CTF4 (CHL15) mutants exhibit defective DNA metabolism in the yeast Saccharomyces cerevisiae.

We have analyzed the CTF4 (CHL15) gene, earlier identified in two screens for yeast mutants with increased rates of mitotic loss of chromosome III and artificial circular and linear chromosomes. Analysis of the segregation properties of circular minichromosomes and chromosome fragments indicated that sister chromatid loss (1:0 segregation) is the predominant mode of chromosome destabilization in ctf4 mutants, though nondisjunction events (2:0 segregation) also occur at an increased rate. Both inter- and intrachromosomal mitotic recombination levels are elevated in ctf4 mutants, whereas spontaneous mutation to canavanine resistance was not elevated. A genomic clone of CTF4 was isolated and used to map its physical and genetic positions on chromosome XVI. Nucleotide sequence analysis of CTF4 revealed a 2.8-kb open reading frame with a 105-kDa predicted protein sequence. The CTF4 DNA sequence is identical to that of POB1, characterized as a gene encoding a protein that associates in vitro with DNA polymerase alpha. At the N-terminal region of the protein sequence, zinc finger motifs which define potential DNA-binding domains were found. The C-terminal region of the predicted protein displayed similarity to sequences of regulatory proteins known as the helix-loop-helix proteins. Data on the effects of a frameshift mutation suggest that the helix-loop-helix domain is essential for CTF4 function. Analysis of sequences upstream of the CTF4 open reading frame revealed the presence of a hexamer element, ACGCGT, a sequence associated with many DNA metabolism genes in budding yeasts. Disruption of the coding sequence of CTF4 did not result in inviability, indicating that the CTF4 gene is nonessential for mitotic cell division. However, ctf4 mutants exhibit an accumulation of large budded cells with the nucleus in the neck. ctf4 rad52 double mutants grew very slowly and produced extremely high levels (50%) of inviable cell division products compared with either single mutant alone, which is consistent with a role for CTF4 in DNA metabolism.     

Noncomplementary DNA double-strand-break rejoining in bacterial and human cells.

We examined the rejoining of noncomplementary restriction enzyme-produced DNA double-strand breaks in Escherichia coli and in cultured human cells. The enzymes used in this study, ClaI, BamHI and SalI, produce double-strand breaks with 5 protruding single strands. The joining of a ClaI-produced DNA end to a BamHI-produced end or to a SalI-produced end was examined at the DNA sequence level. End rejoining in E.coli was studied by transforming cultures with linear plasmid DNA that was gel purified from restriction digests, and end rejoining in cultured human cells was studied by introducing enzymes into the cells by electroporation. The human cells used contain an Epstein-Barr virus (EBV)-based shuttle vector, pHAZE, that was recovered and introduced into E.coli for further analysis. The major products of DNA end-joining processes observed in linear plasmid-transformed E.coli and in the human cells exposed to restriction enzymes were identical. Furthermore, the deletions observed in both systems and in the spontaneous mutant plasmids in untreated human cells had a common underlying feature: short stretches of directly repeated DNA at the junction sites.     

Studies on mammalian mutants defective in rejoining double-strand breaks in DNA

Mutants with defects in the rejoining of DNA double-strand breaks (dsbs) have been identified and characterised from E. coli and the yeast, Saccharomyces cerevisiae. More recently, 3 mammalian cell mutants with defective dsb rejoining have also been described. These mutants are xrs, XR-1 and L5178Y/S, and they are derived from at least two distinct complementation groups. The aim of this article is to review the current status of the studies with these mammalian cell mutants which are defective in dsb rejoining and, in particular, to compare their properties with those mutants identified from lower organisms. Possible mechanistic differences in the process of dsb rejoining between prokaryotes and lower and higher eukaryotes are discussed. All the mammalian mutants defective in dsb rejoining, are sensitive primarily to ionising radiation with little cross-sensitivity to UV-radiation. This is similar to the rad52 mutants of S. cerevisiae but contrasts to the majority of the E. coli mutants with defective dsb rejoining. Where studied, the mammalian cell mutants show enhanced resistance to ionizing radiation in late S/G2 phase, which, in one case, correlates with an enhanced ability to rejoin dsbs. This, together with other evidence, suggests that two mechanisms of dsb rejoining may exist in higher eukaryotes, one which operates uniquely in S/G2 phase and a second mechanism operating throughout the cell cycle and dependent upon the xrs and XR-1 gene products (although whether the xrs and XR-1 dependent pathways are distinct cannot at present be ascertained). Since duplicate homologues will be present in late S/G2 phase cells, this pathway may involve a recombinational mechanism. The xrs-dependent pathway might involve illegitimate recombination, but the xrs mutants do not appear to have a major defect in homologous recombination (involving plasmid DNA) and in this respect are distinct from rad52 mutants.     

