Vector-mediated DNA double-strand break repair analysis in normal, and radiation-sensitive, Chinese hamster V79 cells

DNA double-strand break repair was assessed in 2 new radiation-sensitive V79 hamster cell lines (irs1 and irs2) by their ability to rejoin restriction endonuclease cuts in a transferred selectable SV40--E. coli gpt recombinant gene. The studied gene was carried in the vector pPMH16 which also contained a second selectable HSVtk-neo recombinant gene which acted as a control for DNA transformation. The parental V79 cells showed correct rejoining of KpnI and EcoRV double-strand breaks in approximately 18% and 36% of transformants respectively (correcting for the expression of undamaged gpt in neo+ transformants). irs1 shows a significantly reduced (approximately 3-fold) ability to rejoin correctly such double-strand scissions. However, irs2 rejoined such lesions as correctly as the V79 cells. The data are discussed in the context of the assay and the possible repair deficiencies of these radiosensitive mutant cells.     

V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair.

V(D)J recombination has been examined in several X-ray-sensitive and double-strand break repair-deficient Chinese hamster cell mutants. Signal joint formation was affected in four mutants (xrs 5, XR-1, V-3, and XR-V9B cells, representing complementation groups 1 through 4, respectively) defective in DNA double-strand break rejoining. Among these four, V-3 and XR-V9B were the most severely affected. Only in V-3 was coding joint formation also affected. Ataxia telangiectasia-like hamster cell mutants (V-E5 and V-G8), which are normal for double-strand break repair but are X ray sensitive, were normal for all aspects of the V(D)J recombination reaction, indicating that X-ray sensitivity is not the common denominator but that the deficiency in double-strand break repair appears to be. The abnormality at the signal joints consisted of an elevated incidence of nucleotide loss from each of the two signal ends. Interestingly, in complementation groups 1 (xrs 5) and 2 (XR-1), signal joint formation was within the normal range under some transfection conditions. This suggests that the affected gene products in these two complementation groups are not catalytic components. Instead, they may be either secondary or stochiometric components involved in the later stages of both the V(D)J recombination reaction and double-strand break repair. The fact that such factors can affect the precision of the signal joint has mechanistic implications for V(D)J recombination.     

X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining

Six CHO mutants have previously been described as being sensitive to ionizing radiation and bleomycin treatment, with little or no cross sensitivity to UV-radiation (Jeggo and Kemp, 1983). Their ability to rejoin single- and double-strand breaks has been examined here. Using two techniques, gradient sedimentation and alkaline elution, no difference could be observed between wild-type and mutant strains in the initial number of single-strand breaks induced, the rate of rejoining, or the final level of single-strand breaks rejoined. Thus, a major inability to rejoin single-strand breaks is not the basis for sensitivity in these mutants. In contrast, all 6 mutants showed a decreased ability to rejoin the double-strand breaks induced by gamma-irradiation as measured by neutral elution. Rejoining of half of the breaks occurred in 37 min in wild-type cells and reached a maximum level of 72% after 2 h. All the mutants showed a decreased rate of rejoining, and the final level was 17% of that observed in the wild-type in the most defective mutant, and ranged from 35 to 69% in the other 5 mutants. These are the first mammalian cell mutants to be described with a defect in double-strand break rejoining.     

Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line

Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiation-induced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.     

Mammalian X-ray-sensitive mutants which are defective in non-homologous (illegitimate) DNA double-strand break repair

In all organisms multiple pathways to repair DNA double-strand breaks (DSB) have been identified. In mammalian cells DSB are repaired by two distinct pathways, homologous and non-homologous (illegitimate) recombination. X-ray-sensitive mutants have provided a tool for the identification and understanding of the illegitimate recombination pathway in mammalian cells. Two (sub-)pathways can be distinguished, the first mediated by DNA-PK-dependent protein kinase (DNA-PK), and the second directed by the hMre11/hRad50 complex. A variety of mutants impaired in DSB repair by illegitimate recombination, with mutations in Ku, DNA-PKcs, XRCC4 or nibrin, have been described. Herein, the characterization of these mutants with respect to the impaired cellular function and the molecular defect is provided. Further studies on these mutants, as well as on new mutants impaired in as-of-yet unidentified pathways, should be helpful to a better understanding of DSB repair and of the processes leading to genome instability and cancer.     

Identification and characterization of a third complementation group of emetine-resistant Chinese hamster cell mutants.

