DNA-dependent protein kinase (DNA-PK) consists of three polypeptide subunits: Ku70, Ku80, and the DNA-PK catalytic subunit (DNA-PKcs). Mammalian mutants deficient in either Ku80 or DNA-PKcs function have been shown to be lacking in DNA double-strand break repair and V(D)J recombination, respectively. The precise role of the Ku70 gene in this process has not yet been determined, in part because no cell lines, animals, or human diseases involved with deficiencies in this gene have yet been identified. Both the human and the mouse Ku70 cDNAs have been cloned, and the human gene has been mapped to chromosome 22q13. The original mouse cDNA clones, however, lacked a complete 5'-region, and none of the mammalian Ku70 genomic sequences have been characterized. This report contains an analysis of the 5'-region of the mouse cDNA sequence, a characterization of the mouse Ku70 genomic structure, and fluorescence in situ hybridization data that map the mouse gene to chromosome 15. The deduced amino acid sequence of the mouse gene consists of 608 amino acids compared to 609 for the human gene. The genomic sequence is 24 kb and consists of 13 exons, including an untranslated first exon. Sequences from the upstream region of exon 1 revealed four consensus GC box sequences and a strong transcription initiation site at a reasonable location. The assignment of the mouse Ku70 gene to chromosome 15 is consistent with the syntenic relationship of this gene in human (chromosome 22q13) and mouse and adds to the comparative mapping data for the genes involved in the SCID phenotype.

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair processes, especially nonhomologous DNA-end joining (NHEJ), is critical for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, such as human Ku70 and Ku80, might play critical roles in controlling NHEJ activity. XRCC4-like factor (XLF) is a core NHEJ factor and plays a key role in the Ku-dependent NHEJ repair process in human cells. Recently, companion animals, such as canines, have been proposed to be a good model for many aspects of cancer research, including the development of chemotherapeutics. However, the localization and regulation of core NHEJ factors in canine cells have not been elucidated. Here, we show that the localization of canine XLF changes dynamically during the cell cycle. EYFP-canine XLF localizes in the nuclei of interphase cells and accumulates immediately at microirradiated DSB sites. The structure of a putative human XLF nuclear localization signal (NLS) and a putative 14-3-3 binding motif are evolutionarily conserved in canine, chimpanzee and mouse XLF. However, the putative β-TRCP-recognizable degron of human XLF is not conserved in canine and mouse. Additionally, some vital human XLF phosphorylation sites, including the ATM major phosphorylation site (S251), are not conserved in canine XLF. Our findings might be useful for the study of the molecular mechanisms of NHEJ in canine cells and for the development of new radiosensitizers that target XLF.

The Ku heterodimer binds to the ends of double-stranded breaks (DSBs) in DNA, and is involved in nonhomologous end joining. HDF1 and HDF2, which have been identified in Saccharomyces cerevisiae as homologues of the Ku70 and Ku80 proteins of mammals, reduce radiosensitivity only when homologous recombination repair is impaired and, therefore, affect DSB repair via nonhomologous recombination. Although it has been reported that homologous recombination is defective in the hdf1 null mutant, the roles of HDF1 and HDF2 in this process are not completely clear. We investigated the effect of HDF1 and HDF2 on intrachromosomal recombination by measuring rates of deletion between direct repeats caused by spontaneous and DNA damage-induced events (DEL recombination). We found a decrease in spontaneous DEL recombination in both TCY5 (hdf1delta) and TCY6 (hdf2delta) strains, suggesting that HDF1 and HDF2 play a role in homologous recombination. As DEL recombination events may occur by sister chromatid conversion and/or single-strand annealing, which is initiated by DSBs, HDF1 and HDF2 may be required to recruit proteins to the damaged ends so as to promote single-strand annealing. The strains TCY5 and TCY6 are also defective in methylmethane sulfonate (MMS)- and X-ray-induced, but not in UV-induced DEL recombination. This confirms that HDF1 and HDF2 are required for the completion of DEL recombination by single strand annealing.