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.     

Decreased repair of gamma ray damaged DNA in progeria.

A sensitive host-cell reactivation technique was used to examine the DNA repair ability of fibroblasts from two patients with classical progeria. Fibroblasts were infected with either non-irradiated or gamma-irradiated adenovirus type 2 and at 48 hrs after infection cells were examined for the presence of viral structural antigens using immunofluorescent staining. The production of viral structural antigens was considerably reduced in the progeria lines as compared to normal fibroblasts when gamma-irradiated virus was used, indicating a defect in the repair of gamma ray damaged DNA in the progeria cells.     

Selective excision of gamma ray damaged thymine from the DNA of cultured mammalian cells.

Three mammalian cell lines (WI-38, SV40-transformed WI-38 and Chinese hamster ovary) were exposed to high doses of 137-Cs gamma rays and their DNA analysed, following various periods of postirradiation incubation, for products of the 5,6-dihydroxy-dihydrothymine type. Within fifteen minutes of incubation at 37 degrees C 70 to 90 percent of these radiation products were removed from acid-precipitable material in all three cell lines. The amount of DNA degradation induced by radiation varied from approximately one percent in WI-38 cells to 15 percent in SV40-transformed WI-38 cells. Comparison of DNA degradation with the amount of thymine radiation product removed indicates that a selective gamma ray-induced excision repair capability exists in mammalian cells. Because of its more rapid kinetics, gamma ray excision repair is probably a distinct process as compared with ultraviolet-induced pyrimidine dimer excision.     

Quiescent cells but not cycling cells exhibit enhanced actin synthesis before they synthesize DNA

Major proteins synthesized by Swiss 3T3 cells at different stages of the cell cycle have been analyzed using double isotope labeling and one-dimensional SDS-polyacrylamide slab gels. The synthesis of actin was previously shown to be markedly enhanced a few hours after quiescent cells initiated growth following addition of serum. In contrast, the synthesis of actin remained at a constant rate, similar to that in quiescent cells, relative to synthesis of other proteins during the entire cell cycle. We conclude that enhanced actin synthesis is a process specific for the G0 to S transit, and may serve as a marker event during this interval. In contrast, three other proteins (90,000, 57,000, and 33,000 daltons) were synthesized throughout the cell cycle at higher rates than in G0 cells, and thus, are markers characteristic of cells traversing the cell cycle. A transient increase, such as seen for actin synthesis, by cells emerging from quiescence, may represent a process that these cells must perform before they can enter the G1 portion of the cell cycle. A transient event such as this need not be a periodic event that occurs during each cycle.     

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.     

Human osteogenic sarcoma cells exhibit enhanced protein phosphorylation

Protein phosphorylation was compared in normal human cells and human osteogenic sarcoma cells. The phosphorylation of endogenous cellular protein substrates was measured by two independent methods, incubation of homogenized cells with [γ-³²P]ATP or labeling of intact cells with Na₂H³²PO₄. Phosphorylated proteins were identified by SDS-polyacrylamide gel electrophoresis and autoradiography. The stained protein bands of all four osteosarcoma cell lines were nearly identical to those of the normal cells. However, each of the osteosarcoma cell lines showed autoradiographic evidence of enhanced phosphorylation in many different protein bands which was neither cyclic AMP-dependent nor a function of cellular growth rate or density. When normal and tumor cell homogenates were mixed prior to incubation with [γ-³²P]ATP, the resulting phosphoprotein patterns resembled those obtained with the tumor cells alone. In addition, a surgically derived osteogenic sarcoma was cultured and an established line obtained; another portion of the fresh tumor was immediately homogenized and used in a phosphorylation assay. The same enhanced phosphorylation pattern was obtained with the homogenized fresh tumor as with the cell line established from it. These results suggest thathuman osteogenic sarcoma cells are able to perform a significantly increased amount of phosphorylation of endegenous cellular protein substrates when compared to normal human cells.     

