Non-homologous End Joining Proteins Research Articles

BMI1 Confers Radioresistance to Normal and Cancerous Neural Stem Cells through Recruitment of the DNA Damage Response Machinery

Glioblastoma multiforme (GBM) is an aggressive brain tumor that is resistant to all known therapies. Within these tumors, a CD133-positive cancer-initiating neural stem cell (NSC) population was shown to be resistant to gamma radiation through preferential activation of the DNA double-strand break (DSB) response machinery, including the ataxia-telangiectasia-mutated (ATM) kinase. The polycomb group protein BMI1 is enriched in CD133-positive GBM cells and required for their self-renewal in an INK4A/ARF-independent manner through transcriptional repression of alternate tumor suppressor pathways. We report here that BMI1 copurifies with DNA DSB response and nonhomologous end joining (NHEJ) repair proteins in GBM cells. BMI1 was enriched at the chromatin after irradiation and colocalized and copurified with ATM and the histone gammaH2AX. BMI1 also preferentially copurified with NHEJ proteins DNA-PK, PARP-1, hnRNP U, and histone H1 in CD133-positive GBM cells. BMI1 deficiency in GBM cells severely impaired DNA DSB response, resulting in increased sensitivity to radiation. In turn, BMI1 overexpression in normal NSCs enhanced ATM recruitment to the chromatin, the rate of gammaH2AX foci resolution, and resistance to radiation. BMI1 thus displays a previously uncharacterized function in controlling DNA DSB response and repair. Pharmacological inhibition of BMI1 combined with radiation therapy may provide an effective mean to target GBM stem cells.

Abstract 688: Acetylation of poly-ADP-ribose polymerase and Ku70 by histone deacetylase inhibitors promote abnormal binding to DNA double strand breaks and decrease repair efficiency in leukemia cells

Abstract Histone Deacetylase inhibitors (HDACi) affect gene expression through acetylation of histone and non-histone proteins which could alter their cellular activity. We have previously shown that HDACi trigger DNA damage responses in leukemia cells, and actual DNA Double Strand Breaks (DSBs) which are significantly increased and persist longer compared with normal cells. The mechanism by which this DNA damage occurs is still unknown. Non Homologous End Joining (NHEJ) is one of the main pathways for the repair of DSBs in mammalian cells. We have recently shown that NHEJ activity is abnormal in myeloid leukemias in that classical NHEJ that is Ku and DNA-PKcs dependent, is down regulated, and compensatory repair of DSBs occurs via alternative (Alt) NHEJ pathways involving Poly-ADP-Ribose Polymerase-1 (PARP-1) and DNA LigaseIIIα. Alt NHEJ is known to generate large deletions and translocations in cancers and leukemias. To determine whether HDACi may decrease the capacity of leukemic cells to repair DSBs by direct acetylation of NHEJ proteins, K562 and HL60 leukemia cell lines were treated with Trichostatin A (TSA, 300nM for 1 and 6 hours) followed by immunoprecipitation and western blot analysis for acetylated NHEJ proteins. TSA treatment of K562 and HL60 show differential acetylation of classical NHEJ protein Ku70 and Alt NHEJ protein PARP-1, compared with controls. To investigate whether TSA treatment alters the binding of Ku70 and PARP-1 to DSBs, we used a chromatin immunoprecipitation (CHIP) assay in K562 cell line stably transfected with DRNeo that can be induced to express a single DSB. Strikingly, CHIP analysis shows that PARP-1 is increased at the DSB after TSA treatment while Ku70 is not. To determine whether DSB repair activity is altered after HDACi treatment, we performed a plasmid based NHEJ repair assay in our leukemia cell lines after treatment with TSA. Both leukemia cell lines demonstrate a significant decrease in the capacity to repair DSBs following HDACi treatment. The same result can be obtained by the use of another HDACi entinostat. These data suggest that HDACi treatment leads to an increased presence of PARP-1 at DSBs, preventing subsequent critical repair steps in leukemia cells. Importantly, AML cells from patients treated with entinostat in vivo show significantly increased co-localization of PARP-1 and γH2A.x, a marker for DSBs, compared with pretreatment controls, confirming our in vitro data in leukemia cell lines. Overall, our results suggest that HDACi result in both a physical and functional alteration of proteins participating in both classical and Alt NHEJ, leading to a loss of binding of Ku70 and an abnormal persistence of PARP-1 at DSBs. Our data supports the idea that acetylated PARP-1 prevents repair of DSBs by Alt NHEJ, leading to a persistence of DSBs in leukemia cells treated with HDACi. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 688.

