Enhanced oligonucleotide-directed gene targeting in mammalian cells following treatment with DNA damaging agents

Targeted gene repair, a form of oligonucleotide-directed mutagenesis, employs end-modified single-stranded DNA oligonucleotides to mediate single-base changes in chromosomal DNA. In this work, we use a specific 72-mer to direct the repair of a mutated eGFP gene stably integrated in the genome of DLD-1 cells. Corrected cells express eGFP that can be identified and quantitated by FACS. The repair of this mutant gene is dependent on the presence of a specifically designed oligonucleotide and the frequency with which the mutation is reversed is affected by the induction of DNA damage. We used hydroxyurea, VP16 (etoposide), and thymidine to modulate the rate of DNA replication through the stalling of the replication forks or the introduction of lesions. Addition of hydroxyurea or VP16 before the electroporation of the oligonucleotide, results in an accumulation of double-strand breaks (DSB) whose repair is facilitated by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The addition of thymidine results in DNA damage within replication forks, damage that is repaired through the process of homologous recombination. Our data suggest that gene repair activity is elevated when DNA damage induces or activates the homologous recombination pathway.     

BCCIP regulates homologous recombination by distinct domains and suppresses spontaneous DNA damage

Homologous recombination (HR) is critical for maintaining genome stability through precise repair of DNA double-strand breaks (DSBs) and restarting stalled or collapsed DNA replication forks. HR is regulated by many proteins through distinct mechanisms. Some proteins have direct enzymatic roles in HR reactions, while others act as accessory factors that regulate HR enzymatic activity or coordinate HR with other cellular processes such as the cell cycle. The breast cancer susceptibility gene BRCA2 encodes a critical accessory protein that interacts with the RAD51 recombinase and this interaction fluctuates during the cell cycle. We previously showed that a BRCA2- and p21-interacting protein, BCCIP, regulates BRCA2 and RAD51 nuclear focus formation, DSB-induced HR and cell cycle progression. However, it has not been clear whether BCCIP acts exclusively through BRCA2 to regulate HR and whether BCCIP also regulates the alternative DSB repair pathway, non-homologous end joining. In this study, we found that BCCIP fragments that interact with BRCA2 or with p21 each inhibit DSB repair by HR. We further show that transient down-regulation of BCCIP in human cells does not affect non-specific integration of transfected DNA, but significantly inhibits homology-directed gene targeting. Furthermore, human HT1080 cells with constitutive down-regulation of BCCIP display increased levels of spontaneous single-stranded DNA (ssDNA) and DSBs. These data indicate that multiple BCCIP domains are important for HR regulation, that BCCIP is unlikely to regulate non-homologous end joining, and that BCCIP plays a critical role in resolving spontaneous DNA damage.     

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Abstract

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1?keV/μm, α-particles 116?keV/μm and ³⁶Ar ions 270?keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.     

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.     

Induction of a mutant phenotype in human repair proficient cells after overexpression of a mutated human DNA repair gene.

Antisense and mutated cDNA of the human excision repair gene ERCC-1 were overexpressed in repair proficient HeLa cells by means of an Epstein-Barr-virus derived cDNA expression vector. Whereas antisense RNA did not influence the survival of the transfected cells, a mutated cDNA generating an ERCC-1 protein with two extra amino acids in a conserved region of its C-terminal part resulted in a significant sensitization of the HeLa transfectants to mitomycin C-induced damage. These results suggest that overexpression of the mutated ERCC-1 protein interferes with proper functioning of the excision repair pathway in repair proficient cells and is compatible with a model in which the mutated ERCC-1 protein competes with the wild-type polypeptide for a specific step in the repair process or for occupation of a site in a repair complex. Apparently, this effect is more pronounced for mitomycin C induced crosslink repair than for UV-induced DNA damage.     

Down-regulation of DNA polymerase beta accompanies somatic hypermutation in human BL2 cell lines

Somatic hypermutation (SHM) is a fundamental process in immunoglobulin gene maturation that results in increased affinity of antibodies toward antigens. In one hypothesis explaining SHM in human B cells, the process is initiated by enzymatic deamination of cytosine to uracil in the immunoglobulin gene V-region and this in turn triggers mutation-prone forms of uracil-DNA base excision repair (BER). Yet, an uncertainty with this model is that BER of uracil-DNA in mammalian cells is generally error-free, wherein DNA polymerase beta (pol beta) conducts gap-filling synthesis by insertion of bases according to Watson-Crick rules. To evaluate this inconsistency, we examined pol beta expression in various SHM proficient human BL2 cell line subclones. We report that expression of pol beta in SHM proficient cell lines was strongly down-regulated. In contrast, in other BL2 subclones, we found that SHM was deficient and that pol beta expression was much higher than in the SHM proficient subclones. We also found that overexpression of recombinant human pol beta in a SHM proficient subclone abrogated its capacity for SHM. These results suggest that down-regulation of the normal BER gap-filling DNA polymerase, pol beta, accompanies induced SHM in BL2 cells. This is consistent with the hypothesis that normal error-free BER must be silenced to make way for an error-prone BER process that may be required during somatic hypermutation.     