Induction and rejoining of DNA double-strand breaks in bladder tumor cells

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC-3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24). The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this.     

Transient gene expression from yeast artificial chromosome DNA in mammalian cells is enhanced by adenovirus.

The introduction of high molecular weight DNA into mammalian cells is useful for gene expression studies. However, current transfection strategies are inefficient, necessitating propagation of stable DNA transformants prior to analysis of gene expression. Here we demonstrate that transient lipid-mediated DNA transfection can be used to assess gene expression from yeast artificial chromosomes (YACs) containing the 230 kb cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and Escherichia coli lacZ . We also show that psoralen-UV inactivated adenovirus significantly enhances transfection efficiency. The ability to deliver high molecular weight DNA using lipid-mediated transfection should expedite the analysis of large human genes contained within artificial chromosome vectors.     

DNA strand break and rejoining in cultured human fibroblasts exposed to fast neutrons or gamma rays

The production and rejoining of DNA single-strand and double-strand breaks have been monitored in monolayer cultures of proliferating human skin fibroblasts by means of sensitive techniques. Cells were irradiated with low doses of either 60Co gamma-rays or 14.6 MeV neutrons at 0 degrees C (0-5 Gy for measurement of single-strand breaks by alkaline elution and 0-50 Gy for double-strand breaks measured by neutral elution). The yield of single-strand breaks induced by neutrons was 30 per cent of that produced by the same dose of gamma-rays; whilst in the induction of double-strand breaks neutrons were 1.6 times as effective as gamma-rays. Upon post-irradiation incubation of cells at 37 degrees C, neutron-induced single-strand and double-strand breaks were rejoined with a similar time-course to gamma-induced breaks. Rejoining followed biphasic kinetics; of the single-strand breaks, 50 per cent disappeared within 2 min after gamma-rays and 6-10 min after neutrons. Fifty per cent of the double-strand breaks disappeared within 10 min, after gamma-rays and neutrons. Cells derived from patients suffering from ataxia-telangiectasia showed the same capacity for repair of single- and double-strand breaks induced by 14.6 MeV neutrons, as cells established from normal donors. The comparison of neutrons and gamma-rays in the induction of DNA breaks did not explain the elevated r.b.e. on high LET radiation. However, a study of the variation in the spectrum of lesions induced by different radiation sources will probably contribute to the clarification of the relative importance of other radio products.     

Normal DNA strand rejoining and absence of DNA crosslinking in progeroid and aging human cells

We have measured by alkaline elution and alkaline sedimentation the rate of rejoining of X-ray induced DNA single-strand breaks in terminally senescent cultured WI-38 cells. Using the alkaline elution method, we have also measured the rate of ligation in cultured progeroid cells. In both cells and by both methods of measurement the rates of strand rejoining were normal. Alkaline elution failed to disclose any DNA crosslinking in these cells.     

X-irradiation sensitivity in Escherichia coli defective in DNA replication

Summary A mutant of Escherichia coli with a temperature sensitive defect in DNA replication is sensitive to X-irradiation but not to UV-irradiation. After UV-irradiation, dark-repair processes—dimer excision, DNA breakdown, repair synthesis and DNA strand joining—appear normal at the restrictive temperature. After X-irradiation, DNA degradation exceeds that in the wild type, and irradiation-dependent DNA synthesis does not occur. Single-strand breaks introduced into the DNA by the irradiation are nor repaired. The data indicate that the mutation results in a defect in repair of X-ray induced single-strand breaks as well as a defect in DNA replication. They provide evidence for the existence of a repair pathway for X-irradiated DNA similar to, but at least partially independent from, that postulated for the dark-repair of UV-irradiated DNA, viz., degradation at the site of the lesion, resynthesis of the degraded DNA complement and ligation of the DNA strand.This material has been published as an abstract in Genetics 64, p. 18 (1970).     