We have isolated emetine-resistant cell lines from Chinese hamster peritoneal fibroblasts and have shown that they represent a third distinct class or complementation group of emetine-resistant mutants, as determined by three different criteria. These mutants, like those belonging to the two other complementation groups we have previously defined, which were isolated from Chinese hamster lung and Chinese hamster ovary cells, have alterations that directly affect the protein biosynthetic machinery. So far, there is absolute cell line specificity with respect to the three complementation groups, in that all the emetine-resistant mutants we have isolated from Chinese hamster lung cells belong to one complementation group, all those we have isolated from Chinese hamster ovary cells belong to a second complementation group, and all those isolated from Chinese hamster peritoneal cells belong to a third complementation group. Thus, in cultured Chinese hamster cells, mutations in at least three different loci, designated emtA, emtB, and emtC, encoding for different components of the protein biosynthetic machinery, can give rise to the emetine-resistant phenotype.     

Newly identified Chinese hamster ovary cell mutants defective in peroxisome assembly represent complementation group A of human peroxisome biogenesis disorders and one novel group in mammals

We isolated peroxisome biogenesis-defective mutants from rat PEX2-transformed Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet method. A total of 18 mutant cell clones showing cytosolic localization of catalase were isolated. By complementation group (CG) analysis by means of PEX cDNA transfection and cell fusion, cell mutants, ZP124 and ZP126, were found to belong to two novel CGs of CHO mutants. Mutants, ZP135 and ZP167, were also classified to the same CG as ZP124. Further cell fusion analysis using 12 CGs fibroblasts from patients with peroxisome deficiency disorders such as Zellweger syndrome revealed that ZP124 belonged to human CG-A, the same group as CG-VIII in the United States. ZP126 could not be classified to any of human and CHO CGs. These mutants also showed typical peroxisome assembly-defective phenotypes such as severe loss of catalase latency and impaired biogenesis of peroxisomal enzymes. Collectively, ZP124 represents CG-A, and ZP126 is in a newly identified CG distinct from the 14 mammalian CGs previously characterized.     

Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells

Interstitial Telomeric Repeat Sequence (ITRS) blocks are recognized as hot spots for spontaneous and ionizing radiation-induced chromosome breakage and recombination. Background and ionizing radiation-induced DNA breaks in large blocks of ITRS from Chinese hamster cell lines were analyzed using the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. Our results indicate an extremely alkali-sensitivity of ITRS. Furthermore, it appears that ITRS blocks exhibit a particular chromatin structure, being enriched in short unpaired DNA segments. These segments could be liable to severe topological stress in highly compacted areas of the genome resulting in their spontaneous fragility and thus explaining their alkali-sensitivity. The induction and repair kinetics of DNA single-strand breaks (ssb) and DNA double-strand breaks (dsb) induced by ionizing radiation were assessed by DBD-FISH on neutral comets using Chinese hamster cells deficient in either DNA-PKcs or Rad51C. Our results indicate that the initial rejoining rate of dsb within ITRS is slower than that in the whole genome, in wild-type cells, demonstrating an intragenomic heterogeneity in dsb repair. Interestingly, in the absence of DNA-PKcs activity, the rejoining rate of dsb within ITRS is not modified, unlike in the whole genome. This was also found in the case of Rad51C mutant cells. Our results suggest the possibility that different DNA sequences or chromatin organizations may be targeted by specific dsb repair pathways. Furthermore, it appears that additional unknown dsb repair pathways may be operational in mammalian cells.     

Chinese hamster ovary cell mutants defective in myo-inositol transport

By means of an in situ colony autoradiographic assay for the incorporation of [14C]inositol into the trichloroacetic acid-insoluble fraction, we have isolated a mutant of cultured Chinese hamster ovary cells defective in inositol transport, named mutant 648. Through comparison of the inositol uptake activity of 648 cells with that of the parental cells with various concentrations of inositol and sodium, it has been demonstrated that Chinese hamster ovary cells possess a sodium-dependent transport system for inositol, and that 648 cells lack this system. The sodium-dependent uptake is inhibited by 2,4-dinitrophenol and ouabain, and the intracellular concentration of inositol exceeds the extracellular concentration during the uptake period, indicating that it is active transport, at least partially driven by the sodium gradient generated by Na+,K(+)-ATPase. The apparent Km for inositol has been estimated to be 12.0 microM. It is inhibited by hyperglycemic concentration of D-glucose in a competitive fashion.     

Chromatin dynamics in DNA double-strand break repair

DNA double-strand breaks (DSBs) occur in the context of a highly organized chromatin environment and are, thus, a significant threat to the epigenomic integrity of eukaryotic cells. Changes in break-proximal chromatin structure are thought to be a prerequisite for efficient DNA repair and may help protect the structural integrity of the nucleus. Unlike most bona fide DNA repair factors, chromatin influences the repair process at several levels: the existing chromatin context at the site of damage directly affects the access and kinetics of the repair machinery; DSB induced chromatin modifications influence the choice of repair factors, thereby modulating repair outcome; lastly, DNA damage can have a significant impact on chromatin beyond the site of damage. We will discuss recent findings that highlight both the complexity and importance of dynamic and tightly orchestrated chromatin reorganization to ensure efficient DSB repair and nuclear integrity. This article is part of a Special Issue entitled: Chromatin in time and space.     

A cell-cycle stage-related chromosomal X-ray hypersensitivity in larval neuroblasts of Drosophila mei-9 and mei-41 mutants suggesting defective DNA double-strand break repair

We have examined the chromosomal X-ray hypersensitivity in relation to the cell cycle in larval neuroblasts of the mutagen-sensitive and excision repair-defective mutant mei-9 and of the mutagen-sensitive and post-replication repair-defective mutant mei-41 of Drosophila melanogaster. When compared to wild-type cells, cells bearing the mei-9L1 allele produced unusually high levels in particular of chromatid deletions and to a lesser extent also of isochromatid deletions, but virtually no exchange aberrations. The chromosomal hypersensitivity is apparent at M1 when cells are irradiated in S or G2 but not when irradiated in G1. On the other hand, following irradiation cells bearing the mei-41D5 allele predominantly produce chromosome deletions. Also dicentric and chromatid exchange formation is enhanced with a moderate increase in chromatid deletions. The phases of major sensitivity are the S and G1. Mei-9 and mei-41 mutants have been classified to date as proficient in DNA double-strand break repair. The data presented in this paper revealed an S-independent clastogenic hypersensitivity of mei-9 and mei-41 cells. They are interpreted as indicative evidence for the presence of impaired DNA double-strand break repair. The cell-cycle-related difference in the ratio of chromatid- versus chromosome-type deletions in both mutants suggests repair defects at partially different phases of the cell cycle in mei-9 and mei-41 mutant cells.     

Histone H2A phosphorylation in DNA double-strand break repair

DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process. One of the best-characterized chromatin modification events in DNA-damage responses is the phosphorylation of the SQ motif found in histone H2A or the H2AX histone variant in higher eukaryotes. This modification is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved function. One function that histone modifications can have is to provide a unique binding site for interacting factors. Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implications of these interactions.     

Chinese hamster ovary cell mutants defective in the internalization of ricin.

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.     

A role for small RNAs in DNA double-strand break repair

Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ～21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.     

Phenotypic heterogeneity within the first complementation group of UV-sensitive mutants of Chinese hamster cell lines

A DNA-repair mutant was characterized that has the extraordinary and interesting properties of extreme sensitivity to UV killing combined with a high level of nucleotide excision repair. The mutant V-H1 isolated from the V79 Chinese hamster cell line appeared very stable, with a reversion frequency of about 3.5 × 10⁻⁷. Genetic complementation analysis indicates that V-H1 belongs to the first complementation group of UV-sensitive Chinese hamster ovary (CHO) mutants described by Thompson et al. (1981). This correponds with data on cross-sensitivity and mutation induction after UV irradiation published by this group. Surprisingly, the mutant V-H1 shows only slightly reduced (to ∼ 70%) unscheduled DNA synthesis (UDS) after UV exposure, while the other two mutants of this complementation group are deficient in UDS after UV. In agreement with the high residual UDS, in V-H1 also the amount of repair replication in response to UV treatment is relatively high (∼ 50%). It has also been shown that the incision step of the nucleotide excision pathway takes place in V-H1 (with a lower rate than observed in wild-type cells), whereas another mutant (UV5) of the same complementation group is deficient in incision.This heterogeneity within the first complementation group indicates that the repair gene of this complementation group may have more than one functionally domain or that the gene is not involved in the incision per se but is involved in e.g. preferential repair of active genes.