAD51 foci formation and double-strand break-induced homologous recombination (HR) was decreased by 3- and 10-fold, respectively, when RAD52 was knocked down in BRCA1- or PALB2-depleted cells, with minimal effect in BRCA1- or PALB2-proficient cells. RAD52 function was independent of BRCA1 status, as evidenced by the lack of any defect in RAD52 foci formation in BRCA1-depleted cells. Collectively, these findings suggest that RAD52 is an alternative repair pathway of RAD51-mediated HR, and a target for therapy in cells deficient in the BRCA1-PALB2-BRCA2 repair pathway.

The Fanconi Anemia Ortholog FANCM Ensures Ordered Homologous Recombination in Both Somatic and Meiotic Cells in Arabidopsis

The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

A platform fed-batch process for various CEMAX® producer cell lines

The random nature of transgene integration harbours various pitfalls for development of production cell lines including clonal variation in expression level and growth characteristics. The CEMAX system is an expression system for targeted integration of expression cassettes via DNA double-strand break induced homologous recombination. Stable high producers are available within 4 weeks without the need of extensive clone screening and process development. Stable and high expression rates are ensured with the CEMAX system due to targeted integration of a single copy of the gene of interest at a transcriptionally highly active site in the host cell genome. Producer cell lines for various therapeutic product candidates were established. These cell lines produced antibodies and highly glycosylated antibody fusion proteins and showed high clonal similarity. This made a platform fed-batch process profitable. The CEMAX expression system is based on a genetically modified CHO cell line bearing a tag at a highly active genomic transcription site. The gene of interest (GOI) is integrated via site-directed DNA double-strand break-induced homologous recombination. The target site comprises a screening and selection cassette, which was used for initial development of the high producer host cell. This exchangeable cassette is flanked by elements that facilitate site-directed integration by homologous recombination. These elements include regions of homology for recombination with the CEMAX vector, rudimentary selection markers that are activated during recombination, and cleavage sites for the homing endonuclease I-SceI (Meganuclease). Transfection of the CEMAX vector comprising the GOI along with transient Meganuclease activity triggers a chain of events after cotransfection: DNA gets cleaved by I-SceI and cellular DNA repair machinery is induced by free DNA double-strand breaks. The CEMAX vector comprising the GOI functions as a repair matrix using recombination between homologous elements flanking the DNA lesion. The GOI gets integrated and the rudimentary selection markers become activated upon homologous recombination. CEMAX producer cells were selected at multi-well plate scale followed by analysis for outgrowth of stable producer cells. The platform process for fed-batch cultivation of various CEMAX producer cells was applied after expansion to the scale needed for production of protein material. The idea of a platform process that is suitable for all CEMAX producer cells was based on theoretical consideration about similarity of cells due to site-directed integration and the observation that cell growth and metabolism of CEMAX cell lines were comparable. This was verified by the results of this study. Applying the fed-batch process cell growth of CEMAX host cells and CEMAX producer cells was comparable (Figure ​(Figure1).1). The development of the platform fed-batch process was based on three small scale development steps: (1) basal media screening, (2) feed medium optimization, and (3) improvement of feeding regime. Process development in 1 L bioreactors is currently ongoing. Figure 1 Platform fed-batch process for CEMAX cell lines. Cells were cultivated in comparison to the CEMAX host cell line in shake flask scale. The basal medium was a commercially available chemically defined medium and was used in combination with the proprietary ... The fed-batch process comprises a chemically defined and commercially available basal medium and the chemically defined feed solution CeloFeed (Celonic) supplemented according to the improved feeding regime. The glucose level was maintained between 4.5 g/L and 7.5 g/L. Glutamine was kept at a concentration above 0.8 mM. By applying the platform fed-batch process product concentrations up to 690 mg/L were achieved with CEMAX cell lines producing a glycosylated Fc fusion protein after site-directed integration of the GOI (Figure ​(Figure1).1). Achievable productivity for different protein products varied from product to product. This was most probably due to different requirements in protein processing and secretion. The platform fed-batch process was developed by optimization based on commercial available and proprietary basal and feed media formulations. It was suitable for all producer cell lines derived of a particular CEMAX host cell due to highly similar growth behavior after site-directed integration of the gene of interest. The platform process made product concentrations of up to 0.7 g/L achievable. Animal derived component free chemically defined medium and feed solution ensure a robust and regulatory compliant process. By combining the platform process with site-directed integration timelines in cell line development and process development were shortened for faster entry in first-in-man studies.

Abstract CN05-04: Opportunities for targeting homologous recombination in human cancers.

Abstract BRCA2 mediates homologous recombination (HR) via its direct interaction with Rad51. There is ample genetic and biological evidence that BRCA1 and BRCA2 are functioning in the same pathway of DNA repair, but the functional connection between the two proteins is poorly understood. Interestingly, a mutation of BRCA1 (S988A) localized normally to ionizing radiation induced nuclear foci (IRIF), but the BRCA2 protein could not be recruited to BRCA1 at the damage site. Phosphorylation of BRCA1 at the S988 site is carried out by Chk2, which was also required for BRCA2 to localize to sites of DNA damage and support homologous recombination. These observations suggest that CHK2-BRCA1-BRCA2-Rad51 function in a single pathway of homologous recombinational repair to prevent the development of breast cancer. Loss of function of BRCA1/BRCA2 is a tumor-specific finding, making the role of alternative repair pathways in these cells critical for their survival. We show that loss of Rad52 function is synthetic lethal with BRCA1, PALB2 or BRCA2 deficiency, whereas there is no impact on cell growth in wild-type cells. The frequency of both spontaneous and double-strand break-induced homologous recombination, plus ionizing radiation induced Rad51 foci, decreased when Rad52 was depleted in BRCA2-deficient cells, with no effect in BRCA2-complemented cells. The observation that Rad52-inactivation is synthetic lethal with BRCA2 makes Rad52 a target for therapy in these tumors. The BRCA1-BRCA2 pathway can be functionally inactivated in sporadic breast cancer, perhaps as frequently as 20% of cases, suggesting that the pool of patients with defects in homologous recombinational repair is larger than initially expected. The functional assessment of defects in homologous recombination was assessed by ionizing-radiation (ex-vivo exposure) induced Rad51 foci and confirmed by characteristic patterns of array comparative genomic hybridization. The inactivation of the BRCA1-BRCA2 pathway allows the use of specific therapies designed to exploit the defects in DNA repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN05-04.

Rad52 inactivation is synthetically lethal with BRCA2 deficiency

Synthetic lethality is a powerful approach to study selective cell killing based on genotype. We show that loss of Rad52 function is synthetically lethal with breast cancer 2, early onset (BRCA2) deficiency, whereas there was no impact on cell growth and viability in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination and ionizing radiation-induced Rad51 foci decreased by 2-10 times when Rad52 was depleted in BRCA2-deficient cells, with little to no effect in BRCA2-complemented cells. The absence of both Rad52 and BRCA2 resulted in extensive chromosome aberrations, especially chromatid-type aberrations. Ionizing radiation-induced and S phase-associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double-strand breaks and replication stalling independently of BRCA2. Rad52 thus is an independent and alternative repair pathway of homologous recombination and a target for therapy in BRCA2-deficient cells.

Abstract SY28-02: Connections in the BRCA1-BRCA2 pathway of homologous recombination: Implications for breast cancer development and treatment

Abstract The breast cancer susceptibility gene, BRCA2, mediates homologous recombination (HR) via its direct interaction with the Rad51 recombinase. There is ample genetic and biological evidence that BRCA1 and BRCA2 are functioning in the same pathway of DNA repair, but the functional connection between the two proteins is poorly understood. Interestingly, a mutation of BRCA1 (S988A) localized normally to ionizing radiation induced nuclear foci (IRIF), but the BRCA2 protein could not be recruited to BRCA1 at the damage site. Phosphorylation of BRCA1 at the S988 site is carried out by Chk2, which was also required for BRCA2 to localize to sites of DNA damage and support homologous recombination. These observations suggest that CHK2-BRCA1-BRCA2-Rad51 function in a single pathway of homologous recombinational repair to prevent the development of breast cancer. In familial breast cancer, one allele of BRCA1 or BRCA2 is germline mutant and the second allele is inactivated to allow tumor development. Thus, loss of function of BRCA1/BRCA2 is a tumor-specific finding, making the role of alternative repair pathways in these cells critical for their survival. We show that loss of Rad52 function is synthetic lethal with BRCA2 deficiency, whereas there is no impact on cell growth in BRCA2-complemented cells. The frequency of both spontaneous and double-strand break-induced homologous recombination, and ionizing radiation induced Rad51 foci, decreased by greater than 50%, when Rad52 was depleted in BRCA2-deficient cells, with no effect in BRCA2-complemented cells. Ionizing radiation-induced and S-phase associated Rad52-Rad51 foci form equally well in the presence or absence of BRCA2, indicating that Rad52 can respond to DNA double strand breaks and replication stalling independently of BRCA2. The observation that Rad52-inactivation is synthetic lethal with BRCA2 and that Rad52 acts independently of BRCA2 makes Rad52 a target for therapy in these tumors. Finally, we have evidence that the BRCA1-BRCA2 pathway can be functionally inactivated in sporadic breast cancer, suggesting that the pool of patients with defects in homologous recombinational repair is larger than initially expected. Citation Format: Simon N. Powell. Connections in the BRCA1-BRCA2 pathway of homologous recombination: Implications for breast cancer development and treatment [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY28-02

Homologous recombination: a basis for targeted genome optimization in crop species such as maize

In an attempt to understand the feasibility of future targeted genome optimization in agronomic crops, we tested the efficiency of homologous recombination-mediated sequence insertion upon induction of a targeted DNA double-strand break at the desired integration site in maize. By the development of an efficient tissue culture protocol, and with the use of an I-SceI gene optimized for expression in maize, large numbers of precisely engineered maize events were produced in which DNA integration occurred very accurately. In a subset of events examined in detail, no additional deletions and/or insertions of short filler DNA at the integration site were observed. In 30%-40% of the recovered events, no traces of random insertions were observed. This was true for DNA delivery by both Agrobacterium and particle bombardment. These data suggest that targeted double-strand break-induced homologous recombination is a superior method to generate specific desired changes in the maize genome, and suggest targeted genome optimization of agronomic crops to be feasible.

Dual Roles for DNA Polymerase η in Homologous DNA Recombination and Translesion DNA Synthesis

Chicken B lymphocyte precursors and DT40 cells diversify their immunoglobulin-variable (IgV) genes through homologous recombination (HR)-mediated Ig gene conversion. To identify DNA polymerases that are involved in Ig gene conversion, we created DT40 clones deficient in DNA polymerase eta (poleta), which, in humans, is defective in the variant form of xeroderma pigmentosum (XP-V). Poleta is an error-prone translesion DNA synthesis polymerase that can bypass UV damage-induced lesions and is involved in IgV hypermutation. Like XP-V cells, poleta-disrupted (poleta) clones exhibited hypersensitivity to UV. Remarkably, poleta cells showed a significant decrease in the frequency of both Ig gene conversion and double-strand break-induced HR when compared to wild-type cells, and these defects were reversed by complementation with human poleta. Our findings identify a DNA polymerase that carries out DNA synthesis for physiological HR and provides evidence that a single DNA polymerase can play multiple cellular roles.

Factors Affecting Double-Strand Break-Induced Homologous Recombination in Mammalian Cells

Double-strand break (DSB)-induced homologous recombination (HR) of direct repeats is a powerful means to achieve gene excision, a critical step in genome engineering. In this report we have used an extrachrmosomal reporter system to monitor the impact of different parameters on meganuclease-induced HR in CHO-K1 cells. We found that repeat homology length is critical. Virtually no HR could be detected with a 15-bp duplication, while, with repeats larger than 400 bp, recombination efficiency became less dependent on homology length. The presence of an intervening sequence between the duplications dramatically impairs HR, independent of the cleavage position; by 3 kb of insertion, HR is virtually undetectable. Efficient HR can be restored by positioning cleavage sites at both ends of the intervening sequence, allowing a constant level of excision with up to 10 kb of intervening sequences. Using similar constructs, 2.8-kb inserts could be efficiently removed from several chromosomal loci, illustrating the wide potential of this technology. These results fit current models of direct repeat recombination and identify DSB-induced HR as a powerful tool for gene excision.

Overexpression of Rad51 inhibits double-strand break-induced homologous recombination but does not affect gene conversion tract lengths

DNA double-strand breaks (DSBs) in yeast are repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad51 forms nucleoprotein filaments at processed broken ends that effect strand exchange, forming heteroduplex DNA (hDNA) that gives rise to a gene conversion tract. We hypothesized that excess Rad51 would increase gene conversion tract lengths. We found that excess Rad51 reduced DSB-induced HR but did not alter tract lengths or other outcomes including rates of crossovers, break-induced replication, or chromosome loss. Thus, excess Rad51 appears to influence DSB-induced HR at an early stage. MAT heterozygosity largely mitigated the inhibitory effect of excess Rad51 on allelic HR, but not direct repeat HR. Excess Rad52 had no effect on DSB-induced HR efficiency or outcome, nor did it mitigate the dominant negative effects of excess Rad51. Excess Rad51 had little effect on DSB-induced lethality in wild-type cells, but it did enhance lethality in yku70Δ mutants. Interestingly, dnl4Δ showed marked DSB-induced lethality but this was not further enhanced by excess Rad51. The differential effects of yku70Δ and dnl4Δ indicate that the enhanced killing with excess Rad51 in yku70Δ is not due to its NHEJ defect, but may reflect its defect in end-protection and/or its inability to escape from checkpoint arrest. Srs2 displaces Rad51 from nucleoprotein filaments in vitro, suggesting that excess Rad51 might antagonize Srs2. We show that excess Rad51 does not reduce survival of wild-type cells treated with methylmethane sulfonate (MMS), or cells suffering a single DSB. In contrast, excess Rad51 sensitized srs2Δ cells to both MMS and a single DSB. These results support the idea that excess Rad51 antagonizes Srs2, and underscores the importance of displacing Rad51 from nucleoprotein filaments to achieve optimum repair efficiency.

Homologous recombination in extrachromosomal plasmid substrates is not suppressed by p53.

We and others reported previously that the tumor suppressor p53 down-regulates spontaneous homologous recombination in chromosomally integrating plasmid substrates, but how p53 affects homology-dependent repair of DNA double-strand breaks has not been established. Furthermore, it has been hypothesized that p53 may suppress homologous recombination by direct interaction with recombination intermediates, but it is not known whether p53 directly acts on extrachromosomal plasmid substrates. In the present study, we asked whether p53 can suppress extrachromosomal spontaneous and double-strand break-induced homologous recombination. A plasmid shuttle assay was employed utilizing episomally replicating substrates, which carried mutated tandem repeats of a CAT reporter gene. Spontaneous homologous recombination and homology-dependent repair of double-strand breaks induced by the I-SceI nuclease led to reconstitution of the reporter. Extrachromosomal homologous recombination was found to proceed independently of the p53 status of isogenic mouse fibroblast lines, contrasting the p53-mediated suppression of chromosomal recombination. The lack of p53 effect applied not only to the dominating single-strand annealing pathway, which is Rad51-independent, but also to Rad51-dependent gene conversion events. Comparison of homologous and non-homologous recombination frequencies revealed similar contributions to the repair of I-SceI-induced breaks irrespective of p53 status. Our results are consistent with a model in which the regulation of homologous recombination by p53 is restricted to the highly ordered chromosomal chromatin structure. These data may serve as a cautionary note for future investigations using solely extrachromosomal model systems to address DNA repair in intact cells.

Overexpression of human RAD51 and RAD52 reduces double-strand break-induced homologous recombination in mammalian cells

Double-strand breaks (DSBs) can be repaired by homologous recombination (HR) in mammalian cells, often resulting in gene conversion. RAD51 functions with RAD52 and other proteins to effect strand exchange during HR, forming heteroduplex DNA (hDNA) that is resolved by mismatch repair to yield a gene conversion tract. In mammalian cells RAD51 and RAD52 overexpression increase the frequency of spontaneous HR, and one study indicated that overexpression of mouse RAD51 enhances DSB-induced HR in Chinese hamster ovary (CHO) cells. We tested the effects of transient and stable overexpression of human RAD51 and/or human RAD52 on DSB-induced HR in CHO cells and in human cells. DSBs were targeted to chromosomal recombination substrates with I-SceI nuclease. In all cases, excess RAD51 and/or RAD52 reduced DSB-induced HR, contrasting with prior studies. These distinct results may reflect differences in recombination substrate structures or different levels of overexpression. Excess RAD51/RAD52 did not increase conversion tract lengths, nor were product spectra otherwise altered, indicating that excess HR proteins can have dominant negative effects on HR initiation, but do not affect later steps such as hDNA formation, mismatch repair or the resolution of intermediates.

Regulation of double-strand break-induced mammalian homologous recombination by UBL1, a RAD51-interacting protein.

Mammalian RAD51 protein plays essential roles in DNA homologous recombination, DNA repair and cell proliferation. RAD51 activities are regulated by its associated proteins. It was previously reported that a ubiquitin-like protein, UBL1, associates with RAD51 in the yeast two-hybrid system. One function of UBL1 is to covalently conjugate with target proteins and thus modify their function. In the present study we found that non-conjugated UBL1 forms a complex with RAD51 and RAD52 proteins in human cells. Overexpression of UBL1 down-regulates DNA double-strand break-induced homologous recombination in CHO cells and reduces cellular resistance to ionizing radiation in HT1080 cells. With or without overexpressed UBL1, most homologous recombination products arise by gene conversion. However, overexpression of UBL1 reduces the fraction of bidirectional gene conversion tracts. Overexpression of a mutant UBL1 that is incapable of being conjugated retains the ability to inhibit homologous recombination. These results suggest a regulatory role for UBL1 in homologous recombination.