Double-strand Break Repair Factor Research Articles

Boric Acid Affects the Expression of DNA Double-Strand Break Repair Factors in A549 Cells and A549 Cancer Stem Cells: An In Vitro Study

DNA double-strand break (DSB) repair genes interact with tumor stemness- and resistance-associated processes in cancer stem cells (CSCs). Therefore, targeting DNA DSB genes in cancer treatment is important for the CSC phenotype. Although the anti-cancer effect of boric acid (BA) has been studied, its effect on DNA DSB is unclear. Moreover, no studies investigate BA’s effects on DNA DSB of lung cancer stem cells (LC-SCs). To fill the gap, we aimed to assess the effects of BA on A549 cancer stem cells. CSCs were isolated from human non-small cell lung cancer cells (A549) and characterized by flow cytometry. Different concentrations of BA (at doses ranging from 1 to 100 mM) were applied to cancer stem cells. Cytotoxic activities were determined using the cell viability assay (MTT assay) at 24 and 48 h. Expression levels of DNA DSB genes that BRCA1, BRCA2, RAD51, KU70/80, ATM, and XRCC4 were evaluated by RT-qPCR. Additionally, immunofluorescence staining analysis was exploited for caspase-3 and E-cadherin. ATM expression increased significantly (p < 0.001). No significant change was observed in the expression of other genes. Moreover, BA up-regulated caspase-3 and E-cadherin expression. Consequently, we can say that BA affects DNA DSB and the apoptotic abilities of LC-SCs.Graphical

Testis electroporation coupled with autophagy inhibitor to treat non-obstructive azoospermia

Infertility affects 10%-20% of the population in most countries, with approximately half of those cases resulting from male infertility. Although millions of infertile men are able to have children with the assistance of reproductive technology, individuals with non-obstructive azoospermia (NOA) syndrome are unable to do so because they lack functional sperm. Therefore, some other strategies for infertile NOA men are still urgently needed. Our current study uses an NOA-like mouse model to optimize microinjection and a subsequent electroporation method to test potential treatment strategies. We showed that the spermatogenetic process could be partially rescued in young Stra8-Rnf20 -/- mice with microinjection and subsequent electroporation of Rnf20 plasmids into the testes. All meiotic prophase I stages could be identified, and programmed DNA double-strand break repair factors could successfully be recruited to Stra8-Rnf20 -/- spermatocytes after electroporation. Moreover, by including an autophagy inhibitor in the treatment, electroporation significantly improved the spermatogenetic rescue efficiency of adult Stra8-Rnf20 -/- mice. Most importantly, infertility caused by Rnf20 depletions could be overcome by electroporation coupled with intracytoplasmic sperm injection. Our studies establish a relative safe and efficient testis electroporation system and provide a promising therapeutic strategy for patients with NOA.

Expression of BRCA1, BRCA2, RAD51, and other DSB repair factors is regulated by CRL4WDR70

Double-strand break (DSB) repair relies on DNA damage response (DDR) factors including BRCA1, BRCA2, and RAD51, which promote homology-directed repair (HDR); 53BP1, which affects single-stranded DNA formation; and proteins that mediate end-joining. Here we show that the CRL4/DDB1/WDR70 complex (CRL4WDR70) controls the expression of DDR factors. Auxin-mediated degradation of WDR70 led to reduced expression of BRCA1, BRCA2, RAD51, and other HDR factors; 53BP1 and its downstream effectors; and other DDR factors. In contrast, cNHEJ factors were generally unaffected. WDR70 loss abrogated the localization of HDR factors to DSBs and elicited hallmarks of genomic instability, although 53BP1/RIF1 foci still formed. Mutation of the DDB1-binding WD40 motif, disruption of DDB1, or inhibition of cullins phenocopied WDR70 loss, consistent with CRL4, DDB1, and WDR70 functioning as a complex. RNA-sequencing revealed that WDR70 degradation affects the mRNA levels of DDR and many other factors. The data indicate that CRL4WDR70 is critical for expression of myriad genes including BRCA1, BRCA2, and RAD51.

