In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional beta-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 x 10(-8)) being only 30% of the recombination rate at 2 dpg (9.14 x 10(-8)). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.

OBJECTIVE

Neoadjuvant castration improves response to radiotherapy of prostate cancer. Here, we determine whether castration therapy impairs nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSB) by downregulating Ku70 protein expression.

METHODS

Twenty patients with locally advanced prostate cancer were enrolled, and 6 to 12 needle core biopsy specimens were taken from the prostate of each patient before treatment. Bilateral orchidectomy was conducted in eight patients and 12 patients were treated with a GnRH agonist. After castration, two to four similar biopsies were obtained, and the levels of Ku70 and γ-H2AX foci were determined by immunofluorescence in verified cancer tissues.

RESULTS

We observed that the androgen receptor binds directly to Ku70 in prostate tissue. We also found a reduction of the Ku70 protein levels in the cell nuclei in 12 of 14 patients (P < 0.001) after castration. The reduction in Ku70 expression correlated significantly with decreased serum prostate-specific antigen (PSA) levels after castration, suggesting that androgen receptor activity regulates Ku70 protein levels in prostate cancer tissue. Furthermore, a significant correlation between the reductions of Ku70 after castration versus changes induced of castration of γ-H2AX foci could be seen implicating a functional linkage of decreased Ku70 levels and impaired DNA repair.

CONCLUSIONS

Castration therapy results in decreased levels of the Ku70 protein in prostate cancer cells. Because the Ku70 protein is essential for the NHEJ repair of DSBs and its downregulation impairs DNA repair, this offers a possible explanation for the increased radiosensitivity of prostate cancer cells following castration.

In Yarrowia lipolytica, targeted gene replacement occurs only with long length (1 kb) homologous flanking fragments, as this yeast preferentially uses the non-homologous end-joining mechanism (NHEJ) for DNA repair over homologous recombination (HR). To improve the frequency of HR, we identified and disrupted the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica. In ku70∆ HR of URA3 marker at the ADE2 locus occurred with 43 % frequency with as little as 50 bp long flanking regions. The number of Ura(+) transformants was reduced to 1 % of the Po1d (ura3-302) wild type-like strain level, regardless of the flanking fragment length. On the contrary, even though HR was not improved in ku80∆, Ura(+) transformants was 60 % lower compared to the wild type.

An efficient gene-targeting system based on impairment of the nonhomologous end-joining pathway and the orotidine monophosphate decarboxylase gene (pyrG) in Aspergillus flavus was established. It was achieved by replacing the ku70 gene with the Aspergillus oryzae pyrithiamine resistance (ptr) gene and by inserting the Aspergillus parasiticus cypA gene into the pyrG locus. The utility of this system was demonstrated by disruption of nine candidate genes for conidial pigment biosynthesis. The gene-targeting frequencies ranged from 80 to 100%. Two linked genes on chromosome 4, wA and olgA, were confirmed to be involved in pigment formation. In contrast to the parental strain which produced yellowish-green conidia, the knockout mutants produced white and olive-green conidia, respectively. The system was further refined by restoring the pyrithiamine sensitivity and uracil auxotrophy in the A. flavus transformation recipient with an engineered pyrG marker. The improvement allowed gene manipulation using the reusable pyrG marker as shown by the restoration of laeA-mediated aflatoxin production in an A. flavus laeA-deleted mutant.

Telomeres are protected from nonhomologous end-joining (NHEJ) to avoid deleterious chromosome fusions, yet they associate with the Ku heterodimer that is principal in the classical NHEJ (c-NHEJ) pathway. T-loops have been proposed to inhibit Ku's association with telomeric ends, thus inhibiting c-NHEJ; however, deficiencies in the t-loop model suggest additional mechanisms are in effect. We demonstrate that TRF2 interacts with Ku at telomeres and via residues in Ku70 helix 5 (α5), which are vital for NHEJ. We show that Ku's interaction with a TRF2 mutant that induces telomeric fusions is significantly impaired. Additionally, we demonstrate that Ku70 α5 is required for Ku self-association in live cells, which can bridge DNA ends. Together, these findings lead us to propose a model in which telomeres are directly protected from c-NHEJ via TRF2 impeding Ku's ability to synapse telomere ends.

Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.