The 113,463-bp nucleotide sequence of the linear plasmid pSLA2-M of Streptomyces rochei 7434AN4 was determined. pSLA2-M had a 69.7% overall GC content, 352-bp terminal inverted repeats with 91% (321/352) identity at both ends, and 121 open reading frames. The rightmost 14.6-kb sequence was almost (14,550/14,555) identical to that of the coexisting 211-kb linear plasmid pSLA2-L. Adjacent to this homologous region an 11.8-kb CRISPR cluster was identified, which is known to function against phage infection in prokaryotes. This cluster region as well as another one containing two large membrane protein genes (orf78 and orf79) were flanked by direct repeats of 194 and 566 bp respectively. Hence the insertion of circular DNAs containing each cluster by homologous recombination was suggested. In addition, the orf71 encoded a Ku70/Ku80-like protein, known to function in the repair of double-strand DNA breaks in eukaryotes, but disruption of it did not affect the radiation sensitivity of the mutant. A pair of replication initiation genes (orf1-orf2) were identified at the extreme left end. Thus, pSLA2-M proved to be a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L that might have been generated by multiple recombination events.

We have studied mechanisms of HSP70 gene regulation at 37 degrees C by the cellular factors NF-IL6 and Ku70. As both factors repress HSF1, we first examined whether phosphorylation on serine 303 and 307 of HSF1 by MAPK and GSK3, which has known to inhibit HSF1, was involved in the repression. However, repression by NF-IL6 was found using HSF1 mutants S303G and S307G refractory to the effects of MAPK and GSK3. We then examined whether NF-IL6 repressed HSP70B by a mechanism resembling Ku proteins. However, in Ku-deficient cells, NF-IL6 was still able to displace HSF1 from heat shock element (HSE) and repressed HSF1 activation. In addition, activation of the HSP 70B promoter by wild type, S303G, or S307G HSF1 was observed to be much more pronounced in Ku-deficient cells. In vitro translated Ku70 interacted with HSF1 by binding to and displacing it from HSE. These data indicate that the repression of the HSP70B promoter by NF-IL6, Ku70, and MAPK occurs independently of each other and involves three complementary mechanisms.

Ku70/80 nonhomologous end-joining activity is essential for resolving random DNA double-strand breaks, and the Ku70/80 protein complex has been proposed as "caretaker" of genomic stability. We studied the Ku70/80 heterodimer activity in a patient affected by multiple basal cell carcinomas with a personal history of moderate exposure to ionizing radiation. The Ku70/80 DNA-binding activity was analyzed, by electrophoretic mobility shift assay, in five tumor biopsies from different sites and at distinct clinical stages, and in three matched normal skin samples from the same patient. As control normal tissues from healthy individuals were also tested. The five basal cell carcinomas were classified as "non aggressive" and "aggressive" on the basis of morphologic parameters and expression of the molecular markers bcl-2, Ki67/MIB1, and p53. A 62% increase in the Ku70/80 DNA-binding activity was found in normal skin from the patient, compared to unexposed individuals (p<0.0001). The nuclear activity of the heterodimer was further increased in nonaggressive basal cell carcinomas compared to both matched normal skin from the patient (31%, p=0.0001) and tissues from healthy controls (73%, p=0.0001). Strikingly, the two aggressive basal cell carcinomas tested showed very low Ku70/80 DNA-binding activity with a reduction of 87% compared to normal skin from the patient (p<0.0001) and 64% compared to controls (p=0.001). Although these results are limited to only one patient, together with other recent studies they support the hypothesis that downregulation of the nonhomologous end-joining pathway may be associated with tumor progression.