Pathways for Double-strand Break Repair in Genetically Unstable Z-DNA-forming Sequences

DNA can adopt many structures that differ from the canonical B-form, and several of these non-canonical DNA structures have been implicated in genetic instability associated with human disease. Earlier, we found that Z-DNA causes DNA double-strand breaks (DSBs) in mammalian cells that can result in large-scale deletions and rearrangements. In contrast, the same Z-DNA-forming CG repeat in Escherichia coli resulted in only small contractions or expansions within the repeat. This difference in the Z-DNA-induced mutation spectrum between mammals and bacteria might be due to different mechanisms for DSB repair; in mammalian cells, non-homologous end-joining (NHEJ) is a major DSB repair pathway, while E. coli do not contain this system and typically use homologous recombination (HR) to process DSBs. To test the extent to which the different DSB repair pathways influenced the Z-DNA-induced mutagenesis, we engineered bacterial E. coli strains to express an inducible NHEJ system, to mimic the situation in mammalian cells. Mycobacterium tuberculosis NHEJ proteins Ku and ligase D (LigD) were expressed in E. coli cells in the presence or absence of HR, and the Z-DNA-induced mutations were characterized. We found that the presence of the NHEJ mechanism markedly shifted the mutation spectrum from small deletions/insertions to large-scale deletions (from 2% to 24%). Our results demonstrate that NHEJ plays a role in the generation of Z-DNA-induced large-scale deletions, suggesting that this pathway is associated with DNA structure-induced destabilization of genomes from prokaryotes to eukaryotes.

An end-joining repair mechanism in Escherichia coli

Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.

Histone Deacetylase Inhibitors Promote Abnormal Binding of DNA Repair Proteins to DNA Double Strand Breaks: Consequences for DNA Repair and Genomic Instability?.