The Ku heterodimer, consisting of the proteins Ku70 and Ku80, is the central component of the non-homologous end joining (NHEJ) pathway of double strand break (DSB) repair. Ku is able to recognize and bind a DSB by virtue of its ring-like structure. Both pre-repair and topologically trapped post-repair Ku heterodimers are thought to be inhibitory to multiple cellular processes. Thus, a regulated mechanism for the removal of Ku from chromatin was predicted to exist. Recent evidence shows that Ku80 is removed from DNA through a ubiquitin-mediated process. Similar processes have been shown to be involved in the regulated dissociation of a host of other proteins from chromatin, and this appears to be a general and conserved mechanism for the regulation of chromatin-associated factors. A potential mechanism for this pathway is discussed.

DNA-PK is a nuclear, serine/threonine protein kinase required for repairing DNA double-strand breaks and for V(D)J recombination. To determine the distribution of DNA-PK in human tissues, we assayed paraffin-embedded sections of normal and cancerous tissues for DNA-PKcs and Ku80 by immunohistochemistry. We also assayed for Brca2, a human tumor suppressor gene that is implicated in the repair of DNA strand-breaks. Brca2 was strongly expressed in epithelial cells of the breast, endometrium, and thymus, in tingible body macrophages of follicular germinal centers of lymphoid tissue, and in reticuloendothelial cells in the spleen. DNA-PKcs and Ku80 expression was usually parallel, but both were expressed in a highly cell- and tissue-specific manner. The highest levels were observed in spermatogenic cells (but not in spermatozoa), and in neurons and glial cells of the central and autonomic nervous system. Neither protein was consistently expressed in liver nor in resting mammary epithelium, but lactating breast epithelium was strongly positive for DNA-PKcs and Ku80. In contrast to established human cell cultures, expression between cells in the same tissue was highly selective in the epidermis, exocrine pancreas, renal glomeruli, the red pulp of the spleen, and within cellular compartments of tonsils, lymph nodes, and thymus. Most cancerous tissues were consistently positive for DNA-PKcs and Ku80, except invasive carcinoma of the breast. DNA-PKcs, Ku80, and Ku70 mRNAs were expressed in all normal tissues with relatively little variation in levels. Our results suggest that the apparent absence of DNA-PKcs and Ku80 from some cells or tissues is a consequence of post-transcriptional mechanisms that regulate protein levels.

The partitioning-defective 3 (Par3), a key component in the conserved Par3/Par6/aPKC complex, plays fundamental roles in cell polarity. Herein we report the identification of Ku70 and Ku80 as novel Par3-interacting proteins through an in vitro binding assay followed by liquid chromatography-tandem mass spectrometry. Ku70/Ku80 proteins are two key regulatory subunits of the DNA-dependent protein kinase (DNA-PK), which plays an essential role in repairing double-strand DNA breaks (DSBs). We determined that the nuclear association of Par3 with Ku70/Ku80 was enhanced by gamma-irradiation (IR), a potent DSB inducer. Furthermore, DNA-PKcs, the catalytic subunit of DNA-PK, interacted with the Par3/Ku70/Ku80 complex in response to IR. Par3 over-expression or knockdown was capable of up- or downregulating DNA-PK activity, respectively. Moreover, the Par3 knockdown cells were found to be defective in random plasmid integration, defective in DSB repair following IR, and radiosensitive, phenotypes similar to that of Ku70 knockdown cells. These findings identify Par3 as a novel component of the DNA-PK complex and implicate an unexpected link of cell polarity to DSB repair.

Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs-c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142-XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells.

We have recently shown that the in vitro differentiation of human mesenchymal stem cells (hMSCs) was accompanied by an increased sensitivity toward apoptosis; however, the mechanism responsible for this shift is not known. Here, we show that the repair of DNA double-strand breaks (DSBs) was more rapid in undifferentiated hMSCs than in differentiated osteoblasts by quantification of the disappearance of γ-H2AX foci in the nuclei after γ-irradiation-induced DNA damage. In addition, there was a marked and prolonged increase in the level of nuclear Ku70 and an increased phosphorylation of DNA-PKcs. This was accompanied by an augmentation in the phosphorylation of ATM in hMSCs post-irradiation suggesting the nonhomologous end joining repair mechanism. However, when hMSCs were induced to differentiate along the osteogenic or adipogenic pathways; irradiation of these cells caused an expeditious and robust cell death, which was primarily apoptotic. This was in sharp contrast to undifferentiated hMSCs, which were highly resistant to irradiation and/or temozolomide-induced DSBs. In addition, we observed a 95% recovery from DSB in these cells. Our results suggest that apoptosis and DNA repair are major safeguard mechanisms in the control of hMSCs differentiation after DNA damage.

