The DNA helicase BRIP1 is defective in Fanconi anemia complementation group J.

The protein predicted to be defective in individuals with Fanconi anemia complementation group J (FA-J), FANCJ, is a missing component in the Fanconi anemia pathway of genome maintenance. Here we identify pathogenic mutations in eight individuals with FA-J in the gene encoding the DEAH-box DNA helicase BRIP1, also called FANCJ. This finding is compelling evidence that the Fanconi anemia pathway functions through a direct physical interaction with DNA.

[The role of the Fanconi anemia pathway in DNA repair and maintenance of genome stability]. / Rola bialek szlaku niedokrwistosci Fanconiego w naprawie DNA i utrzymaniu stabilnosci genomu.

The Fanconi anemia (FA) pathway is one of the DNA repair systems involved in removal of DNA crosslinks. Proteins which belong to this pathway are crucial to the protection of genetic information, whereas disturbances in their function have serious implications for the whole organism. Biallelic mutations in FA genes are the cause of Fanconi anemia - a genetic disease which manifests itself through numerous congenital abnormalities, chromosomal instability and increased predisposition to cancer. The FA pathway is composed of fifteen proteins. Eight of them, in the presence of DNA interstrand crosslinks (ICLs), form a nuclear core complex responsible for monoubiquitination of FANCD2 and FANCI, which is a key step of ICL repair. FA proteins which are not involved in the monoubiquitination step participate in repair of DNA double strand breaks via homologous recombination. Some of the FA proteins, besides having a direct role in the repair of DNA damage, are engaged in replication, cell cycle control and mitosis. The unperturbed course of those processes determines the maintenance of genome stability.

Warsaw breakage syndrome, a cohesinopathy associated with mutations in the XPD helicase family member DDX11/ChlR1.

The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.

Synthetic lethal targeting of DNA double-strand break repair deficient cells by human apurinic/apyrimidinic endonuclease inhibitors.

An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In this study, we have investigated the ability of APE1 inhibitors to induce synthetic lethality (SL) in a panel of DNA double-strand break (DSB) repair deficient and proficient cells; i) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant [V-C8(Rev1)]. ii) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested SL in CH ovary cells expressing a dominant-negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. SL was also demonstrated in CH cells expressing a dominant-negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising SL target in cancer.

A DNA-PKcs mutation in a radiosensitive T-B- SCID patient inhibits Artemis activation and nonhomologous end-joining.

Radiosensitive T-B- severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.

[Poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2 cancer therapy]. / Inhibitory polimerazy poli(ADP-rybozy) (PARP) w terapii nowotworów z mutacjami BRCA1/2.

 A majority of currently used anticancer drugs belong to a group of chemical agents that damage DNA. The efficiency of the treatment is limited by effective DNA repair systems functioning in cancer cells. Many chemotherapeutic compounds cause strong systemic toxicity. Therefore, there is still a need for new anticancer agents which are less toxic for nontransformed cells and selectively kill cancer cells. One of the most promising molecular targets in cancer therapy is poly(ADP-ribose) polymerases (PARP). PARP play an essential role in repairing DNA strand breaks. Small molecule inhibitors of these enzymes have been developed and have proved to be extremely toxic for cancer cells that lack the functional BRCA1 and BRCA2 proteins that are involved in homologous recombination, a complex repair mechanism of DNA double strand breaks. Mutations in BRCA1/2 genes are associated with genetically inherited breast and ovarian cancers. Therefore PARP inhibitors may prove to be very effective and selective in the treatment of these cancer types. This review is focused on the function of BRCA1/2 proteins and poly(ADP-ribose) polymerases in DNA repair systems, especially in the homologous recombination process. A short history of the studies that led to synthesis of high specificity small molecule PARP inhibitors is also presented, as well as the results of clinical trials concerning the most effective PARP inhibitors in view of their potential application in oncological treatment, particularly breast cancers.

Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage.

The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA(-/-), FANCC(-/-) and FANCA(-/-)C(-/-) cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2(-/-) cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.

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

O(6)-methylguanine (O(6)MeG) is a highly critical DNA adduct induced by methylating carcinogens and anticancer drugs such as temozolomide, streptozotocine, procarbazine and dacarbazine. Induction of cell death by O(6)MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O(6)MeG. Here, we compared the response of cells mutated in HR and NHEJ proteins to temozolomide and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (DNA-PK(cs) mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of O(6)-methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific hypersensitivity was due to O(6)MeG lesions. MNNG provoked H2AX phosphorylation 24-48h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The gammaH2AX foci formed in response to O(6)MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O(6)MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O(6)MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving cancer therapy with methylating agents.

Rad51C is essential for embryonic development and haploinsufficiency causes increased DNA damage sensitivity and genomic instability.

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C(splice) heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C(splice) embryos die during early development (E8.5). Detailed analysis of two CL-V4B revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C(splice/neo) heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation.