Abstract 186Histone Deacetylase inhibitors (HDi) affect gene expression through induction of histone acetylation and lead also to the acetylation of many other proteins which could affect their cellular activity. We have previously shown that HDi trigger in hematopoietic cells not only widespread histone acetylation and DNA damage responses, but actual DNA Double Strand Breaks (DSBs) which are significantly increased and persist for long periods of time compared with normal cells (Gaymes TJ. and al., Mol Cancer Res. 2006). This raises the hypothesis that HDi regulate the capacity of leukemic cells to repair DSBs, and the explanation for the increased and persistent DNA damage in leukemic cells may be that HDi directly acetylates proteins involved in DSB repair, thus decreasing repair activity. Non Homologous End-Joining (NHEJ) is one of the main pathways for the repair of DSBs in mammalian cells. While normal cells use NHEJ that is Ku and DNA-PKcs dependent, an alternative (Alt) NHEJ pathway (DNA-PKcs and Ku independent) involving Poly-ADP-Ribose Polymerase-1 (PARP-1), Werner syndrome helicase (WRN) and DNA LigaseIIIa proteins, has been identified and is responsible for deletions and translocations in cancer. We have recently reported that myeloid leukemia cells repair DSBs using this Alt NHEJ pathway (Sallmyr A. and al., Blood, 2008). Here we show that HDi treatment by Trichostatin A (300nM) results in differential acetylation of main NHEJ protein Ku70 in acute leukemia K562 cell line. In addition, PARP-1, active in several repair pathways, including single strand break repair and Alt NHEJ is also hyperacetylated in K562 cells after 1 and 6 hours of Trichostatin A treatment compared with control treatment. To investigate whether Trichostatin A treatment alters the binding of DNA repair proteins to DSBs, we used a chromatin immunoprecipitation (CHIP) assay in K562 cell line stably transfected with the DRNeo construct that can be induced to express a single DSB. Strikingly, CHIP analysis shows that PARP-1 is increased at the DSB after 1 hour of Trichostatin A treatment, compared with controls. Preliminary CHIP analysis for the protein XRCC1, necessary for the final step of Alt NHEJ repair, shows that it is decreased at the DSB site. Importantly, AML patients treated with the HDi MS-275 in vivo show significantly increased colocalization of PARP-1 and gH2A.x, a marker for DSBs, compared with pretreatment controls, confirming our in vitro data in leukemia cell lines. Altogether, these data suggest that HDi treatment leads to an increased presence of PARP-1 at DSBs, and that this may prevent subsequent critical repair steps, providing a possible explanation for the persistence of DNA damage. Finally, to determine whether DSB repair activity is indeed decreased with HDi treatment, we used an in vivo NHEJ repair assay in K562 and HL60 acute leukemia cell lines before and after treatment with Trichostatin A for 1 hour. Both leukemia cell lines demonstrate a significant decrease in the capacity of the cells to repair DSBs following Trichostatin A treatment. These results suggest that HDi result in both a physical and functional alteration of proteins participating in DNA repair pathways, leading to a decrease in NHEJ activity. The decrease in Alt NHEJ activity may have implications for genomic instability, diminishing abnormal repair following HDi treatment. Disclosures:No relevant conflicts of interest to declare.

Adeno-Associated Virus Site-Specific Integration Is Mediated by Proteins of the Nonhomologous End-Joining Pathway

Adeno-associated virus type 2 (AAV 2) is the only eukaryotic virus capable of site-specific integration; the target site is at chromosome 19q13.4, a site termed AAVS1. The biology of AAV latency has been extensively studied in cell culture, yet the precise mechanism and the required cellular factors are not known. In this study, we assessed the relative frequencies of stable site-specific integration by characterization of cell clones containing integrated AAV vectors. By this assay, two proteins involved in nonhomologous end joining (NHEJ), DNAPKcs and ligase IV, exhibit differential effects on AAV site-specific integration. DNAPKcs is not required; its presence increases the frequency of junction formation indicative of site-specific integration, but seems to reduce the ratio of site-specific integration to random integration (i.e., the latter is even more enhanced). In contrast, site-specific integration is significantly reduced relative to random integration in cells deficient in ligase IV expression. Furthermore, we show that single-stranded AAV vectors are better substrates for site-specific integration than are self-complementary AAV vectors; the absence of DNAPKcs did not affect the targeted integration of these double-stranded AAV vectors. Together, these data suggest that NHEJ proteins participate in site-specific integration, and indicate a role for the single-stranded form of AAV DNA in targeted integration.

Regulation of repair choice: Cdk1 suppresses recruitment of end joining factors at DNA breaks

Cell cycle plays a crucial role in regulating the pathway used to repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, homologous recombination is primarily limited to non-G 1 cells as the formation of recombinogenic single-stranded DNA requires CDK1-dependent 5′ to 3′ resection of DNA ends. However, the effect of cell cycle on non-homologous end joining (NHEJ) is not yet clearly defined. Using an assay to quantitatively measure the contributions of each repair pathway to repair product formation and cellular survival after DSB induction, we found that NHEJ is most efficient at G 1, and markedly repressed at G 2. Repression of NHEJ at G 2 is achieved by efficient end resection and by the reduced association of core NHEJ proteins with DNA breaks, both of which depend on the CDK1 activity. Importantly, repression of 5′ end resection by CDK1 inhibition at G 2 alone did not fully restore either physical association of Ku/Dnl4-Lif1 with DSBs or NHEJ proficiency to the level at G 1. Expression of excess Ku can partially offset the inhibition of end joining at G 2. The results suggest that regulation of Ku/Dnl4-Lif1 affinity for DNA ends may contribute to the cell cycle-dependent modulation of NHEJ efficiency.