The repair of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. In higher eukaryotes, DNA DSBs are predominantly repaired by non-homologous end joining (NHEJ), but DNA ends can also be joined by an alternative error-prone mechanism termed microhomology-mediated end joining (MMEJ). In MMEJ, the repair of DNA breaks is mediated by annealing at regions of microhomology and is always associated with deletions at the break site. In budding yeast, the Mre11/Rad5/Xrs2 complex has been demonstrated to play a role in both classical NHEJ and MMEJ, but the involvement of the analogous MRE11/RAD50/NBS1 (MRN) complex in end joining in higher eukaryotes is less certain. Here we demonstrate that in Xenopus laevis egg extracts, the MRN complex is not required for classical DNA-PK-dependent NHEJ. However, the XMRN complex is necessary for resection-based end joining of mismatched DNA ends. This XMRN-dependent end joining process is independent of the core NHEJ components Ku70 and DNA-PK, occurs with delayed kinetics relative to classical NHEJ and brings about repair at sites of microhomology. These data indicate a role for the X. laevis MRN complex in MMEJ.

NHEJ (non-homologous end joining) is the predominant mechanism for repairing DNA double-stranded breaks in human cells. One essential NHEJ factor is the Ku heterodimer, which is composed of Ku70 and Ku86. Here we have generated heterozygous loss-of-function mutations for each of these genes in two different human somatic cell lines, HCT116 and NALM-6, using gene targeting. Previous work had suggested that phenotypic differences might exist between the genes and/or between the cell lines. By providing a side-by-each comparison of the four cell lines, we demonstrate that there are indeed subtle differences between loss-of-function mutations for Ku70 versus Ku86, which is accentuated by whether the mutations were derived in the HCT116 or NALM-6 genetic background. Overall, however, the phenotypes of the four lines are quite similar and they provide a compelling argument for the hypothesis that Ku loss-of-function mutations in human somatic cells result in demonstrable haploinsufficiencies. Collectively, these studies demonstrate the importance of proper biallelic expression of these genes for NHEJ and telomere maintenance and they provide insights into why these genes are uniquely essential for primates.

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.

Repair of DNA double-strand breaks (DSBs) in mammalian cells by nonhomologous end-joining (NHEJ) is initiated by the DNA-PK protein complex. Recent studies have shown inositol hexakisphosphate (InsP6) is a potent cofactor for DNA-PK activity in NHEJ. Specifically, InsP6 binds to the Ku component of DNA-PK, where it induces a conformational change and a corresponding increase in DNA end-joining activity. However, the effect of InsP6 on the dynamics of Ku, such as its mobility in the nucleus, is unknown. Importantly, these dynamics reflect the character of Ku's interactions with other molecules. To address this question, the diffusion of Ku was measured by fluorescence photobleaching experiments using cells expressing green fluorescent protein (GFP)-labeled Ku. InsP6 was depleted by treating cells with calmodulin inhibitors, which included the compounds W7 and chlorpromazine. These treatments caused a 50% reduction in the mobile fraction of Ku-GFP, and this could be reversed by replenishing cells with InsP6. By expressing deletion mutants of Ku-GFP, it was determined that its W7-sensitive region occurred at the N-terminus of the dimerization domain of Ku70. These results therefore show that InsP6 enhances Ku mobility through a discrete region of Ku70, and modulation of InsP6 levels in cells represents a potential avenue for regulating NHEJ by affecting the dynamics of Ku and hence its interaction with other nuclear proteins.

