Nonhomologous DNA End Joining Research Articles

Factors determining DNA double-strand break repair pathway choice in G2 phase.

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

Structural characterization of filaments formed by human Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Structural characterization of filaments formed by human Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining

Structure of a Preternary Complex Involving a Prokaryotic NHEJ DNA Polymerase

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair.

Gene inactivation in the citrus pathogenic fungus Alternaria alternata defect at the Ku70 locus associated with non-homologous end joining

Low frequency of targeted gene disruption in filamentous fungi has been attributed to the predominance of ectopic integration that is controlled by a non-homologous end-joining (NHEJ) DNA repair mechanism. It has been shown in a number of fungi that suppression of NHEJ by inactivating the yeast Ku70 or Ku80 homolog facilitates homologous recombination, thereby enhancing the frequency of gene targeting. A Ku70 gene homolog was cloned from the necrotrophic fungus Alternaria alternata, causing citrus brown spot. The cloned gene, designated AaKu70, was independently inactivated by insertion of an acetolactate synthase gene (SUR) conferring sulfonylurea resistance or of a bacterial phosphotransferase gene (HYG) cassette conferring hygromycin resistance. The AaKu70 deficient strain reduced conidial formation and pigmentation slightly, but unchanged in toxin production and pathogenicity. Unlike many fungal species, mutation of the AaKu70 gene by inserting either SUR or HYG did not facilitate gene disruption, as tested for disruption targeting an AaAP1gene encoding a redox responsive transcription regulator or an AaHSK1 gene encoding a histidine kinase. However, a split marker approach, using truncated DNA fragments overlapping within the HYG, but not SUR, selectable marker, enhanced the frequency of AaAP1 disruption considerably in the progenitor and the ∆AaKu70 strain. Compared to the disruption targeting the AaAP1 gene, the frequency for recovery of the AaHSK1 impaired mutant was much lower, indicating an allele-dependent disruption. The present study indicates that AaKu70 has little or no effect on gene disruption and homologous integration in A. alternata.

ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks.

Classical non-homologous DNA end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Deficiencies for C-NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for C-NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF) is mutated in certain immunodeficient human patients and has been implicated in C-NHEJ3,4,5,6. Yet, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The Ataxia Telangiectasia-Mutated protein (“ATM”) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM-deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX-deficiency does not measurably impact these processes7,8,9. Here, we show that XLF, ATM, and H2AX all have fundamental roles in processing and joining ends during V(D)J recombination; but that these roles have been masked by unanticipated functional redundancies. Thus, combined ATM/XLF-deficiency nearly blocks mouse lymphocyte development due inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs C-NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in C-NHEJ are mediated via ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, suggesting a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX indeed has a role in this process.

Dysfunctional Double-Strand DNA Break Repair In Primary t-AML/t-MDS Myeloblasts.

AbstractAbstract 3366The gross chromosomal aberrations in treatment-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/t-MDS) cells suggest that disease initiation and progression may result from the inability of cells to appropriately respond to double-strand DNA breaks (DSBs) induced by prior exposure to radiation, alkylator, or topoisomerase II inhibitor therapy. We hypothesize that dysregulation of DSB repair by homology-directed repair (HDR) or nonhomologous end joining (NHEJ) contributes to the development of t-AML/t-MDS. Dysregulation of DSB repair in t-AML/t-MDS may result from inherited variants or acquired mutations in HDR/NHEJ pathway genes. To directly test this possibility, we used next-generation sequencing technology and an array CGH platform to identify inherited and somatic genetic variants, including small indels and copy number alterations, in 21 canonical HDR and 9 NHEJ DNA repair genes, as well as a subset of 7 DNA damage response genes using tumor DNA and paired normal DNA obtained from 30 t-AML/t-MDS patients. All the data has been acquired and the analysis is ongoing. Because dysregulation of DNA repair pathways can result from epigenetic changes or post-translational modifications in DNA repair genes and would not be detected using sequencing and array CGH, we are also performing functional studies to interrogate DSB repair using primary bone marrow cells from 16 of these t-AML/t-MDS patients and CD34+ cells from 5 normal healthy controls. We performed a flow-based assay for DSB formation and repair by measuring the phosphorylated form of the variant histone H2AX (pH2AX), which is rapidly phosphorylated upon DSB formation, in myeloblasts (CD45 dim, low side scatter) and lymphocytes (a surrogate for normal cells) from leukemic bone marrow. Baseline measurements of unmanipulated primary bone marrow cells and a time course to measure the kinetics of DSB repair after gamma irradiation are used to assess a cell's basal DSB burden and the response to acute damage. We have validated this assay in a defined genetic system using isogenic cells deficient or not in BRCA2, a gene central to the HDR pathway, and were able to demonstrate that cells lacking BRCA2 have elevated pH2AX levels at 4–24 hours post DSB induction compared to controls (p=0.01). In addition, we have evaluated the ability of the cells to form nuclear foci of pH2AX and RAD51 (a protein central to the repair of DSB by HDR) by quantifying the relative numbers of immunofluorescent nuclear foci upon irradiation. We have performed these functional assays for 8 t-AML/t-MDS samples and 3 normal donors. We identified one t-AML/t-MDS sample whose myeloblasts show 2–3 fold greater pH2AX at baseline, compared to control CD34+ cells (p=0.006). Interestingly, the clearance of pH2AX is significantly faster in this sample compared to controls (p=0.001) despite showing elevated baseline pH2AX levels. This patient had a translocation involving chromosome 11q23. One additional patient in our cohort with an 11q23 translocation also showed similar increased baseline pH2AX levels. In addition, we identified abnormalities in a third t-AML/t-MDS sample that trends towards impaired maximal pH2AX induction (p=0.052). Furthermore, myeloblasts from a fourth t-AML/t-MDS sample, a known heterozygous BRCA2 mutation carrier, show a trend towards delayed pH2AX clearance (p=0.07). As predicted, bone marrow cells from this BRCA2 carrier also have 3.5 fold less RAD51 nuclear foci formation compared to control cells (8.6% vs 30%). Collectively, these results show that primary t-AML bone marrow cells can be used to assess the functional integrity of DSB response, and suggest that a large proportion of samples (4/8) may contain genetic alterations in DNA DSB response and/or repair genes. Functional studies of an additional 8 samples are ongoing. Next generation sequencing and array CGH data for the 37 HDR/NHEJ genes has been completed and will be correlated with the functional studies for each sample. Identification of abnormal DSB repair in leukemic patients may not only elucidate the molecular pathogenesis of the disease, but may provide rationale for testing agents to achieve selective killing of leukemic cells, as defects in one DNA repair pathway may increase reliance on another, making these cancer cells particularly susceptible to killing by inhibitors targeting the extant pathway(s). Disclosures:No relevant conflicts of interest to declare.

Endless Forms Most Viral

Endless Forms Most Viral

Coilin interacts with Ku proteins and inhibits in vitro non-homologous DNA end joining

Coilin is a nuclear protein that plays a role in Cajal body formation. The function of nucleoplasmic coilin is unknown. Here we report that coilin interacts with Ku70 and Ku80, which are major players in the DNA repair process. Ku proteins compete with SMN and SmB′ proteins for coilin interaction sites. The binding domain on coilin for Ku proteins cannot be localized to one discrete region, and only full-length coilin is capable of inhibiting in vitro non-homologous DNA end joining (NHEJ). Since Ku proteins do not accumulate in CBs, these findings suggest that nucleoplasmic coilin participates in the regulation of DNA repair. Structured summary MINT- 8052983: coilin (uniprotkb: P38432) physically interacts (MI: 0915) with SmB′ (uniprotkb: P14678) by pull down (MI: 0096)MINT- 8052941: coilin (uniprotkb: P38432) physically interacts (MI: 0915) with Ku70 (uniprotkb: P12956) by competition binding (MI: 0405)MINT- 8052765: coilin (uniprotkb: P38432) physically interacts (MI: 0915) with Ku80 (uniprotkb: P13010) by pull down (MI: 0096)MINT- 8052971: coilin (uniprotkb: P38432) physically interacts (MI: 0915) with SMN (uniprotkb: Q16637) by pull down (MI: 0096)MINT- 8052957: coilin (uniprotkb: P38432) physically interacts (MI: 0915) with Ku80 (uniprotkb: P13010) by competition binding (MI: 0405)MINT- 8052894, MINT- 8052908: coilin (uniprotkb: P38432) binds (MI: 0407) to Ku80 (uniprotkb: P13010) by pull down (MI: 0096)MINT- 8052804: coilin (uniprotkb: P38432) physically interacts (MI: 0915) with Ku80 (uniprotkb: P13010) by anti bait coimmunoprecipitation (MI: 0006)MINT- 8052925: coilin (uniprotkb: P38432) binds (MI: 0407) to Ku70 (uniprotkb: P12956) by pull down (MI: 0096)MINT- 8052786: Ku80 (uniprotkb: P13010) physically interacts (MI: 0914) with coilin (uniprotkb: P38432) and Ku70 (uniprotkb: P12956) by anti bait coimmunoprecipitation (MI: 0006)MINT- 8052776: coilin (uniprotkb: P38432) physically interacts (MI: 0915) with Ku70 (uniprotkb: P12956) by pull down (MI: 0096)

Evidence of an Adaptive Response Targeting DNA Nonhomologous End Joining and Its Transmission to Bystander Cells

Adaptive response (AR) is a term describing resistance to ionizing radiation-induced killing or formation of aberrant chromosomes that is mediated by pre-exposure to low ionizing radiation doses. The mechanism of AR remains elusive. Because cell killing and chromosome aberration formation derive from erroneous processing of DNA double-strand breaks (DSB), AR may reflect a modulation of DSB processing by nonhomologous end joining (NHEJ) or homologous recombination repair. Here, we use plasmid end-joining assays to quantify modulations induced by low ionizing radiation doses to NHEJ, the dominant pathway of DSB repair in higher eukaryotes, and investigate propagation of this response through medium transfer to nonirradiated bystander cells. Mouse embryo fibroblasts were conditioned with 10 to 1000 mGy and NHEJ quantified at different times thereafter by challenging with reporter plasmids containing a DSB. We show robust increases in NHEJ efficiency in mouse embryo fibroblasts exposed to ionizing radiation >100 mGy, irrespective of reporter plasmid used. Human tumor cells also show AR of similar magnitude that is compromised by caffeine, an inhibitor of DNA damage signaling acting by inhibiting ATM, ATR, and DNA-PKcs. Growth medium from pre-irradiated cells induces a caffeine-sensitive AR in nonirradiated cells, similar in magnitude to that seen in irradiated cells. In bystander cells, γH2AX foci are specifically detected in late S-G(2) phase and are associated with Rad51 foci that signify the function of homologous recombination repair, possibly on DNA replication-mediated DSBs. The results point to enhanced NHEJ as a mechanism of AR and suggest that AR may be transmitted to bystander cells through factors generating replication-mediated DSBs.

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Cancer cells are often associated with secondary chromosomal rearrangements, such as deletions, inversions, and translocations, which could be the consequence of unrepaired/misrepaired DNA double strand breaks (DSBs). Nonhomologous DNA end joining is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we studied end joining (EJ) in different cancer cell lines. We found that the efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower levels of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or the addition of purified BCL2 led to the down-regulation of EJ. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute toward increased chromosomal abnormalities in cancer.

The Role of the DNA Damage Response Mechanisms after Low-Dose Radiation Exposure and a Consideration of Potentially Sensitive Individuals

Here I consider whether radiation-sensitive individuals might exist in the population and the potential impact of low-dose/dose-rate radiation exposure. Radiation induces DNA double-strand breaks (DSBs), which cause lethality if they are unrepaired and enhance genomic instability if they are misrepaired. DNA damage response (DDR) mechanisms play a vital role in protecting cells from the harmful effects of DSB formation. The DDR encompasses DSB repair pathways, of which DNA nonhomologous end joining is the most significant, and a signal transduction process involving ATM. Patients defective in DDR proteins have been described, and some have shown clinical radiosensitivity. However, such patients are rare and belong to defined syndromes. The critical question is whether heterozygosity or mild defects in DDR proteins confer low-dose radiosensitivity. While it is unlikely that low-dose radiation will dramatically enhance cell killing in such patients, it is possible that there could be an impact on stem cell turnover, leading to stem cell depletion with age. More importantly, it is likely that such patients could have increased misrepair of radiation damage and hence an elevated risk of radiation-induced carcinogenesis. Evidence in support of this and the potentially important genes in this context are discussed.

Efficiency of nonhomologous DNA end joining varies among somatic tissues, despite similarity in mechanism.

Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia. Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer. DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia. We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

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.

Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest

ATM-dependent initiation of the radiation-induced G(2)/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G(2) phase are repaired by DNA nonhomologous end joining (NHEJ), while approximately 15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G(2)/M checkpoint is maintained in irradiated G(2) cells, in light of our current understanding of G(2) phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1(-/-) and MDC1(-/-) cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.

Crystal Structure of the Human Hsmar1-Derived Transposase Domain in the DNA Repair Enzyme Metnase,

Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway

Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). The various causes of double-strand breaks (DSBs) result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, DNA polymerases, and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during variable (diversity) joining [V(D)J] recombination and class switch recombination (CSR). Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation (IR), but also severely immunodeficient.

A natural compound, methyl angolensate, induces mitochondrial pathway of apoptosis in Daudi cells

Natural products discovered from medicinal plants have played an important role in the treatment of cancer. In an effort to identify novel small molecules which can affect the proliferation of lymphoma cells, we tested methyl angolensate (MA), a plant derived tetranortriterpenoid, purified from the crude extract of the root callus of Soymida febrifuga commonly known as Indian red wood tree. We have tested MA for its cytotoxic properties on Burkitt's lymphoma cell lines, using various cellular assays. We observed that MA induces cytotoxicity in Daudi cells in a dose-dependent manner using trypan blue, MTT and LDH assays. We find that the treatment with MA led to activation of DNA double-strand break repair proteins including KU70 and KU80, suggesting the activation of nonhomologous DNA end joining pathway in surviving cells. Further, we find that methyl angolensate could induce apoptosis by cell cycle analysis, annexin V-FITC staining, DNA fragmentation and PARP cleavage. Besides, MA treatment led to reactive oxygen species generation and loss of mitochondrial transmembrane potential. These results suggest the activation of mitochondrial pathway of apoptosis. Hence, we identify MA as a potential chemotherapeutic agent against Daudi cells.

DNA-PKcs regulates a single-stranded DNA endonuclease activity of Artemis

Human nuclease Artemis belongs to the metallo-beta-lactamase protein family. It acquires double-stranded DNA endonuclease activity in the presence of DNA-PKcs. This double-stranded DNA endonuclease activity is critical for opening DNA hairpins in V(D)J recombination and is thought to be important for processing overhangs during the nonhomologous DNA end joining (NHEJ) process. Here we show that purified human Artemis exhibits single-stranded DNA endonuclease activity. This activity is proportional to the amount of highly purified Artemis from a gel filtration column. The activity is stimulated by DNA-PKcs and modulated by purified antibodies raised against Artemis. Moreover, the divalent cation-dependence and sequence-dependence of this single-stranded endonuclease activity is the same as the double-stranded DNA endonuclease activity of Artemis:DNA-PKcs. These findings further expand the range of DNA substrates upon which Artemis and Artemis:DNA-PKcs can act. The findings are discussed in the context of NHEJ.