Efficient rejoining of radiation-induced DNA double-strand breaks in centromeric DNA of human cells

Although major efforts in elucidating different DNA double-strand break (DSB) repair pathways and their contribution to accurate repair or misrepair have been made, little is known about the influence of chromatin structure on the fidelity of DSB repair. Here, the repair of ionizing radiation-induced DSBs was investigated in heterochromatic centromeric regions of human cells in comparison with other genomic locations. A hybridization assay was applied that allows the quantification of correct DSB rejoining events in specific genomic regions by measuring reconstitution of large restriction fragments. We show for two primary fibroblast lines (MRC-5 and 180BR) and an epithelial tumor cell line that restriction fragment reconstitution is considerably more efficient in the centromere than in average genomic locations. Importantly, however, DNA ligase IV-deficient 180BR cells show, compared with repair-proficient MRC-5 cells, impaired restriction fragment reconstitution both in average DNA and in the centromere. Thus, the efficient repair of DSBs in centromeric DNA is dependent on functional non-homologous end joining. It is proposed that the condensed chromatin state in the centromere limits the mobility of break ends and leads to enhanced restriction fragment reconstitution by increasing the probability for rejoining correct break ends.     

MicroRNA-deficient NK cells exhibit decreased survival but enhanced function

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3'UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3'UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.     

Spontaneously hypertensive rats exhibit an enhanced mydriatic response to clonidine. Evidence for enhanced sensitivity of central alpha 2-adrenoceptors

Comparisons among spontaneously hypertensive (SHR), Kyoto Wistar (KW), and Wistar (W) rats were made of the functional states of central nervous system (CNS) alpha 2-adrenoceptors (clonidine-induced mydriasis) and nonvascular peripheral presynaptic alpha 2-adrenoceptors (clonidine-induced inhibition of the neurogenic twitch of the isolated vas deferens). While there were no differences among the strains of rats in the concentration of clonidine required to produce a 50% inhibition of the electrically evoked contractile response of the vas deferens, there was a significant reduction in the mean effective concentration (ED50) of clonidine to induce mydriasis in SHR as compared with KW and W rats. These observations indicate that CNS alpha 2-adrenoceptors may be functionally more sensitive in SHR. The data also suggest that the sensitivity of nonvascular presynaptic alpha 2-adrenoceptors, at least in the vas deferens, is not altered in hypertensive animals.     

DNA repair in human cells: in Cockayne syndrome cells rejoining of DNA strands is impaired

Fibroblasts from patients with Cockayne Syndrome (CS) are hypersensitive to UV light. DNA repair was analyzed in these cells by sedimentation behaviour of DNA nucleoids in sucrose gradients and compared to normal control cells. The initiation of repair, the incision of the DNA strand next to the UV lesion appeared to be normal. The rejoining of DNA stretches, however, is retarded in CS cells. DNA repair synthesis of UV damages was measured by autoradiography of [14C]thymidine incorporation into resting cells. Up to 4 h the DNA repair synthesis was comparable with normal cells. From 4 to 7 h the incorporation of radioactive precursors declined in CS cells. Besides a defective DNA polymerase this could be due to accelerated excorporation of radioactive nucleotides as a consequence of delayed ligation. In ligation the enzyme itself could be affected as well as its activation by ADP-ribosylation. Nicotine adenine dinucleotide (NAD+) is needed for the ADP ribosylation process. The cellular NAD+ content, however, was found to be the same in normal and in CS fibroblasts. Increase of the extracellular NAD+ supply accelerated the rejoining of UV damaged DNA in CS cells.     

Serratia liquefaciens swarm cells exhibit enhanced resistance to predation by Tetrahymena sp.

Tetrahymena sp. was found to graze extensively on Serratia liquefaciens MG1 swim cells (1.5–3 μm long rods) resulting in the rapid elimination of the bacterial strain. However, when S. liquefaciens cells are exposed to certain surfaces they differentiate into elongated, highly motile swarm cells and these cells were found to be grazing-resistant provided their length exceeded 15 μm.