One of the functions of the abundant heterodimeric nuclear protein, Ku (Ku70/Ku80), is its involvement in the initiation of DNA replication through its ability to bind to chromosomal replication origins in a sequence-specific and cell cycle dependent manner. Here, using HCT116 Ku80+/- cells, the effect of Ku80 deficiency on cell cycle progression and origin activation was examined. Western blot analyses revealed a 75% and 36% decrease in the nuclear expression of Ku80 and Ku70, respectively. This was concomitant with a 33% and 40% decrease in chromatin binding of both proteins, respectively. Cell cycle analysis of asynchronous and late G1 synchronized Ku80+/- cells revealed a prolonged G1 phase. Furthermore, these Ku-deficient cells had a 4.5-, 3.4- and 4.3-fold decrease in nascent strand DNA abundance at the lamin B2, beta-globin and c-myc replication origins, respectively. Chromatin immunoprecipitation (ChIP) assays showed that the association of Ku80 with the lamin B2, beta-globin and c-myc origins was decreased by 1.5-, 2.3- and 2.5-fold, respectively, whereas that of Ku70 was similarly decreased (by 2.1-, 1.5- and 1.7-fold, respectively) in Ku80+/- cells. The results indicate that a deficiency of Ku80 resulted in a prolonged G1 phase, as well as decreased Ku binding to and activation of origins of DNA replication.

Loss of telomere equilibrium and associated chromosome-genomic instability might effectively promote tumour progression. Telomere function may have contrasting roles: inducing replicative senescence and promoting tumourigenesis and these roles may vary between cell types depending on the expression of the enzyme telomerase, the level of mutations induced, and efficiency/deficiency of related DNA repair pathways. We have identified an alternative telomere maintenance mechanism in mouse embryonic stem cells lacking telomerase RNA unit (mTER) with amplification of non-telomeric sequences adjacent to existing short stretches of telomere repeats. Our quest for identifying telomerase-independent or alternative mechanisms involved in telomere maintenance in mammalian cells has implicated the involvement of potential DNA repair factors in such pathways. We have reported earlier on the telomere equilibrium in scid mouse cells which suggested a potential role of DNA repair proteins in telomere maintenance in mammalian cells. Subsequently, studies by us and others have shown the association between the DNA repair factors and telomere function. Mice deficient in a DNA-break sensing molecule, PARP-1 (poly [ADP]-ribopolymerase), have increased levels of chromosomal instability associated with extensive telomere shortening. Ku80 null cells showed a telomere shortening associated with extensive chromosome end fusions, whereas Ku80+/- cells exhibited an intermediate level of telomere shortening. Inactivation of PARP-1 in p53-/- cells resulted in dysfunctional telomeres and severe chromosome instability leading to advanced onset and increased tumour incidence in mice. Interestingly, haploinsufficiency of PARP-1 in Ku80 null cells causes more severe telomere shortening and chromosome abnormalities compared to either PARP-1 or Ku80 single null cells and Ku80+/-PARP-/- mice develop spontaneous tumours. This overview will focus mainly on the role of DNA repair/recombination and DNA damage signalling molecules such as PARP-1, DNA-PKcs, Ku70/80, XRCC4 and ATM which we have been studying for the last few years. Because the maintenance of telomere function is crucial for genomic stability, our results will provide new insights into the mechanisms of chromosome instability and tumour formation.

In vertebrate cells, DNA double-strand breaks are efficiently repaired by homologous recombination or nonhomologous end-joining (NHEJ). The latter pathway relies on Ku (the Ku70/Ku86 heterodimer), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). Here, we show that a human pre-B cell line nullizygous for Lig4 exhibits hypersensitivity to topoisomerase II (Top2) inhibitors, demonstrating a crucial role for the NHEJ pathway in repair of Top2-induced DNA damage in vertebrates. We also show that in the chicken DT40 cell line, all NHEJ mutants (i.e., Ku70-, Lig4-, and DNA-PKcs-null cells) are equally hypersensitive to the Top2 inhibitor ICRF-193, indicating that the drug-induced damage is repaired by NHEJ involving DNA-PKcs. Intriguingly, however, DNA-PKcs-null cells display considerably less severe phenotype than other NHEJ mutants in terms of hypersensitivity to VP-16, a Top2 poison that stabilizes cleavable complexes. The results indicate that two distinct NHEJ pathways, involving or not involving DNA-PKcs, are important for the repair of VP-16-induced DNA damage, providing additional evidence for the biological relevance of DNA-PKcs-independent NHEJ. Our results provide significant insights into the mechanisms of repair of Top2-mediated DNA damage, with implications for chemotherapy involving Top2 inhibitors.

Cernunnos-XLF is the most recently identified core component in the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double strand breaks (DSBs) in mammals. It associates with the XRCC4/ligase IV ligation complex and stimulates its activity in a still unknown manner. NHEJ also requires the DNA-dependent protein kinase that contains a Ku70/Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit. To understand the interplay between Cernunnos-XLF and the other proteins implicated in the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with DNA double strand-breaking agents by means of a detergent-based cellular fractionation protocol. We report that Cernunnos-XLF is corecruited with the core NHEJ components on chromatin damaged with DSBs in human cells and is phosphorylated by the DNA-dependent protein kinase catalytic subunit. Our data show a pivotal role for DNA ligase IV in the NHEJ ligation complex assembly and recruitment to DSBs because the association of Cernunnos-XLF with the XRCC4/ligase IV complex relies primarily on the DNA ligase IV component, and an intact XRCC4/ligase IV complex is necessary for Cernunnos-XLF mobilization to damaged chromatin. Conversely, a Cernunnos-XLF defect has no apparent impact on the XRCC4/ligase IV association and recruitment to the DSBs or on the stimulation of the DNA-dependent protein kinase on DNA ends.

Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G1 phase and exposed to very low fluences of alpha particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4-9% of wild-type cells and 36-55% of Xrcc5-/- cells, whereas only 2-3% of the nuclei were traversed by an alpha particle and thus received any radiation exposure. G22p1-/- cells responded similarly to Xrcc5-/- cells, whereas Prkdc-/- and Adprt-/- cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5-/- and G22p1-/- cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc-/- cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.

The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogaster strain deficient in the RAD54 homolog DmRAD54 is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54(-/-) flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, a DmRad54(-/-)/DmKu70(-/-) double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements in Drosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3' overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.

Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5β, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome) or premature aging (Werner Syndrome). RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining.

The homologous recombination mechanism for DNA-repair is not predominant in most filamentous fungi, resulting in extremely low targeting efficiencies for molecular engineering. To increase the gene targeting efficiency, it is becoming common practice to inactivate the non-homologous end-joining (NHEJ) pathway that causes random integration, by deleting the fungal homologs of the human KU70 and KU80 genes that encode proteins functioning in the NHEJ pathway. This has been described for several filamentous fungi, but limited knowledge on the physiological consequences is available. In this study we characterized targeting efficiency and physiology of penicillinG producing Penicillium chrysogenum strains, in which the KU70 or KU80 homologues hdfA and hdfB had been deleted. Targeting efficiency was increased from ca. 1% in the reference strain to 47% and 56% in the hdfA and hdfB mutant strains, respectively, using an ends-out construct. Physiological and transcriptome analysis of glucose-limited chemostat cultures of the hdfA deletion strain and the reference strain showed minimal differences. Although, in a direct competition experiment to assess strain fitness, the reference strain had a clear advantage over the deletion strain, the results demonstrate the potential of DeltahdfAP. chrysogenum strains for the functional analysis of the recently completed P. chrysogenum genome sequence and in further metabolic engineering of antibiotics production.

To determine the patterns of gene expression responsible for the radiosensitivity of glioblastoma cells, we analyzed transcriptional changes after ionizing radiation in different cell lines. After completing clonogenic survival assays, we selected two glioblastoma cell lines with different radiosensitivities. Subsequently, they were investigated by using the technique of DNA microarray, and we then categorized the upregulated genes into 10 groups. Between the two cell lines, the difference in the percentage of DNA repair/replication category was the largest, and this category was present at a greater percentage with radioresistant cell line U87MG. Moreover, among the commonly upregulated genes, the DNA repair/replication category was present in the largest percentage. These genes included G22P1 (Ku70) and XRCC5 (Ku80) genes known as important members of the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Furthermore, cell line that specifically upregulated genes included the members of major pathways of DNA DSB or single strand damage repair. These pathways were not only NHEJ, but also homologous recombination (HR) and postreplication repair (PRR). In conclusion, the distribution of genes involved in the DNA repair/replication category was most different between two human glioblastoma cell lines of different radiosensitivities. Among commonly upregulated genes, the DNA repair/replication category was present in the largest percentage.