DNA damage-induced degradation of Sp1 promotes cellular senescence

Persistent DNA damage (genotoxic stress) triggers signaling cascades that drive cells into apoptosis or senescence to avoid replicating a damaged genome. Sp1 has been found to play a role in double strand break (DSB) repair, and a link between Sp1 and aging has also been established, where Sp1 protein, but not RNA, levels decrease with age. Interestingly, inhibition ATM reverses the age-related degradation of Sp1, suggesting that DNA damage signaling is involved in senescence-related degradation of Sp1. Proteasomal degradation of Sp1 in senescent cells is mediated via sumoylation, where sumoylation of Sp1 on lysine 16 is increased in senescent cells. Taking into consideration our previous findings that Sp1 is phosphorylated by ATM in response to DNA damage and that proteasomal degradation of Sp1 at DSBs is also mediated by its sumoylation and subsequent interaction with RNF4, we investigated the potential contribution of Sp1’s role as a DSB repair factor in mediating cellular senescence. We report here that Sp1 expression is decreased with a concomitant increase in senescence markers in response to DNA damage. Mutation of Sp1 at serine 101 to create an ATM phospho-null mutant, or mutation of lysine 16 to create a sumo-null mutant, prevents the sumoylation and subsequent proteasomal degradation of Sp1 and results in a decrease in senescence. Conversely, depletion of Sp1 or mutation of Sp1 to create an ATM phosphomimetic results in premature degradation of Sp1 and an increase in senescence markers. These data link a loss of genomic stability with senescence through the action of a DNA damage repair factor.

Elevated Expression of the RAGE Variant-V in SCLC Mitigates the Effect of Chemotherapeutic Drugs.

Simple SummaryRadiomimetic drugs induce extensive genotoxic insults to their target cells. Irreparable DNA damage leaves cells with the choice between a program leading to cell death or senescence, but not DNA repair. Among the challenges of an advanced stage of small cell lung carcinoma (SCLC), the resistance to radiomimetic drugs is the most prominent one. In SCLC, the initial chemotherapeutic treatment primes cell to modify their DNA repair and cell cycle regulatory systems, using alternative but highly efficient forms of DNA repair and auxiliary factors. This modulated system now bypasses several regulatory controls. Thus, at this stage, cells become resistant to any beneficial effects of chemotherapeutic drugs. In the present study, we observed that variant-V of the receptor for advanced glycation end-products (RAGE) is abundantly expressed in advancing and metastasizing SCLC. Therefore, it may serve as a potential target for specific therapeutic interventions directed to SCLC.Small cell lung carcinoma (SCLC) is a highly aggressive malignancy with a very high mortality rate. A prominent part of this is because these carcinomas are refractory to chemotherapies, such as etoposide or cisplatin, making effective treatment almost impossible. Here, we report that elevated expression of the RAGE variant-V in SCLC promotes homology-directed DNA DSBs repair when challenged with anti-cancer drugs. This variant exclusively localizes to the nucleus, interacts with members of the double-strand break (DSB) repair machinery and thus promotes the recruitment of DSBs repair factors at the site of damage. Increased expression of this variant thus, promotes timely DNA repair. Congruently, the tumor cells expressing high levels of variant-V can tolerate chemotherapeutic drug treatment better than the RAGE depleted cells. Our findings reveal a yet undisclosed role of the RAGE variant-V in the homology-directed DNA repair. This variant thus can be a potential target to be considered for future therapeutic approaches in advanced SSLC.

Ku80-Targeted pH-Sensitive Peptide-PNA Conjugates Are Tumor Selective and Sensitize Cancer Cells to Ionizing Radiation.

The development of therapeutic agents that specifically target cancer cells while sparing healthy tissue could be used to enhance the efficacy of cancer therapy without increasing its toxicity. Specific targeting of cancer cells can be achieved through the use of pH-low insertion peptides (pHLIP), which take advantage of the acidity of the tumor microenvironment to deliver cargoes selectively to tumor cells. We developed a pHLIP-peptide nucleic acid (PNA) conjugate as an antisense reagent to reduce expression of the otherwise undruggable DNA double-strand break repair factor, KU80, and thereby radiosensitize tumor cells. Increased antisense activity of the pHLIP-PNA conjugate was achieved by partial mini-PEG sidechain substitution of the PNA at the gamma position, designated pHLIP-αKu80(γ). We evaluated selective effects of pHLIP-αKu80(γ) in cancer cells in acidic culture conditions as well as in two subcutaneous mouse tumor models. Fluorescently labeled pHLIP-αKu80(γ) delivers specifically to acidic cancer cells and accumulates preferentially in tumors when injected i.v. in mice. Furthermore, pHLIP-αKu80(γ) selectively reduced KU80 expression in cells under acidic conditions and in tumors in vivo. When pHLIP-αKu80(γ) was administered to mice prior to local tumor irradiation, tumor growth was substantially reduced compared with radiation treatment alone. Furthermore, there was no evidence of acute toxicity associated with pHLIP-αKu80(γ) administration to the mice. These results establish pHLIP-αKu80(γ) as a tumor-selective radiosensitizing agent. IMPLICATIONS: This study describes a novel agent, pHLIP-αKu80(γ), which combines PNA antisense and pHLIP technologies to selectively reduce the expression of the DNA repair factor KU80 in tumors and confer tumor-selective radiosensitization.

Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination.

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a ‘cut-and-close’ mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/β domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.

Ginsenoside Rg1 impairs homologous recombination repair by targeting CtBP-interacting protein and sensitizes hepatoblastoma cells to DNA damage.

The ginsenoside Rg1, the primary pharmacologically active ingredient of the traditional Chinese herb ginseng, is widely used in the clinical treatment of diseases of the immune and nervous systems. Recent studies have shown that it also has an antitumor effect. In this study, we explored the effects of Rg1 on hepatoblastoma (HB) and its underlying mechanisms. We demonstrated that Rg1 significantly inhibited HB cell growth both in vivo and in vitro. Mechanistic studies revealed that Rg1 impaired homologous recombination and triggered double-strand breaks in HB cells by directly targeting CtBP-interacting protein (CtIP), a key double-strand break repair factor, which is highly expressed in HB tissues. Moreover, we also demonstrated that Rg1 sensitized HB cells to DNA-damaging agents both in vitro and in vivo. In conclusion, our data not only demonstrate the potential clinical application of Rg1 as a novel chemotherapeutic candidate but also offer a mechanism-based therapeutic option by which DNA-damaging agents can be used in combination with Rg1 to target HB.

Saccharomyces cerevisiae Mhr1 can bind Xho I-induced mitochondrial DNA double-strand breaks in vivo

Mitochondrial DNA (mtDNA) double-strand break (DSB) repair is essential for maintaining mtDNA integrity, but little is known about the proteins involved in mtDNA DSB repair. Here, we utilize Saccharomyces cerevisiae as a eukaryotic model to identify proteins involved in mtDNA DSB repair. We show that Mhr1, a protein known to possess homologous DNA pairing activity in vitro, binds to mtDNA DSBs in vivo, indicating its involvement in mtDNA DSB repair. Our data also indicate that Yku80, a protein previously implicated in mtDNA DSB repair, does not compete with Mhr1 for binding to mtDNA DSBs. In fact, C-terminally tagged Yku80 could not be detected in yeast mitochondrial extracts. Therefore, we conclude that Mhr1, but not Yku80, is a potential mtDNA DSB repair factor in yeast.

H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation.

Meiotic recombination is essential for fertility in most sexually reproducing species, but the molecular mechanisms underlying this process remain poorly understood in mammals. Here, we show that RNF20-mediated H2B ubiquitination is required for meiotic recombination. A germ cell-specific knockout of the H2B ubiquitination E3 ligase RNF20 results in complete male infertility. The Stra8-Rnf20−/− spermatocytes arrest at the pachytene stage because of impaired programmed double-strand break (DSB) repair. Further investigations reveal that the depletion of RNF20 in the germ cells affects chromatin relaxation, thus preventing programmed DSB repair factors from being recruited to proper positions on the chromatin. The gametogenetic defects of the H2B ubiquitination deficient cells could be partially rescued by forced chromatin relaxation. Taken together, our results demonstrate that RNF20/Bre1p-mediated H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation, and suggest an old drug may provide a new way to treat some oligo- or azoospermia patients with chromatin relaxation disorders.

Loss of p53-mediated cell-cycle arrest, senescence and apoptosis promotes genomic instability and premature aging

Although p53-mediated cell cycle arrest, senescence and apoptosis are well accepted as major tumor suppression mechanisms, the loss of these functions does not directly lead to tumorigenesis, suggesting that the precise roles of these canonical activities of p53 need to be redefined. Here, we report that the cells derived from the mutant mice expressing p533KR, an acetylation-defective mutant that fails to induce cell-cycle arrest, senescence and apoptosis, exhibit high levels of aneuploidy upon DNA damage. Moreover, the embryonic lethality caused by the deficiency of XRCC4, a key DNA double strand break repair factor, can be fully rescued in the p533KR/3KR background. Notably, despite high levels of genomic instability, p533KR/3KRXRCC4−/− mice, unlike p53−/− XRCC4−/− mice, are not succumbed to pro-B-cell lymphomas. Nevertheless, p533KR/3KR XRCC4−/− mice display aging-like phenotypes including testicular atrophy, kyphosis, and premature death. Further analyses demonstrate that SLC7A11 is downregulated and that p53-mediated ferroptosis is significantly induced in spleens and testis of p533KR/3KRXRCC4−/− mice. These results demonstrate that the direct role of p53-mediated cell cycle arrest, senescence and apoptosis is to control genomic stability in vivo. Our study not only validates the importance of ferroptosis in p53-mediated tumor suppression in vivo but also reveals that the combination of genomic instability and activation of ferroptosis may promote aging-associated phenotypes.

Molecular biology: Unequal opportunity during class switching.

The DNA breakage-and-repair mechanism that generates antibodies of different classes has, in theory, a 50% chance of occurring correctly. But this recombination turns out to be heavily biased towards productive events. See Letter p.134 The process of antibody generation requires rearrangements in the immunoglobulin heavy chain (IgH) locus to juxtapose single V, D and J gene segments, by excising all the remaining segments. In principle, the process making these deletions could also result in inversions. Frederick Alt and colleagues now use high-throughput genome-wide sequencing to examine the long-standing question of why the process at this locus has a directional bias towards deletion rather than inversion. They find that it involves sequences within the IgH locus itself, double-strand DNA breaks initiated by the AID deaminase, and double-strand break repair factors 53BP1 and ATM.

Abstract 3027: Investigating the function of NONO, a novel double strand break repair factor, and exploring its potential role as a biomarker for melanoma

Abstract We investigated the in vivo function of NONO, a protein that has been shown to promote a distinct sub-pathway of nonhomologous end joining (NHEJ) repair in vitro and in cultured cells. We used a gene trap strategy to create a null mutant for Nono, the mouse homolog of the human NONO gene. We investigated hematopoietic stem cells (HSCs), which are known to be sensitive to deficiencies in other DNA repair proteins. Nono-deficient mice showed reduced bone marrow and spleen cellularity. HSCs from Nono-deficient mice showed severe impairment in competitive repopulation assays in primary and secondary recipients. HSCs from Nono-deficient mice also displayed significantly higher levels of reactive oxygen species, bromodeoxyuridine incorporation, and Ki-67 positivity, consistent with hyperproliferation in response to chronic genotoxic stress. To investigate DNA repair competence more directly, we exposed HSCs ex vivo to the DNA crosslinking agent, mitomycin C (MMC), or to ionizing radiation. HSCs from Nono-deficient and wild type mice showed increased sensitivity to both of these agents in colony forming cell assays, that NONO protein is induced after X-ray treatment, and that Nono-deficient HSCs show delayed resolution of γ-H2AX foci and increased apoptosis. Separately, we evaluated changes in the testes, another organ that is frequently affected by DNA repair gene mutations. Testes of Nono-deficient mice showed growth retardation, which became apparent between 18 and 26 days of postnatal development, as well as reduced sperm count. X-irradiation of Nono-deficient mice at 4 Gy led to increased apoptosis in the seminiferous tubules after 24 h, as determined in a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. There was also nearly complete depletion of germ cells at 48 h. Significantly less effects were seen at the same time points in wild type control mice. Similar to the results in HSCs, NONO was up regulated in the testes following X-ray exposure. Taken together, results are consistent with an in vivo defect in DNA repair. Notably, the co-occurrence of hematopoietic and germ cell phenotypes is reminiscent of other DNA repair gene mutations, including Fanconi anemia. Although DNA repair contributes to genomic stability in normal tissues, heightened repair activity can be a cause of treatment resistance in cancer. In this respect, it is interesting that NONO has been reported to be highly expressed in melanoma specimens and cell lines. We confirmed that NONO is expressed in melanoma cell lines and showed that it is additionally upregulated in melanoma cell lines following irradiation. Moreover, siRNA-mediated attenuation of NONO expression led to inhibition of melanoma cell proliferation. We are currently investigating NONO expression patterns of human melanoma specimens (stage I to IV) in order to explore NONO potential role as a useful prognostic biomarker for melanoma. Citation Format: Shuyi Li, Fengjue Shu, Mohammad K. Khan, Brian P. Pollack, Zhentian Li, Morgan McLemore, William S. Dynan. Investigating the function of NONO, a novel double strand break repair factor, and exploring its potential role as a biomarker for melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3027. doi:10.1158/1538-7445.AM2015-3027

Deletion of Nono, a Novel Double-Strand Break Repair Factor, Leads to Hematopoietic Stem Cell Defects

Three genes, SFPQ, NONO, and PSPC1 encode for a small family of tandem RRM domain-containing proteins with diverse functions in RNA processing, transcription, and DNA repair. Our previous work has shown that an SFPQ•NONO complex promotes a distinct sub-pathway of non-homologous end joining (NHEJ) in vitro, implicating it as a novel double-strand break (DSB) repair factor. Consistently, attenuation of NONO function in human fibroblasts leads to partial radiosensitivity, delayed resolution of DNA repair foci, and an increase in radiation-dependent chromosome aberrations. To investigate the in vivo function of this complex, we knocked out Nono, the mouse homolog of the human NONO gene. We hypothesized that a DSB repair deficiency would lead to stem cell exhaustion, sensitivity to DNA damaging agents, or both. First, we investigated hematopoietic stem cells (HSCs), which are known to be sensitive to impairment of DNA repair. Nono-deficient (gt/0) mice showed reduced bone marrow and spleen cellularity, potentially due to reduced body size. The proportion of HSCs (LSK-SLAM) in relation to total bone marrow cells was similar in Nono-deficient and wild type (WT) mice, and bone marrow cell cultures yielded similar numbers and types of colonies. However, HSCs from Nono-deficient mice showed severe impairment in competitive repopulation assays in primary and secondary recipients. HSCs from gt/0 mice also displayed significantly higher levels of ROS and proliferation (accessed via BrdU incorporation), as well as higher percentage of Ki-67 positivity when compared to HSCs from WT mice. Together, these results indicate that Nono-deficient HSCs exhibit impaired capacity for self-renewal that may be caused by excessive HSC proliferation, a primary defect in response to genotoxic stress or a combination of both. To further elucidate this phenomenon, we investigated potential differences in sensitivity to the DNA crosslinking agent mitomycin C (MMC) and radiation. As previously observed in other genomic instability models (e.g., Fanconi anemia), colony-forming-cells from Nono-deficient mice were highly sensitive to MMC and X-rays (2 Gγ). Utilizing FACS-sorted HSCs, we determined that NONO protein levels are induced after X-ray treatment, and that Nono-deficient HSCs show delayed resolution of γ-H2AX foci and increased apoptosis. We also evaluated changes in the testis, another organ frequently affected by DNA repair gene mutations. Accordingly, testes of Nono-deficient mice showed growth retardation that became apparent between 18 and 26 days of postnatal development. The co-occurrence of hematopoietic and germ cell defects is reminiscent of other DNA repair gene defects, including those seen in Fanconi anemia. Collectively, our data identify NONO as a novel and important regulator of both HSC maintenance and germ cell function. Its potential effect in other organ systems and mechanism of action are under investigation. DisclosuresNo relevant conflicts of interest to declare.

Nitric Oxide Induces Ataxia Telangiectasia Mutated (ATM) Protein-dependent γH2AX Protein Formation in Pancreatic β Cells

In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylation (γH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells.

Therapeutic and space radiation exposure of mouse brain causes impaired DNA repair response and premature senescence by chronic oxidant production

Despite recent epidemiological evidences linking radiation exposure and a number of human ailments including cancer, mechanistic understanding of how radiation inflicts long-term changes in cerebral cortex, which regulates important neuronal functions, remains obscure. The current study dissects molecular events relevant to pathology in cerebral cortex of 6 to 8 weeks old female C57BL/6J mice two and twelve months after exposure to a γ radiation dose (2 Gy) commonly employed in fractionated radiotherapy. For a comparative study, effects of 1.6 Gy heavy ion 56Fe radiation on cerebral cortex were also investigated, which has implications for space exploration. Radiation exposure was associated with increased chronic oxidative stress, oxidative DNA damage, lipid peroxidation, and apoptosis. These results when considered with decreased cortical thickness, activation of cell-cycle arrest pathway, and inhibition of DNA double strand break repair factors led us to conclude to our knowledge for the first time that radiation caused aging-like pathology in cerebral cortical cells and changes after heavy ion radiation were more pronounced than γ radiation.

A requirement for polymerized actin in DNA double-strand break repair

Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

Tip60-ing the balance in DSB repair

The tumour suppressor Tip60 is a histone acetyltransferase implicated in transcriptional control and DNA double-strand break repair. Tip60 binds to the heterochromatic histone mark H3K9me3, triggering acetylation and activation of DNA double-strand break repair factors.

Deficiencies of Double-Strand Break Repair Factors and Effects on Mutagenesis in Directly γ-Irradiated and Medium-Mediated Bystander Human Lymphoblastoid Cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells.

Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.