Radioresistant cervical cancer shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and Ku86

Background:Radiotherapy is central in the treatment of cervical cancer. The formation of DNA double-strand breaks is considered to be critical for the radiotherapeutic effect. The non-homologous end joining (NHEJ) proteins DNA–PKcs, Ku70 and Ku86 have a major role in repairing DNA lesions. The objective of this study was to analyse if the expression of DNA–PKcs, Ku70 and Ku86 and their downstream signalling molecules p53, p21 and Mdm-2 are altered in residual cervical tumours after radiotherapy.Methods:Retrospective analysis of 127 patients with cervical cancer stage IB-IIA treated with preoperative radiotherapy and radical surgery, revealed residual tumour in the cervical specimen in 30 patients. In 22 cases tumour material from residual and corresponding primary tumour were retrieved and the expression of DNA–PKcs, Ku86, Ku70, p53, p21 and Mdm-2 were assessed by immunohistochemistry.Results:Residual tumours showed increased frequency of DNA–PKcs (P=0.037), Ku70 (P=0.018), Ku86 (P=0.008) positive cells. A correlation in DNA–PKcs expression between primary and residual tumours was found. The frequency of p21-positive cells was decreased (P=0.007) in residual tumours whereas no change in p53 or Mdm-2-positive cells were observed.Conclusion:Our results show that cervical carcinoma surviving radiotherapy have an increased DNA–PK expression. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of DNA–PK function may be part of a radioresistance mechanism within this tumour type.

AdnAB: a new DSB-resecting motor–nuclease from mycobacteria

The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase-nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase-nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is triggered by dsDNA with free ends and the energy of ATP hydrolysis is coupled to DSB end resection by the AdnAB nuclease. The mycobacterial nonhomologous end-joining (NHEJ) protein Ku protects DSBs from resection by AdnAB. We find that AdnAB incises ssDNA by measuring the distance from the free 5' end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 5' end. The "molecular ruler" of AdnAB is regulated by ATP, which elicits an increase in ssDNA cleavage rate and a distal displacement of the cleavage sites 16-17 nt from the 5' terminus. AdnAB is a dual nuclease with a clear division of labor between the subunits. Mutations in the nuclease active site of the AdnB subunit ablate the ATP-inducible cleavages; the corresponding changes in AdnA abolish ATP-independent cleavage. Complete suppression of DSB end resection requires simultaneous mutation of both subunit nucleases. The nuclease-null AdnAB is a helicase that unwinds linear plasmid DNA without degrading the displaced single strands. Mutations of the phosphohydrolase active site of the AdnB subunit ablate DNA-dependent ATPase activity, DSB end resection, and ATP-inducible ssDNA cleavage; the equivalent mutations of the AdnA subunit have comparatively little effect. AdnAB is a novel signature of the Actinomycetales taxon. Mycobacteria are exceptional in that they encode both AdnAB and RecBCD, suggesting the existence of alternative end-resecting motor-nuclease complexes.

Genetic Evidence That the Non-Homologous End-Joining Repair Pathway Is Involved in LINE Retrotransposition

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs—zebrafish ZfL2-2 and human L1—in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

