DNA Double-strand Break Repair In Cells Research Articles

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro-nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP-23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and γ-IR. CP-8 and CP-23 effectively inhibited γ-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23's efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors.

DNA End Resection: Mechanism and Control.

DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genome integrity and cell viability. Typically, cells repair DSBs by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The relative use of these two pathways depends on many factors, including cell cycle stage and the nature of the DNA ends. A critical determinant of repair pathway selection is the initiation of 5'→3' nucleolytic degradation of DNA ends, a process referred to as DNA end resection. End resection is essential to create single-stranded DNA overhangs, which serve as the substrate for the Rad51 recombinase to initiate HR and are refractory to NHEJ repair. Here, we review recent insights into the mechanisms of end resection, how it is regulated, and the pathological consequences of its dysregulation.

Mapping the genetic landscape of DNA double-strand break repair

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.

Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics.

Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms – MENTHU, inDelphi, and Lindel – in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB repair events. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. In contrast, only Lindel correctly predicted predominant single-base insertions. We report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations in this large dataset. We then estimated the frequency of Cas9-targetable homogeneous frameshift-inducing DSBs in vertebrate coding regions for gene discovery using MENdel. 47 out of 54 genes (87%) contained at least one early frameshift-inducing DSB and 49 out of 54 (91%) did so when also considering Cas12a-mediated deletions. We suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

DNA double-strand breaks in telophase lead to coalescence between segregated sister chromatid loci

DNA double strand breaks (DSBs) pose a high risk for genome integrity. Cells repair DSBs through homologous recombination (HR) when a sister chromatid is available. HR is upregulated by the cycling dependent kinase (CDK) despite the paradox of telophase, where CDK is high but a sister chromatid is not nearby. Here we study in the budding yeast the response to DSBs in telophase, and find they activate the DNA damage checkpoint (DDC), leading to a telophase-to-G1 delay. Outstandingly, we observe a partial reversion of sister chromatid segregation, which includes approximation of segregated material, de novo formation of anaphase bridges, and coalescence between sister loci. We finally show that DSBs promote a massive change in the dynamics of telophase microtubules (MTs), together with dephosphorylation and relocalization of kinesin-5 Cin8. We propose that chromosome segregation is not irreversible and that DSB repair using the sister chromatid is possible in telophase.

P37 Induction of DNA Double Strand Breaks in Human Primary Lung Cells

DNA double strand breaks (DSB) are the most detrimental effects on cells by which ionizing radiation (IR) causes cancer. The frequency of DSB induced decreases with time, indicative of DSB repair. One of the earliest responses to DSB damage is the recruitment of the repair protein, 53BP1, to DSB sites. Fluorescent-tagged antibodies specific for 53BP1 can therefore be exploited to detect DSB lesions as discrete ‘fluorescent foci’. This project aimed to assess the frequency of induction and kinetics of DNA DSB repair in cells exposed to IR. Primary human bronchial epithelial cells were cultured, irradiated and fixed at different time-points. Radiation induced DNA DSB were identified by immunofluorescence technique using 53BP1 as a marker protein. Images were captured and analyzed by fluorescence microscope and axiovision software. After exposure to 2Gy radiation, the highest number of foci was visible within 10min (6.613foci/nucleus). The score at 10min in unirradiated and IR cells were significantly different (P=0.00001). After then the trend declined. Average number of 53BP1 foci decreased up to 6hr (2.83 foci/nucleus at 6hr). The score at 10min was statistically different from that of 6hr (P=0.0008). Data stayed the same from 6hr to 16hr (P at 6hr=0.552, at 8hr=0.615 and at 16hr=0.768). After 16hr the foci reduced up to 24hr where it returned to nearly baseline but significant variation remained from that of unirradiated cells at 24hr-point (P=0.001). In case of unirradiated cells, difference was not observed over the whole duration (P>0.05). Initially, after irradiation, a wide variation existed among the proportion of small, medium and large foci which was, however, getting closer with time, and by 24hr it appeared like a cluster of proportion where all fractions of foci remained the same. In unirradiated cells, an average of 1 DSB was seen in each nucleus for a 24hr duration whereas radiation of a dose of 2Gy produced as many as 6 DSB within 10min. DSB were quickly repaired for the first 6hr, and more than half of DSB got repaired within this period. Few DNA lesions persisted even after 24hr. Changes in the proportional size distribution of foci may be indicative of migration and clustering of radiation-induced foci representing slow DSB repair, leading to chance of chromosomal rearrangement, and subsequently increase the risk of cancer cell formation. If this hypothesis is valid and we can locate these clustering time-points specifically, specific time-points of transforming cancer cells after irradiation may be identified.