The biological consequences of low levels of radiation exposure and their effects on human health are unclear. Ionizing radiation induces a variety of lesions of which DNA double-strand breaks (DSBs) are the most biologically significant, because unrepaired or misrepaired DSBs can lead to genomic instability and cell death. Using repair-proficient mice as an in vivo system we monitored the accumulation of DNA damage in normal tissues exposed to daily low-dose radiation of 100mGy or 10mGy. Radiation-induced foci in differentiated and tissue-specific stem cells were quantified by immunofluorescence microscopy after 2, 4, 6, 8, and 10 weeks of daily low-dose radiation and DNA lesions were characterized using transmission electron microscopy (TEM) combined with immunogold-labeling. In brain, long-living cortical neurons had a significant accumulation of foci with increasing cumulative doses. In intestine and skin, characterized by constant cell renewal of their epithelial lining, differentiated enterocytes and keratinocytes had either unchanged or only slightly increased foci levels during protracted low-dose radiation. Significantly, analysis of epidermal stem cells in skin revealed a constant increase of 53BP1 foci during the first weeks of low-dose radiation even with 10mGy, suggesting substantial accumulations of DSBs. However, TEM analysis suggests that these remaining 53BP1 foci, which are predominantly located in compact heterochromatin, do not co-localize with phosphorylated Ku70 or DNA-PKcs, core components of non-homologous end-joining. The biological relevance of these persistent 53BP1 foci, particularly their contribution to genomic instability by genetic and epigenetic alterations, has to be defined in future studies.

OBJECTIVE

The E3 ubiquitin ligase RNF8 regulates the accumulation or removal of a number of proteins at DNA lesions, thereby playing a critically important role in DNA damage response. The present study investigated the possibility of using RNF8 as a new target in the radiation treatment of human non-small cell lung cancer.

METHODS

We used RNA interference technology to silence the expression of RNF8 in A549 cells, and then detected the radiation response by colony forming assays. DNA repair was monitored by γ-H2AX foci formation after RNF8 depletion. Expression of Ku70 and Rad51 were assessed by immunofluorescent staining and Western blotting. Cell cycle and apoptosis were measured by flow cytometry assays.

RESULTS

After lentivirus-mediated siRNA transfection, expression of RNF8 in A549 cells downregulated which led to an increased radiosensitivity and impaired DNA repair. RNF8 knockdown did not affect Ku70 expression, however, Rad51, a key player in homologous recombination (HR) repair, was abrogated at sites of DNA damage. Furthermore, we observed an extended G2/M arrest and an increased induction of apoptosis after ionizing radiation in the absence of RNF8.

CONCLUSIONS

RNF8 silencing effectively downregulates Rad51 therefore maybe impairing HR repair, and prolongs the G2/M accumulation as well as cell apoptosis upon radiation, which all suggest an enhanced radiosensitivity on A549 cells.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a distinct factor in the non-homologous end-joining (NHEJ) pathway involved in DNA double-strand break (DSB) repair. We examined the crosstalk between key proteins in the DSB NHEJ repair pathway and cell cycle regulation and found that mouse embryonic fibroblast (MEF) cells deficient in DNA-PKcs or Ku70 were more vulnerable to ionizing radiation (IR) compared with wild-type cells and that DSB repair was delayed. γH2AX was associated with phospho-Ataxia-telangiectasia mutated kinase (Ser1987) and phospho-checkpoint effector kinase 1 (Ser345) foci for the arrest of cell cycle through the G2/M phase. Inhibition of DNA-PKcs prolonged IR-induced G2/M phase arrest because of sequential activation of cell cycle checkpoints. DSBs were introduced, and cell cycle checkpoints were recruited after exposure to IR in nasopharyngeal carcinoma SUNE-1 cells. NU7441 radiosensitized MEF cells and SUNE-1 cells by interfering with DSB repair. Together, these results reveal a mechanism in which coupling of DSB repair with the cell cycle radiosensitizes NHEJ repair-deficient cells, justifying further development of DNA-PK inhibitors in cancer therapy.

OBJECTIVE

Ku protein stimulates DNA repair and signals the damage/stress responses, which may affect apoptosis and cell proliferation. The expression of Ku70 has been reported to be related to cell proliferation in a human gastric cancer cell line. However, in colorectal carcinoma, the clinical significance of Ku70 expression remains unclear. We examined the expression pattern of Ku70 in sporadic colorectal cancer and its relation to clinicopathological parameters and survival.