A new type of radiosensitive T-B-NK+ severe combined immunodeficiency caused by a LIG4 mutation.

V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T-B-NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual D(H)-J(H) junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed D(H)-J(H) ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development.

Brca2 (XRCC11) deficiency results in radioresistant DNA synthesis and a higher frequency of spontaneous deletions.

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.

Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle.

We analyzed the phenotype of cells derived from SCID patients with different mutations in the Artemis gene. Using clonogenic survival assay an increased sensitivity was found to X-rays (2-3-fold) and bleomycin (2-fold), as well as to etoposide, camptothecin and methylmethane sulphonate (up to 1.5-fold). In contrast, we did not find increased sensitivity to cross-linking agents mitomycin C and cis-platinum. The kinetics of DSB repair assessed by pulsed-field gel electrophoresis and gammaH2AX foci formation after ionizing irradiation, indicate that 15-20% of DSB are not repaired in Artemis-deficient cells. In order to get a better understanding of the repair defect in Artemis-deficient cells, we studied chromosomal damage at different stages of the cell cycle. In contrast to AT cells, Artemis-deficient cells appear to have a normal G(1)/S-block that resulted in a similar frequency of dicentrics and translocations, however, frequency of acentrics fragments was found to be 2-4-fold higher compared to normal fibroblasts. Irradiation in G(2) resulted in a higher frequency of chromatid-type aberrations (1.5-3-fold) than in normal cells, indicating that a fraction of DSB requires Artemis for proper repair. Our data are consistent with a function of Artemis protein in processing of a subset of complex DSB, without G(1) cell cycle checkpoint defects. This type of DSB can be induced in high proportion and persist through S-phase and in part might be responsible for the formation of chromatid-type exchanges in G(1)-irradiated Artemis-deficient cells. Among different human radiosensitive fibroblasts studied for endogenous (in untreated samples) as well as X-ray-induced DNA damage, the ranking order on the basis of higher incidence of spontaneously occurring chromosomal alterations and induced ones was: ligase 4> or =AT>Artemis. This observation implicates that in human fibroblasts following exposure to ionizing radiation a lower risk might be created when cells are devoid of endogenous damage.

Distinct cellular phenotype linked to defective DNA interstrand crosslink repair and homologous recombination.

Repair of DNA interstrand crosslinks (ICLs) predominantly involves the Fanconi anemia (FA) pathway and homologous recombination (HR). The HR repair system eliminates DNA double strand breaks (DSBs) that emerge during ICLs removal. The current study presents a novel cell line, CL­V8B, representing a new complementation group of Chinese hamster cell mutants hypersensitive to DNA crosslinking factors. CL­V8B exhibits increased sensitivity to various DNA­damaging agents, including compounds leading to DSBs formation (bleomycin and 6­thioguanine), and is extremely sensitive to poly (ADP-ribose) polymerase inhibitor (>400­fold), which is typical for HR­defective cells. In addition, this cell line exhibits a reduced number of spontaneous and induced sister chromatid exchanges, which suggests likely impairment of HR in CL­V8B cells. However, in contrast to other known HR mutants, CL­V8B cells do not show defects in Rad51 foci induction, but only slight alterations in the focus formation kinetics. CL­V8B is additionally characterized by a considerable chromosomal instability, as indicated by a high number of spontaneous and MMC­induced chromosomal aberrations, and a twice as large proportion of cells with abnormal centrosomes than that in the wild type cell line. The molecular defect present in CL­V8B does not affect the efficiency and stabilization of replication forks. However, stalling of the forks in response to replication stress is observed relatively rarely, which suggests an impairment of a signaling mechanism. Exposure of CL­V8B to crosslinking agents results in S­phase arrest (as in the wild type cells), but also in larger proportion of G2/M­phase cells and apoptotic cells. CL­V8B exhibits similarities to HR­ and/or FA­defective Chinese hamster mutants sensitive to DNA crosslinking agents. However, the unique phenotype of this new mutant implies that it carries a defect of a yet unidentified gene involved in the repair of ICLs.

Does tumorigenesis select for or against mutations of the DNA repair-associated genes BRCA2 and MRE11?: considerations from somatic mutations in microsatellite unstable (MSI) gastrointestinal cancers.

BACKGROUND: The BRCA2 and MRE11 proteins participate in the repair of double-strand DNA breaks by homologous recombination. Germline BRCA2 mutations predispose to ovarian, breast and pancreatic cancer, while a germline MRE11 mutation is associated with an ataxia telangiectasia-like disorder. Somatic mutations of BRCA2 are rare in typical sporadic cancers. In tumors having microsatellite instability (MSI), somatic truncating mutations in a poly [A] tract of BRCA2 are reported on occasion. RESULTS: We analyzed gastrointestinal MSI cancers by whole gene BRCA2 sequencing, finding heterozygous truncating mutations in seven (47%) of 15 patients. There was no cellular functional defect in RAD51 focus-formation in three heterozygously mutated lines studied, although other potential functions of the BRCA2 protein could still be affected. A prior report of mutations in primary MSI tumors affecting the IVS5-(5-15) poly [T] tract of the MRE11 gene was confirmed and extended by analysis of the genomic sequence and protein expression in MSI cancer cell lines. Statistical analysis of the published MRE11 mutation rate in MSI tumors did not provide evidence for a selective pressure favoring biallelic mutations at this repeat. CONCLUSION: Perhaps conflicting with common suspicions, the data are not compatible with selective pressures during tumorigenesis promoting the functional loss of BRCA2 and MRE11 in MSI tumors. Instead, these data fit closely with an absence of selective pressures acting on BRCA2 and MRE11 gene status during tumorigenesis.