Abstract 1353: Role of TLK1B/NEK1 axis in DNA DSB repair: Implications for prostate cancer progression

Abstract Defects in DNA Damage Response and Repair are linked to the majority of cancers, including Prostate Cancer (PCa). Common genotoxic stresses that cause damage to the DNA, or replication fork collapse are generated when PCa cells face Androgen Deprivation Therapy (ADT), and must be bypassed to allow continuous replication and division. Tousled like kinase (TLK1) splice variant TLK1B is implicated in DNA damage repair pathways and is translationally increased following various stresses, including the DDR. We found that the expression of TLK1B is rapidly increased following a shift of LNCaP cells to charcoal-stripped serum (ADT), via an activation of mTOR and phosphorylation of 4EBP1 (inhibitor of the translation factor eIF4E). We recently uncovered the existence of the important DDR axis TLK1B> NEK1> ATR>Chk1.(1). TLK1 phosphorylates NEK1 at T141 and activates its autophosphorylation of Y315. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks (DSB) after low dose ionizing radiation (IR).(2). Cells repair DSB either via HR or NHEJ. We investigated whether TLK1 phosphorylation is essential for NEK1 activity in HR repair. siRNA-mediated NEK1 deficient cells have decreased kinetics of Rad54-S572 phosphorylation and persistent Rad51 foci when induced with Doxorubicin that creates DSB.(3). We tested a NEK1-T141A overexpressing (Hek293) mutant for its effect on Rad54-S572 phosphorylation (pRad54) during a time course of recovery from Doxorubicin. There was no significant delay in pRad54 kinetics compared to the control, unless endogenous (WT) NEK1 was knocked down, in which case we observed a significant delay in pRad54 in NEK1 mutant cells. When DSBs occur during DNA damage, Rad51 becomes localized at DSB repair foci. Delay in Rad54 phosphorylation results in persistent Rad51 foci since aspects of HR subsequent to Rad51 filament formation and strand invasion are impaired. We observed increased Rad51 foci that persisted in NEK1-T141A mutants following two hours of Doxorubicin treatment and 14 hours of recovery. A quantitative approach to monitor HR in NEK1-T141A mutants was obtained with the generation of a stable DR-GFP recombination reporter substrate (4) in HSG cell line. Transfection with the megabase cutter endonuclease I-SceI, creates a DSB in a defective double GFP cassette which would generate a functional GFP gene conversion product. Results indicate that approximately 14-17 % cells from parent population undergoes HR in WT, NEK1 overexpressing, and NEK1-T141A mutants. We would expect statistically significant conclusion from improvising GFP reporter experiments with cells in which endogenous Nek1 and Rad54 is knocked down, consistent with the results obtained with pRad54. Another approach would be inhibiting TLK1B interaction with NEK1 to block HR repair which is thought to be critical in Prostate Cancer progression to Castration Resistant Prostate Cancer. Citation Format: Ishita Ghosh, Arrigo DeBenedetti, Gulshan Sunavala. Role of TLK1B/NEK1 axis in DNA DSB repair: Implications for prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1353.

Assays to Study Repair of Inducible DNA Double-Strand Breaks at Telomeres.

The ends of linear chromosomes are constituted of repetitive DNA sequences called telomeres. Telomeres, nearby regions called subtelomeres, and their associated factors prevent chromosome erosion over cycles of DNA replication and prevent chromosome ends from being recognized as DNA double-strand breaks (DSBs). This raises the question of how cells repair DSBs that actually occur near chromosome ends. One approach is to edit the genome and engineer cells harboring inducible DSB sites within the subtelomeric region of different chromosome ends. This provides a reductionist and tractable genetic model system in which mechanisms mediating repair can be dissected via genetics, molecular biology, and microscopy tools.

Downregulation of eukaryotic initiation factor 4A1 improves radiosensitivity by delaying DNA double strand break repair in cervical cancer.

Expression of eukaryotic initiation factor 4A1 (eIF4A1) following brachytherapy has been reported to predict improved radiosensitivity and tumor-specific survival in cervical cancer. Therefore, the present study investigated the function of eIF4A1 in cervical cancer and the mechanism by which eIF4A1 regulates cervical cancer radiosensitivity. It was determined that the downregulation of eIF4A1 in HeLa and SiHa cells notably attenuated cell proliferation, in addition to repressing cervical cancer migration and invasion, and promoting cell apoptosis. In vitro and in vivo studies have demonstrated that silencing eIF4A1 improves cervical cancer radiosensitivity. Detection of γ-H2AX using western blot analysis at 0, 0.5, 1, 6 and 24 h following the exposure of cervical cancer cells to X-rays illustrated that eIF4A1-knockdown results in postponed radiation-induced DNA double strand break (DSB) repair. Overall, the results of the present study demonstrated that downregulated eIF4A1 improves cervical cancer radiosensitivity by delaying cancer cell DSB repair. In conclusion, the data indicated that eIF4A1 performs a vital role in cervical cancer progression and radiosensitivity. Therefore, eIF4A1 may be a potential therapeutic target in patients with cervical cancer.

CRISPR/Cas9-mediated targeted mutagenesis in grape.

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

Gamma-irradiated quiescent cells repair directly induced double-strand breaks but accumulate persistent double-strand breaks during subsequent DNA replication.

H2AX is expressed at very low levels in quiescent normal cells invivo and invitro. Such cells repair DNA double-strand breaks (DSBs) induced by γ-irradiation through a transient stabilization of H2AX. However, the resultant cells accumulate small numbers of irreparable (or persistent) DSBs via an unknown mechanism. We found that quiescent cells that had repaired DSBs directly induced by γ-rays were prone to accumulate DSBs during the subsequent DNA replication. Unlike directly induced DSBs, secondary DSBs were not efficiently repaired, although Rad51 and 53BP1 were recruited to these sites. H2AX was dramatically stabilized in response to DSBs directly caused by γ-rays, enabling γH2AX foci formation and DSB repair, whereas H2AX was barely stabilized in response to secondary DSBs, in which γH2AX foci were small and DSBs were not efficiently repaired. Our results show a pathway that leads to the persistent DSB formation after γ-irradiation.

Homologous recombination pathway may play a major role in high-LET radiation-induced DNA double-strand break repair

Purpose: Particle beams are increasingly applied to various cancer treatments because of their excellent dose localization to tumors while preserving surrounding normal tissues. However, characteristic of DNA damages induced by particle beams and their repair mechanisms are not fully understood. It is known that the majority of DNA double-strand breaks (DSBs) induced by ionizing radiation are repaired either by non-homologous end-joining (NHEJ) or by homologous recombination (HR) pathways. However, it has not been clarified how NHEJ and HR pathways contribute to the repair of DSBs induced by various particle beams [1, 2]. Thus, the purpose of this study is to clarify how these repair pathways contribute to the DSB repair in cells after irradiation with various radiation qualities.Material and methods: A control Chinese hamster ovary (CHO) cell line (AA8), its mutant cell line deficient of DNA-PKcs (V3), XRCC4 (XR1) and Chinese hamster lung fibroblast cell line deficient of XRCC2 (IRS1) were exposed to gamma rays, protons, carbon ions and iron ions. V3 and XR1 lack NHEJ pathway, while IRS1 lacks HR pathway. After each irradiation, colony survival and gross-chromosome aberration were examined.Results: It was demonstrated that colony survival was clearly dependent on the presence of NHEJ or HR pathways as well as radiation qualities. Although HR-deficient cells (IRS1) became more sensitive as LET value increased, NHEJ-deficient cells (V3 and XR1) did not further sensitized as LET value increased (Fig. 1). In addition, values of relative biological effectiveness of iron beams were higher in HR-deficient cells than in NHEJ-deficient cells (3.2 in AA8; 2.7 in IRS1; 1.8 in XR1 and V3). These may suggest that HR plays an important role in repairing DNA lesions induced by high-LET radiation. As for the incidence of total chromosomal aberration, we found that its incidence increased as LET values increased in wild-type (AA8) and NHEJ-deficient cells (V3, XR1), but not in HR-deficient cells (IRS1) (Table 1). However, occurrence of chromosome-type aberrations increased as LET values increased in all cell lines analysed here. This may indicate that the chromosomal aberrations occur from not only unrepaired damages but also the repair process of error-prone NHEJ pathway, suggesting that limited capacity of NHEJ to repair DSBs induced by high-LET irradiation may cause increased number of chromosome-type aberrations.Conclusions: Taken together, although NHEJ pathway is the major pathway to repair DSBs induced by various types of radiation, HR pathway may play more important roles as LET value increases. Fig. 1.Radio sensitivity after gamma ray and iron beam. Clonogenic survival curves of AA8 (closed circle); XR1 (open circle); V3 (closed square) and IRS1 (open square) after irradiation with gamma ray (dashed) and iron beam (dotted). NHEJ-deficient cells are more sensitive to gamma ray, but HR-deficient cells are most sensitive to iron beam. Table 1.Chromatid and chromosome type aberration per chromosomeCell linesTypeControlProtonCarbonIronAA8 (wild-type)Chromatid0.860.240.611.07Chromosome0.051.180.743.44XR1 (NHEJ)Chromatid0.053.181.553.41Chromosome02.592.504.96V3 (NHEJ)Chromatid0.483.372.417.89Chromosome0.165.264.978.01IRS1 (HR)Chromatid0.028.2610.357.59Chromosome0.014.247.438.18

NBN Phosphorylation regulates the accumulation of MRN and ATM at sites of DNA double-strand breaks

In response to ionizing radiation, the MRE11/RAD50/NBN (MRN) complex re-distributes to the sites of DNA double strand breaks (DSBs) where each of its individual components is phosphorylated by the serine-threonine kinase, ATM. ATM phosphorylation of NBN is required for activation of the S-phase checkpoint, but the mechanism whereby these phosphorylation events signal the checkpoint machinery remains unexplained. Here, we describe the use of direct protein transduction of the homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of the endonuclease in live cells, facilitating comparisons across multiple cell lines. We demonstrate the utility of this system by introducing I-PpoI into isogenic cell lines carrying mutations at the ATM phosphorylation sites in NBN and assaying the effects of these mutations on the spatial distribution and temporal accumulation of NBN and ATM at DSBs by chromatin immunoprecipitation, as well as timing and extent of DSB repair. Although the spatial distribution of NBN and ATM recruited to the sites of DSBs was comparable between control cells and those expressing phosphorylation mutants of NBN, the timing of accumulation of NBN and ATM was altered. Serine to alanine mutations that blocked phosphorylation resulted in delayed recruitment of both NBN and ATM to DSBs. Serine to glutamic acid substitutions that mimicked the phosphorylation event resulted in both increased and prolonged accumulation of both NBN and ATM at DSBs. The repair of DSBs in cells lacking full-length NBN was significantly delayed compared to control cells while blocking phosphorylation of NBN resulted in a more modest delay in repair. These data indicate that following the induction of DSBs, phosphorylation of NBN regulates its accumulation, and that of ATM, at sites of DNA DSB as well as the timing of the repair of these sites.

Involvement of p54(nrb), a PSF partner protein, in DNA double-strand break repair and radioresistance

Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a γ-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.

Effects of IL-10 and IL-6 Gene Polymorphisms and Atomic-bomb Radiation Exposure on Gastric Cancer Risk

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.

ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin

Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that < or =25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.

Rag mutations reveal robust alternative end joining

Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1-Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, 'alternative NHEJ', appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell's overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.

Analysis of DNA-Dependent Protein Kinase-Mediated DNA End Joining by Two-Photon Fluorescence Cross-Correlation Spectroscopy

Nonhomologous end joining (NHEJ) is the primary mechanism by which mammalian cells repair DNA double-strand breaks (DSBs). Proteins known to play a role in NHEJ include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the Ku 70/Ku 80 heterodimer (Ku), XRCC4, and DNA ligase IV. One of the main roles of the DNA-PKcs-Ku complex is to bring the ends of the DSB together in a process termed synapsis, prior to end joining. Synapsis results in the autophosphorylation of DNA-PKcs, which is required to make the DNA ends available for ligation. Here, we describe a novel assay using two-photon fluorescence cross-correlation spectroscopy that allows for the analysis of DNA synapsis and end joining in solution using purified proteins. We demonstrate that although autophosphorylation-defective DNA-PKcs does not support DNA ligase-mediated DNA end joining, like wild-type (WT) DNA-PKcs, it is capable of Ku-dependent DNA synapsis in solution. Moreover, we show that, in the presence of Ku, both WT DNA-PKcs and autophosphorylation-defective DNA-PKcs promote the formation of multiple, large multi-DNA complexes in solution, suggesting that, rather than align two opposing DNA ends, multiple DNA-PK molecules may serve to bring multiple DNA ends into the NHEJ complex.

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells.

Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination. We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells. A plasmid containing the neo(R) expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells. Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells. In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells. Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neo(R) gene flanked by exon 3 of HPRT: The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells. However, all three enzymes decreased the absolute frequency of homologous integration approximately 2-fold, and the percentage of homologous integration decreased >10-fold. This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search.

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.