METHODS

We studied 101 consecutive cases of advanced colorectal carcinoma. In resected specimens, the number of cells stained positive for Ku70 protein was evaluated by immunohistochemistry.

RESULTS

The percentage of cells expressing Ku70 in different tumors ranged from 4.2-99.7%. The elevated Ku70 expression group comprised 52 of 101 cases (52%). No significant correlation was seen between Ku expression pattern and the clinical parameters except depth of invasion. pTNM stage, histopathological grade and Ku70 were significant variables for prognosis of survival in the multivariate analysis.

CONCLUSIONS

This is the first report correlating reduced survival with elevated expression of Ku protein in colorectal adenocarcinoma. Ku may be a new prognostic marker useful for selecting adjuvant treatment strategies.

OBJECTIVE

To characterize DNA-PKcs and Ku70 expressions in hepato- and cholangio-neoplastic tissues and the association with the degree of malignancy and invasiveness of the tumors.

METHODS

The expression of DNA-PKcs and Ku70 was examined in 47 cases of hepato- or cholangio-neoplasm by immunohistochemistry.

RESULTS

Ku70 was expressed in all of the neoplastic tissues examined and with a little variation in levels. The highest expression was observed in adenocarcinomas and adenomas. There was no statistically significant association between Ku70 expression level and the degree of their malignancy extent or invasiveness. In contrast to Ku 70, a wide variation in expression levels of DNA-Pkcs was observed among different types of neoplastic tissues. The highest ratio of positive expressing cells was detected in hepatocellular carcinomas (92.1%), which was significantly higher than that in cholangioadeno carcinomas (65.3%) and biliary cystadenocarcinomas (51.9%). Low or no expression level was detected in papillary adenoma cases. DNA-PKcs expression of invasive adenomas and adeno-carcinomas (61.2%) was significantly higher than that of non-invasive adenomas and adeno-carcinomas (30.4%). There was no expression observed in the normal tissues adjacent to the tumors.

CONCLUSIONS

DNA-PKcs is expressed in hepato- and cholangio-neoplasms and its variable level of expression is associated with the types of the tumor and their degree of malignancy and invasiveness. DNA-PKcs could be recognized as a new biomarker for liver neoplasm.

BACKGROUND

While investigating estrogen response element (ERE) binding properties of human estrogen receptor-α (hERα) in breast cancer cytosols, other ERE-binding proteins (ERE-BP) were observed.

METHODS

Recognition properties of ERE-BP were evaluated by electrophoretic mobility shift assays (EMSA) with ERE sequences of the 5'-flanking region of the estrogen responsive gene vitellogenin A2 (VitA2). Cytosols were incubated 16 h, 4 °C with [32P]ERE sequences and separated by EMSA. A method of estimating ERE-BP levels was developed by measuring band intensity from EMSA profiles, expressed in digital light units (DLU)/μg protein and normalized to total DLU. ERE-BP were purified by affinity chromatography and EMSA, and then identified by mass spectrometry.

RESULTS

ERE-BP in cytosols did not supershift in the presence of anti-hERα or anti-hERβ antibodies recognizing different ER epitopes suggesting that they are not fragments of either receptor isoform. ERE-BP competed with hERα for binding to VitA2-ERE. Increased levels of ERE-BP DNA-binding activities measured in 310 cytosols prepared from breast cancer biopsies correlated with decreased patient survival. Strikingly, breast cancer patients with ER negative status and high ERE-BP expression exhibited the poorest disease-free and overall survival. After purification, ERE-BP were identified as Ku70 (XRCC6) and Ku80 (XRCC5) using mass spectrometry. ERE-BP were confirmed to be Ku70/80 by supershift assay.

CONCLUSIONS

Presence of these novel ERE-binding proteins in a breast carcinoma is a strong predictor of poor prognosis. Our results suggest that ERE-BP, identified as Ku70/Ku80, in cytosols prepared from breast carcinoma biopsies are useful biomarkers for assessing risk of breast cancer recurrence.

Radiosurgery is used increasingly upon recurrence of high-grade gliomas to deliver a high dose of focused radiation to a defined target. The purpose of our study was to compare intermittent irradiation (IIR) by using a CyberKnife (CK) with continuous irradiation (CIR) by using a conventional linear accelerator (LINAC). A significant decrease in surviving fraction was observed after IIR irradiation compared with after CIR at a dose of 8 Gy. Three hours after irradiation, most of the DNA damage was repaired in U87. Slightly higher basal levels of Ku70/80 mRNA were found in U87 compared with A172, while radiation treatment induced only minor regulation of Ku70/80 and Rad51 transcription in either cell lines. IIR treatment using CK significantly decreased the survival in U87 and A172 compared with CIR. Although the two cell lines differed in DNA repair capability, the role of Ku70/80 and Rad51 in the cell line radiosensitivity seemed marginal.

A comparative approach in biology is needed to assess the universality of rules governing this discipline. In plant telomere research, most of the key principles were established based on studies in only single model plant, Arabidopsis thaliana. These principles include the absence of telomere shortening during plant development and the corresponding activity of telomerase in dividing (meristem) plant cells. Here we examine these principles in Physcomitrella patens as a representative of lower plants. To follow telomerase expression, we first characterize the gene coding for the telomerase reverse transcriptase subunit PpTERT in P. patens, for which only incomplete prediction has been available so far. In protonema cultures of P. patens, growing by filament apical cell division, the proportion of apical (dividing) cells was quantified and telomere length, telomerase expression and activity were determined. Our results show telomere stability and demonstrate proportionality of telomerase activity and expression with the number of apical cells. In addition, we analyze telomere maintenance in mre11, rad50, nbs1, ku70 and lig4 mutants of P. patens and compare the impact of these mutations in double-strand-break (DSB) repair pathways with earlier observations in corresponding A. thaliana mutants. Telomere phenotypes are absent and DSB repair kinetics is not affected in P. patens mutants for DSB factors involved in non-homologous end joining (NHEJ). This is compliant with the overall dominance of homologous recombination over NHEJ pathways in the moss, contrary to the inverse situation in flowering plants.

SC-1, the aqueous phase of soybean fermentation products by bacteria (Bacillus subtilis and Bacillus brevis), significantly inhibited the growth and clonogenesity of human hepatocellular (Hep 3B), mouse hepatocellular (ML-1), and human colorectal (HCT 116 and HT-29) carcinoma cells. Cytotoxicity of SC-1 in Hep 3B cells was through the process of apoptosis characterizing by increase in cell population of sub-G(1) phase, fragmentation of DNA, and change of nuclear morphology. Treatment of Hep 3B cells with SC-1 activated caspase 8 and caspase 3. Elevation of nuclear DNA fragmentation factor 40 (DFF40) and cleavage form of poly(ADP-ribose) polymerase (PARP) were also observed. SC-1 also activated intrinsic pathway via increase of pro-apoptotic (tBid, Bak and Bax) and decrease of anti-apoptotic (Bcl-2 and Bcl-x(L)) proteins on mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspase/direct IAP binding protein with low PI) from mitochondria, and activation of caspase 9. Inhibition on protein expression of Ku70 in cytosol and cyclooxygenase (COX)-2, but not COX-1, in whole cell lystes were revealed in SC-1-treated Hep 3B cells. These results suggest caspase 8, Ku70 and mitochondria are involved in the antitumor mechanism of SC-1 in Hep 3B cells.

Ku70, a DNA repair factor in the nucleus, also regulates cell death by binding to the apoptotic protein Bax in the cytoplasm. Acetylation of Ku70 triggers Bax release resulting in Bax dependent cell death. Thus dissociating Bax from Ku70, either by inhibiting histone deacetylase 6 (HDAC6) that deacetylates Ku70 or by increasing Ku70 acetylation induces cell death. Our results showed that in neuroblastoma cells, the depletion of Ku70 results in Bax-dependent cell death. This model provides a rationale for screening Ku70 acetylation modulators that can be tested in clinical trials, either alone or in combination with radiotherapy or DNA-damaging agents for the treatment of cancer.

Fluorescence immunostaining for the phosphorylated H2AX histone (gammaH2AX) in the grasshopper Eyprepocnemis plorans has shown abundance of gammaH2AX in the nuclei of round and elongating spermatids, suggesting that DNA double-strand breaks (DSBs) occur regularly during spermiogenesis. Immunofluorescence patterns for Ku70, a DNA-repair protein participating in the non-homologous end-joining (NHEJ) pathway, showed that this protein is present in round and elongating spermatids, implying that the NHEJ DNA-repair pathway operates during chromatin compaction in spermiogenesis. In addition, during the final stages of spermiogenesis, the Ku70 protein concentrates on the region forming the sperm tail. Since Ku70 was also abundant in spermatid tails, it is reasonable to assume that Ku70 might play a novel function in sperm-tail formation. The analysis of Ku70 immunofluorescence patterns in 13 other grasshopper species also showed the presence of this protein in the nucleus and tail of elongating spermatids, indicating that this is a general characteristic in grasshoppers.

BACKGROUND

Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances the Ig and TcR gene diversity in the N region at the junctions of variable (V), diversity (D) and joining (J) segments in B- and T-cells. TdT synthesizes the N region in concert with many proteins including DNA-PKcs, Ku70 and Ku86. To elucidate the molecular mechanism of the N region synthesis, we first attempted to isolate the genes with products that directly interact with TdT.

RESULTS

Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 2 (TdIF2). The confined region of the C-terminal in TdIF2 is involved in specific interaction with the entire C-terminal in TdT. TdIF2 contains an acidic region comprised of 42 residues. TdIF2 was shown to bind specifically to a core histone by pull down assay using specific antibodies against TdIF2. When a TdT/TdIF2 complex was applied on to a DNA-cellulose column, only TdT bound to the column while TdIF2 passed through. TdIF2 reduces the TdT activity to 46% of its maximum value in vitro assay system using activated DNA as primer.

CONCLUSIONS

TdIF2 binds directly to TdT and core histone. Furthermore, TdT, TdIF2 and core histone form a ternary complex. TdIF2 liberates H2A/H2B from a core histone in correlation with PCNA. The enzymatic consequence of the TdIF2/TdT complex is the reduction of TdT activity in vitro. TdIF2 would function as a chromatin remodeling protein at the N region synthesis.

Normally, gene targeting by homologous recombination occurs rarely during a transformation process since non-homologous recombination is predominant in filamentous fungi. In our previous researches, the average gene replacement frequency (GRF) in Monascus ruber M7 was as low as 15 %. To develop a highly efficient gene targeting system for M. ruber M7, two M. ruber M7 null mutants of ku70 (MrΔku70) and ku80 (MrΔku80) were constructed which had no apparent defects in the development including vegetative growth, colony phenotype, microscopic morphology and spore yield compared with M. ruber M7. In addition, the production of some significant secondary metabolites such as pigments and citrinin had no differences between the two disruptants and the wild-type strain. Further results revealed that the GRFs of triA (encoding a putative acetyltransferase) were 42.2 % and 61.5 % in the MrΔku70 and MrΔku80 strains, respectively, while it was only about 20 % in M. ruber M7. Furthermore, GRFs of these two disruptants at other loci (the pigE, fmdS genes in MrΔku70 and the ku70 gene in MrΔku80) were investigated, and the results indicated that GRFs in the MrΔku70 strain and the MrΔku80 strain were doubled and tripled compared with that in M. ruber M7, respectively. Therefore, the ku70 and ku80 null mutants of M. ruber M7, especially the ku80-deleted strain, will be excellent hosts for efficient gene targeting.

Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C. lusitaniae is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of C. lusitaniae. We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in C. lusitaniae strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in C. lusitaniae and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. IMPORTANCE The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology.

Numerous strains of Aspergillus oryzae are industrially used for Japanese traditional fermentation and for the production of enzymes and heterologous proteins. In A. oryzae, deletion of the ku70 or ligD genes involved in non-homologous end joining (NHEJ) has allowed high gene targeting efficiency. However, this strategy has been mainly applied under the genetic background of the A. oryzae wild strain RIB40, and it would be laborious to delete the NHEJ genes in many A. oryzae industrial strains, probably due to their low gene targeting efficiency. In the present study, we generated ligD mutants from the A. oryzae industrial strains by employing the CRISPR/Cas9 system, which we previously developed as a genome editing method. Uridine/uracil auxotrophic strains were generated by deletion of the pyrG gene, which was subsequently used as a selective marker. We examined the gene targeting efficiency with the ecdR gene, of which deletion was reported to induce sclerotia formation under the genetic background of the strain RIB40. As expected, the deletion efficiencies were high, around 60~80%, in the ligD mutants of industrial strains. Intriguingly, the effects of the ecdR deletion on sclerotia formation varied depending on the strains, and we found sclerotia-like structures under the background of the industrial strains, which have never been reported to form sclerotia. The present study demonstrates that introducing ligD mutation by genome editing is an effective method allowing high gene targeting efficiency in A. oryzae industrial strains.

Nonhomologous DNA end joining (NHEJ) is one of the major DNA double-strand break (DSB) repair pathways in eukaryotes. The well-known critical proteins involved in NHEJ include Ku70/80, DNA-PKcs, Artemis, DNA pol λ/μ, DNA ligase IV-XRCC4, and XLF. Recent studies have added a number of new proteins to the NHEJ repertoire namely paralog of XRCC4 and XLF (PAXX), modulator of retroviral infection (MRI)/ cell cycle regulator of NHEJ (CYREN), transactivation response DNA-binding protein (TARDBP) of 43 kDa (TDP-43), intermediate filament family orphan (IFFO1), ERCC excision repair 6 like 2 (ERCC6L2), and RNase H2. PAXX acts as a stabilizing factor for the main NHEJ components. MRI/CYREN seems to play a dual role stimulating NHEJ in the G1 phase of the cell cycle, while inhibiting the pathway in the S and G2 phases. TDP-43 can recruit the ligase IV-XRCC4 complex to the DSB sites and stimulate ligation in neuronal cells. RNase H2 excises out the ribonucleotides inserted during repair by DNA polymerase μ/TdT. This review provides a brief glimpse into how these new partners were discovered and their contribution to the mechanism and regulation of NHEJ.

Keywords: DSB repair; NHEJ machinery; cell cycle; double-strand break; end joining; genomic instability; homologous recombination.

Ku70, a known nonhomologous end-joining (NHEJ) factor, also functions in tumor suppression, although this molecular mechanism remains uncharacterized. Previously, we showed that mice deficient for DNA ligase IV (Lig4), another key NHEJ factor, succumbed to aggressive lymphoma in the absence of tumor suppressor p53. However, the tumor phenotype is abrogated by the introduction of a hypomorphic mutant p53(R172P), which impaired p53-mediated apoptosis but not cell-cycle arrest. However, Lig4(-/-)p53(R172P) mice succumbed to severe diabetes. To further elucidate the role of NHEJ and p53-mediated apoptosis in vivo, we bred Ku70(-/-) p53(R172P) mice. Unexpectedly, these mice were free of diabetes, although 80% of the mutant mice had abnormally enlarged colons with pronounced inflammation. Remarkably, most of these mutant mice progressed to dysplasia, adenoma and adenocarcinoma; this is in contrast to the Lig4(-/-)p53(R172P) phenotype, strongly suggesting an NHEJ-independent function of Ku70. Significantly, our analyses of Ku70(-/-)p53(R172P) colonic epithelial cells show nuclear stabilization of β-catenin accompanied by higher expression of cyclin D1 and c-Myc in affected colon sections than in control samples. This is not due to the p53 mutation, as Ku70(-/-) mice share this phenotype. Our results not only unravel a novel function of Ku70 essential for colon homeostasis, but also establish an excellent in vivo model in which to study how chronic inflammation and abnormal cellular proliferation underlie tumorigenesis and tumor progression in the colon.

Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.