Isolation of mutagen-sensitive Chinese hamster cell lines by replica plating.

Mutant rodent cell lines hypersensitive to DNA-damaging agents have provided a useful tool for the characterization of DNA repair pathways and have contributed to a better understanding of the mechanisms involved in the cellular responses to mutagenic treatment. Here we present a detailed description of how to isolate mutagen-sensitive mutants from hamster "wild-type" cell lines. First, cells are treated with ethyl nitrosourea, and then the mutagenized cell populations are screened for cells with an increased sensitivity to various mutagens using a replica-plating method. Mutagen-sensitive clones are identified and then characterized by assessing their stability, degree of sensitivity to various mutagens, and by genetic complementation analysis.

Inhibition of DNA synthesis by ionizing radiation: a marker for an S-phase checkpoint.

Inhibition of replicative DNA synthesis by ionizing radiation is partly caused by an active, signal-mediated response termed the "S-phase checkpoint." Defects in this checkpoint were first discovered in the human inherited disorder ataxia-telangiectasia (AT). gamma-Irradiated cells from AT patients consistently display a diminished inhibition of DNA synthesis, a feature called "radioresistant DNA synthesis" (RDS). RDS has been widely used as a diagnostic marker for AT, in postnatal as well as prenatal material. The regulation and control of the S-phase checkpoint is complex and multifaceted; it is not restricted to ionizing radiation, but can occur after many genotoxic stressors. Defects in both upstream control functions, such as ATM, NBS1, and MRE11, as well as downstream modulators can provoke an RDS phenotype. Here a simple, accurate and highly reproducible experimental protocol is presented for the generation of DNA synthesis inhibition curves from cells in culture.

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function.

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.

Inducibility of nuclear Rad51 foci after DNA damage distinguishes all Fanconi anemia complementation groups from D1/BRCA2.

Fanconi anemia (FA) is a cancer susceptibility disorder characterized by chromosomal instability and hypersensitivity to DNA cross-linking agents. So far 11 complementation groups have been identified, from which only FA-D1/BRCA2 and FA-J are defective downstream of the central FANCD2 protein as cells from these groups are capable of monoubiquitinating FANCD2. In this study we show that cells derived from patients from the new complementation groups, FA-I, FA-J and FA-L are all proficient in DNA damage induced Rad51 foci formation, making the cells from FA-D1/BRCA2 patients that are defective in this process the sole exception. Although FA-B patient HSC230 was previously reported to also have biallelic BRCA2 mutations, we found normal Rad51 foci formation in cells from this patient, consistent with the recent identification of an X-linked gene being mutated in four unrelated FA-B patients. Thus, our data show that none of the FA proteins, except BRCA2, are required to sequester Rad51 into nuclear foci. Since cells from the FA-D1 and FA-J patient groups are both able to monoubiquitinate FANCD2, the "Rad51 foci phenotype" provides a convenient assay to distinguish between these two groups. Our results suggest that FANCJ and FANCD1/BRCA2 are part of the integrated FANC/BRCA DNA damage response pathway or, alternatively, that they represent sub-pathways in which only FANCD1/BRCA2 is directly connected to the process of homologous recombination.

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2.

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway.

A single amino acid substitution in DNA-PKcs explains the novel phenotype of the CHO mutant, XR-C2.

We recently described a CHO DSBR mutant belonging to the XRCC7 complementation group (XR-C2) that has the interesting phenotype of being radiosensitive, but having only a modest defect in VDJ recombination. This cell line expresses only slightly reduced levels of DNA-PKcs but has undetectable DNA-PK activity. Limited sequence analyses of DNA-PKcs transcripts from XR-C2 revealed a point mutation that results in an amino acid substitution of glutamic acid for glycine six residues from the C-terminus. To determine whether this single substitution was responsible for the phenotype in XR-C2 cells, we introduced the mutation into a DNA-PKcs expression vector. Whereas transfection of this expression vector significantly restores the VDJ recombination deficits in DNA-PKcs-deficient cells, radioresistance is not restored. Thus, expression of this mutant form of DNA-PKcs in DNA-PKcs- deficient cells substantially recapitulates the phenotype observed in XR-C2, and we conclude that this single amino acid substitution is responsible for the non-homologous end joining deficits observed in XR-C2.