Telomere Dysfunction and DNA-PKcs Deficiency: Characterization and Consequence

The mechanisms by which cells accurately distinguish between DNA double-strand break (DSB) ends and telomeric DNA ends remain poorly defined. Recent investigations have revealed intriguing interactions between DNA repair and telomeres. We were the first to report a requirement for the nonhomologous end-joining (NHEJ) protein DNA-dependent protein kinase (DNA-PK) in the effective end-capping of mammalian telomeres. Here, we report our continued characterization of uncapped (as opposed to shortened) dysfunctional telomeres in cells deficient for the catalytic subunit of DNA-PK (DNA-PKcs) and shed light on their consequence. We present evidence in support of our model that uncapped telomeres in this repair-deficient background are inappropriately detected and processed as DSBs and thus participate not only in spontaneous telomere-telomere fusion but, importantly, also in ionizing radiation-induced telomere-DSB fusion events. We show that phosphorylation of DNA-PKcs itself (Thr-2609 cluster) is a critical event for proper telomere end-processing and that ligase IV (NHEJ) is required for uncapped telomere fusion. We also find uncapped telomeres in cells from the BALB/c mouse, which harbors two single-nucleotide polymorphisms that result in reduced DNA-PKcs abundance and activity, most markedly in mammary tissue, and are both radiosensitive and susceptible to radiogenic mammary cancer. Our results suggest mechanistic links between uncapped/dysfunctional telomeres in DNA-PKcs-deficient backgrounds, radiation-induced instability, and breast cancer. These studies provide the first direct evidence of genetic susceptibility and environmental insult interactions leading to a unique and ongoing form of genomic instability capable of driving carcinogenesis.

Brca2/Xrcc2 dependent HR, but not NHEJ, is required for protection against O 6-methylguanine triggered apoptosis, DSBs and chromosomal aberrations by a process leading to SCEs

O 6-methylguanine (O 6MeG) is a highly critical DNA adduct induced by methylating carcinogens and anticancer drugs such as temozolomide, streptozotocine, procarbazine and dacarbazine. Induction of cell death by O 6MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O 6MeG. Here, we compared the response of cells mutated in HR and NHEJ proteins to temozolomide and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (DNA-PK cs mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of O 6-methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific hypersensitivity was due to O 6MeG lesions. MNNG provoked H2AX phosphorylation 24–48 h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The γH2AX foci formed in response to O 6MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O 6MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O 6MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving cancer therapy with methylating agents.

Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells

Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and γ-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.

Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells

The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell's genome is important to advance strategies for biotechnology and genetic medicine.

Aberrant V(D)J Recombination in Ataxia Telangiectasia Mutated-Deficient Lymphocytes Is Dependent on Nonhomologous DNA End Joining

During lymphocyte Ag receptor gene assembly, DNA cleavage by the Rag proteins generates pairs of coding and signal ends that are normally joined into coding joints and signal joints, respectively, by the classical nonhomologous end-joining (NHEJ) pathway of DNA double strand break repair. Coding and signal ends can also be aberrantly joined to each other, generating hybrid joints, through NHEJ or through NHEJ-independent pathways, such as Rag-mediated transposition. Hybrid joints do not participate in the formation of functional Ag receptor genes and can alter the configuration of Ag receptor loci in ways that limit subsequent productive rearrangements. The formation of these nonfunctional hybrid joints occurs rarely in wild type lymphocytes, demonstrating that mechanisms exist to limit both the NHEJ-dependent and the NHEJ-independent joining of a signal end to a coding end. In contrast to wild-type cells, hybrid joint formation occurs at high levels in ataxia telangiectasia mutated (Atm)-deficient lymphocytes, suggesting that Atm functions to limit the formation of these aberrant joints. In this study, we show that hybrid joint formation in Atm-deficient cells requires the NHEJ proteins Artemis, DNA-PKcs, and Ku70, demonstrating that Atm functions primarily by modulating the NHEJ-dependent, and not the NHEJ-independent, joining of coding ends to signal ends.