Modulation of error-prone double-strand break repair in mammalian chromosomes by DNA mismatch repair protein Mlh1

We assayed error-prone double-strand break (DSB) repair in wild-type and isogenic Mlh1-null mouse embryonic fibroblasts containing a stably integrated DSB repair substrate. The substrate contained a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene; the tk-neo fusion gene was disrupted in the tk portion by a 22bp oligonucleotide containing the 18 bp recognition site for endonuclease I-SceI. Following DSB-induction by transient expression of I-SceI endonuclease, cells that repaired the DSB by error-prone nonhomologous end-joining (NHEJ) and restored the correct reading frame to the tk-neo fusion gene were recovered by selecting for G418-resistant clones. The number of G418-resistant clones induced by I-SceI expression did not differ significantly between wild-type and Mlh1-deficient cells. While most DSB repair events were consistent with simple NHEJ in both wild-type and Mlh1-deficient cells, complex repair events were more common in wild-type cells. Furthermore, genomic deletions associated with NHEJ events were strikingly larger in wild-type versus Mlh1-deficient cells. Additional experiments revealed that the stable transfection efficiency of Mlh1-null cells is higher than that of wild-type cells. Collectively, our results suggest that Mlh1 modulates error-prone NHEJ by inhibiting the annealing of DNA ends containing noncomplementary base pairs or by promoting the annealing of microhomologies.     

Molecular-genetic analysis of dual-stranded DNA break repair in saccharomyces yeasts

DNA double-strand breaks (DSB) are the most dangerous damage to genetic material caused by ionizing radiation and some chemical agents. Nonrestored DSB lead to chromosomal rearrangements, genetic instability, and cell death. On the other hand, DSB normally occur in cells in the course of normal gene functioning. DSB repair not only protects cells from adverse consequences and maintains stability of genetic material but is directly involved in the most important processes of cell life, such as meiosis and humoral immunity in vertebrates. The diverse mechanisms of homologous and nonhomologous recombination underlie DSB repair. In this respect, yeast are the best-studied object. In this review, genetic control and molecular models of the recombination DNA DSB repair in Saccharomyces cerevisiae are considered. Evidence has accumulated that indicates the higher eukaryotes retained the basic set of the repair pathways characteristic of bacteria and lower eukaryotes. However, different repair mechanisms predominate in yeast as compared to higher eukaryotes. Therefore, the results obtained in yeast experiments may be applicable to higher eukaryotes.     

Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.     

Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange

Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.     

Analysis of DNA double-strand break repair pathways in mice

During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.     

Low levels of DNA ligases III and IV sufficient for effective NHEJ

Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non-homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA-PKcs, Ku, LIG4, XRCC4, XLF/Cernunnos, Artemis as well as DNA polymerase lambda (termed D-NHEJ). Mutants with defects in these proteins remove a large proportion of DSBs from their genome utilizing an alternative pathway of NHEJ that operates as a backup (B-NHEJ). While D-NHEJ relies exclusively on DNA ligase IV, recent work points to DNA ligase III as a component of B-NHEJ. Here, we use RNA interference (RNAi) to further investigate the activity requirements for DNA ligase III and IV in the pathways of NHEJ. We report that 70-80% knock down of LIG3 expression has no detectable effect on DSB rejoining, either in D-NHEJ proficient cells, or in cells where D-NHEJ has been chemically or genetically compromised. Surprisingly, also LIG4 knock down has no effect on repair proficient cells, but inhibits DSB rejoining in a radiosensitive cell line with a hypomorphic LIG4 mutation that severely compromises its activity. The results suggest that complete coverage for D-NHEJ or B-NHEJ is afforded by very low ligase levels and demonstrate residual end joining by DNA ligase IV in cells of patients with mutations in LIG4.     

Rapid assessment of two major repair activities against DNA double-strand breaks in vertebrate cells

A linearized plasmid DNA, in which tandem repeats of 400bp flank the breakpoints, was transfected into vertebrate cells, and breakpoint junctions of plasmid DNA circularized in the cells were analyzed to assess the repair activities against DNA double-strand break (DSB) by non-homologous end joining and homology-directed repair (i.e., homologous recombinational repair and single-strand annealing). The circularization by non-homologous end joining repair of the breakpoints depended on the expression of DNA-PKcs, while that by homology-directed repair through the repeats depended on the length of the repeats, indicating that these two DSB repair activities can be rapidly assessed by this assay. Predominance in circularization by either non-homologous end joining or homology-directed repair differed among cells examined, and circularization was exclusively undertaken by homology-directed repair in DT40 cells known to show a high homologous recombination rate against gene-targeting vectors. Thus, this assay will be helpful in studies on mechanisms and inter-cellular variations of DSB repair.     

scid cells efficiently integrate hairpin and linear DNA substrates.

The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination.     

DNA interstrand crosslinks repair in mammalian cells

We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression. It was accompanied by gamma-H2AX foci formation suggesting that DSBs were formed during the process. However, the same amount of gamma-H2AX foci was observed when cells were transfected with native plasmid, which indicated that gamma-H2AX foci appearance could not be used to determine the amount of DSBs connected with the ICL repair in this system. For this reason we further monitored the DSB formation by determining the amount of linearized plasmids, since having one crosslink per plasmid on average, any ICL-driven DSB formation would lead to plasmid linearization. Native and crosslinked plasmids were incubated in repair-competent cell-free extracts from G1 and S phase HeLa cells. Although a considerable part of the ICLs was repaired, no linearization of the plasmids was observed in the extracts, which was interpreted that DSBs were not formed as intermediates in the process of ICL repair. In another set of experiments HR-proficient HeLa and HR-deficient irs3 cells were transfected with native and crosslinked plasmids, and 6 h and 12 h later the plasmid DNA was isolated and analyzed by electrophoresis. The same amount of linear plasmid molecules was observed in both cell lines, regardless of whether they were transfected with native or crosslinked pEGFP-N1, which further confirmed that DSB formation was not an obligatory step in the process of ICL repair by HR.     

Prognostic values of filamin-A status for topoisomerase II poison chemotherapy

Filamin-A, also called Actin Binding Protein-280, is not only an essential component of the cytoskeleton networks, but also serves as the scaffold in various signaling networks. It has been shown that filamin-A facilitates DNA repair and filamin-A proficient cells are more resistant to ionizing radiation, bleomycin, and cisplatin. In this study, we assessed the role of filamin-A in modulating cancer cell sensitivity to Topo II poisons, including etoposide and doxorubicin. Intriguingly, we found that cells with filamin-A expression are more sensitive to Topo II poisons than those with defective filamin-A, and filamin-A proficient xenograft melanomas have better response to etoposide treatment than the filamin-A deficient tumors. This is associated with more potent induction of DNA double strand breaks (DSBs) by Topo II poisons in filamin-A proficient cells than the deficient cells. Although the expression of filamin-A enables cells a slightly stronger capability to repair DSB, the net outcome is that filamin-A proficient cells bear more DSBs due to the significantly enhanced DSB induction by Topo II poisons in these cells. We further found that filamin-A proficient cells have increased drug influx and decreased drug efflux, suggesting that filamin-A modulates the intra-cellular drug kinetics of Topo II poisons to facilitate the generation of DSB after Topo II poison exposure. These data suggest a novel function of filamin-A in regulating the pharmacokinetics of Topo II poisons, and that the status of filamin-A may be used as a prognostic marker for Topo II poisons based cancer treatments.     

Mechanisms of induction and repair of DNA double-strand breaks by ionizing radiation: Some contradictions

The various aspects of formation and repair of radiation-induced double-strand breaks (DSB) are summarized. Concerning the structure of DSB found in irradiated cells, enzymatic and microdosimetric analysis hints at complex damage of the DNA structure at the position of a DSB. With increasing LET, the DSB damage may be more complex than that induced by low-LET irradiation. Most of the DSB are repaired in the irradiated cell; apparently the kinetics of DSB repair and the fraction of unrejoined DSB determine cell survival or cell death. We do not know the details of the complex machinery of DSB repair; certaintly recombination processes are involved, but there are still contradictions between our current knowledge about the mechanisms of recombinational DSB repair and the observed kinetics.Dedicated to Prof. W. Jacobi on the occasion of his 65th birthday     

Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.     

Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells

Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 ± 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 ± 3.5% for MGH-U1; 39.9 ± 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.