The Yku70–Yku80 complex contributes to regulate double‐strand break processing and checkpoint activation during the cell cycle

DNA double-strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR requires 5' DSB end degradation that occurs in the presence of cyclin-dependent kinase (CDK) activity. Here, we show that a lack of any of the NHEJ proteins Yku (Yku70-Yku80), Lif1 or DNA ligase IV (Dnl4) increases 5' DSB end degradation in G1 phase, with ykuDelta cells showing the strongest effect. This increase depends on MRX, the recruitment of which at DSBs is enhanced in ykuDelta G1 cells. DSB processing in G2 is not influenced by the absence of Yku, but it is delayed by Yku overproduction, which also decreases MRX loading on DSBs. Moreover, DSB resection in ykuDelta cells occurs independently of CDK activity, suggesting that it might be promoted by CDK-dependent inhibition of Yku.

Down regulation of BRCA2 causes radio‐sensitization of human tumor cells in vitro and in vivo

In order to study the role of BRCA2 protein in homologous recombination repair and radio-sensitization, we utilized RNA interference strategy in vitro and in vivo with human tumor cells. HeLa cells transfected with small-interfering BRCA2 NA (BRCA2 siRNA) (Qiagen) as well as negative-control siRNA for 48 h were irradiated, and several critical end points were examined. The radiation cell survival level was significantly reduced in HeLa cells with BRCA2 siRNA when compared with mock- or negative-control siRNA transfected cells. DNA double strand break repair as measured by constant field gel-electrophoresis showed a clear inhibition in cells with BRCA2 siRNA, while little inhibition was observed in cells with negative control siRNA. Our immuno-staining experiments revealed a significant delay in Rad51 foci formation in cells with BRCA2 siRNA when compared with the control populations. However, none of the non-homologous end joining proteins nor the phosphorylation of DNA-dependent protein kinase catalytic subunit was affected in cells transfected with BRCA2 siRNA. In addition, the combined treatment with radiation and BRCA2 siRNA in xenograft model with HeLa cells showed an efficient inhibition of in vivo tumor growth. Our results demonstrate down-regulation of BRCA2 leads to radio-sensitization mainly through the inhibition of homologous recombination repair type double-strand break repair; a possibility of using BRCA2 siRNA as an effective radiosensitizer in tumor radiotherapy may arise.

Ku70 prevents genome instability resulting from heterozygosity of the telomerase RNA component in a vertebrate tumour line

Telomere repeat sequences are added to linear chromosome ends by telomerase, an enzyme comprising a reverse transcriptase (TERT) and an RNA template component (TR). We aimed to investigate TR in the DT40 B-cell tumour line using gene targeting, but were unable to generate TR nulls, suggesting a requirement for TR in DT40 proliferation. Disruption of one TR allele reduced telomerase activity and caused a progressive decline in telomere and G-strand overhang length. We then examined the interactions between TR and cellular DNA double-strand break (DSB) repair. Deletion in TR +/− cells of the gene encoding the non-homologous end-joining protein, Ku70, caused rapid loss of G-strand overhangs. Ku70 −/− TR +/− cells proliferated more slowly than either single mutant and showed frequent mitotic aberrations. Activation of the DNA damage response was observed in TR-deficient cells and was exacerbated by Ku deficiency, although frequent telomeric DNA damage signals were not observed until late passages. This activation of the DNA damage response was suppressed by deletion of Rad54, a key homologous recombination gene. These findings suggest that Ku and telomerase cooperate to block homologous recombination from acting on telomeres.

Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in Saccharomyces cerevisiae.

Nonhomologous end joining (NHEJ) is an important DNA double-strand-break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae: the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2), and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO-endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.

In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

Background and purpose The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins. Materials and methods Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. Results The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively in the nucleus with no detectable amount in the cytoplasm. This distribution was not altered upon irradiation. In non-irradiated cells the level of these proteins varied with a CV ranging between 16% and 20%, but there was no correlation with the respective cellular DSB repair capacity. Irradiation (3.5 and 15 Gy) did not alter the expression of these proteins and there was also no change in the DNA-PK activity. These results indicate that the variation in DSB repair capacity determined for these fibroblasts can be ascribed to differences neither in the localisation or expression of Ku70, Ku80 and XRCC4 nor in the activity of the DNA-PK complex induced upon irradiation. Conclusions For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity.