Mutation of Chinese hamster cells by near-UV activation of promutagens

A tissue-culture assay for mutagenesis and cytotoxicity incorporating near ultraviolet (NUV) light activation of polyaromatic hydrocarbons (PAH) has been developed. Cultures of Chinese hamster cells (line CHO) growing in suspension culture were inoculated with benzo[a]pyrene (B[a]P), 7,12-dimethylbenzanthracene (DMBA) or shale-oil retort-water and exposed to light from a high-pressure mercury lamp fitted with a Corning NUV bandpass filter. This light source both permitted activation of PAH and the shale-oil water and preculded detectable damage to DNA. Neither the PAH nor the NUV alone had any effect on cell survival or mutation frequencies but the chemicals plus NUV were extremely effective in producing mutations to 6-thioguanine resistance (hgprt gene).     

Mutation induction and killing of Escherichia coli by DNA adducts and crosslinks: a photobiological study with 8-methoxypsoralen.

Low doses of 350 nm radiation (NUV) in the presence of 8-methoxypsoralen (8-MOP) induce predominantly mono-adducts in bacterial DNA. Further exposure to NUV in the absence of 8-MOP converts a proportion of these mono-adducts to interstrand cross-links. Using this approach the relative effects of adducts and cross-links on bacteria with different repair capacities was studied. Escherichia coli WP100 uvrA recA, believed to be totally deficient in the ability to repair 8-MOP plus NUV damage to DNA, was inactivated on average by a single photon event occurring with a quantum efficiency of about 0.03. We conclude that the inactivating lesion is probably a single mono-adduct. E. coli WP2 uvrA, deficient in excision endonuclease activity, may be inactivated by a very small number of cross-links, probably one. These conclusions are consistent with present knowledge of the repair capabilities of these bacteria. Conversion of mono-adducts to cross-links in WP2 uvrA (which occurs with a quantum efficiency of around 0.3) greatly increases lethality but results in a reduction of the induced mutation frequency presumably because cross-links are (almost) invariably lethal. In the repair-proficient strain WP2 both adducts and cross-links can be repaired but the latter are more likely than the former to lead to either death or mutation.     

Tumor Cell Death Mediated by Peptides That Recognize Branched Intermediates of DNA Replication and Repair

Effective treatments for cancer are still needed, both for cancers that do not respond well to current therapeutics and for cancers that become resistant to available treatments. Herein we investigated the effect of a structure-selective d-amino acid peptide wrwycr that binds replication fork mimics and Holliday Junction (HJs) intermediates of homologous recombination (HR) in vitro, and inhibits their resolution by HJ-processing enzymes. We predicted that treating cells with HJ-binding compounds would lead to accumulation of DNA damage. As cells repair endogenous or exogenous DNA damage, collapsed replication forks and HJ intermediates will accumulate and serve as targets for the HJ-binding peptides. Inhibiting junction resolution will lead to further accumulation of DNA breaks, eventually resulting in amplification of the damage and causing cell death. Both peptide wrwycr and the related wrwyrggrywrw entered cancer cells and reduced cell survival in a dose- and time-dependent manner. Early markers for DNA damage, γH2AX foci and 53BP1 foci, increased with dose and/or time exposure to the peptides. DNA breaks persisted at least 48 h, and both checkpoint proteins Chk1 and Chk2 were activated. The passage of the cells from S to G2/M was blocked even after 72 h. Apoptosis, however, was not induced in either HeLa or PC3 cells. Based on colony-forming assays, about 35% peptide-induced cytotoxicity was irreversible. Finally, sublethal doses of peptide wrwycr (50–100 µM) in conjunction with sublethal doses of several DNA damaging agents (etoposide, doxorubicin, and HU) reduced cell survival at least additively and sometimes synergistically. Taken together, the results suggest that the peptides merit further investigation as proof-of-principle molecules for a new class of anti-cancer therapeutics, in particular in combination with other DNA damaging therapies.     

Surface structure of in vitro assembled bacteriophage lambda polyheads.

Interstrand cross-links induced by psoralen-plus-light are removed from the DNA of Escherichia coli, and this reaction is effected by the uvrA, uvrB, uvrC and polA (5′ → 3′ exonuclease) gene products. During cross-link removal, cellular DNA strands are cut so that, upon denaturation, the DNA dissociates into segments having an average molecular weight about equal to twice the average distance between cross-links. These strand cuts are persistent in cells, having a half-life of more than 20 minutes.The structure of cross-linked DNA undergoing repair was further investigated by use of density and radioactively labeled isotopes. These experiments demonstrate that two strand cuts are made in one DNA strand near each cross-link, one on each side of one arm of the cross-link. A mechanism is proposed for cross-link removal. The endonuclease coded for by the uvrA and B genes makes an incision on the 5′ side of one arm of a cross-link. Polymerase I (5′ → 3′ exonuclease) then makes a second cut on the 3′ side, in the same strand. This allows the strands to be separated during denaturation, but would leave the second arm of the cross-linking structure still attached to the uncut strand. The persistence of strand cuts at cross-links suggests that rejoining, dependent upon repair polymerization and ligation, is blocked by such a partially excised cross-linking residue. Initial stages of cross-link removal appear to be similar to pyrimidine dimer excision, but intermediates generated by these processes differ substantially in structure and repair must be completed by different mechanisms.     

Repair in E. coli of transforming plasmid DNA damaged by psoralen plus near-ultraviolet irradiation

Treatment of DNA with psoralen plus near-ultraviolet irradiation gives rise to both monoadducts and cross-links. We have examined the repair of plasmid NTP16 DNA treated in this way in vitro and then used to transform E. coli. Monoadducts are found to be potentially lethal, and can be repaired by uvr-dependent and recA-dependent pathways. The presence of a related resident plasmid in the transformed cells can enhance the survival of the incoming damaged NTP16 DNA. This effect is not recA-dependent, and a similar effect (designated "resident enhanced repair") has been observed previously with UV-irradiated plasmids of this particular incompatibility group. Removal of unbound psoralen from the plasmid DNA and exposure to further NUV is known to increase the ratio of cross-links to monoadducts, and we demonstrate that such cross-linked plasmid DNA is not readily repaired following transformation. However in the presence of homologous DNA (related resident plasmid) there is evidence for the repair, and hence uptake by the cell, of cross-linked DNA.     

Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation

Although Dienococcus radiodurans is notoriously resistant to far-ultraviolet radiation (FUV; 254 nm), it is highly sensitive to near-ultraviolet radiation (NUV; 300-400 nm), thus demonstrating that the mechanisms of damage (and/or recovery) by the two types of irradiation are different. This observed difference between FUV and NUV effects in D. radiodurans agrees with previous studies with Escherichia coli. Near-ultraviolet radiation produces DNA damage which is presumed to be single-strand breaks (SSB) in the DNA of D. radiodurans. Unique lesions, such as DNA-protein crosslinks could not be demonstrated in this study. Cells that were pre-irradiated with a small dose of NUV were subsequently protected against inactivating doses of NUV. The data presented are consistent with induced DNA repair following NUV damage in D. radiodurans; this is in contrast to FUV damage where DNA repair is constitutive but not induced.     

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc⁻) DT40 cells arrest in G₂ phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc⁻ cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G₂ checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G₂ arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G₂ phase.     

Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks

During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.     

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Mammalian cells: Damage in late replicating DNA as the most efficient cause of reproductive death

Chinese hamster cells were synchronized by mitotic shake off, labeled in the first S period with ¹²⁵IUdR, cooled to 4 °C in the G2 stage and then stored up to 4 days to accumulate damage due to ¹²⁵I disintegrations in cell DNA. There was a large difference in the efficiency of induction of reproductive death when damage accumulated in the DNA, which replicated in the second half of the DNA synthesis period, was compared with damage accumulated in the DNA, which replicated in the first half of the DNA synthesis period. Damage accumulated in the late replicated DNA appears to be the most critical. This result suggests that the mammalian cell nucleus is not homogeneous with respect to the damaging events leading to reproductive death and may stress the importance for cell survival of the integrity of the late-replicating, heterochromatic DNA near the nucleus membrane.     

Radiation response of haematopoietic cell lines of human origin

Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radiosensitivity of the cells varied widely, the D0s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivites.     

Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50–70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.     

Split-dose recovery and protein synthesis in X-irradiated CHO cells

A temperature-sensitive mutant for protein synthesis, CHO-tsH1, has been compared to the wild-type (WT) cell, CHO-SC1, in single- and split-radiation-dose schemes. When the exponentially growing mutant and the wild-type cells were treated at 40 degrees C for up to 3 h prior to split doses of X rays, survival was progressively reduced in the mutant compared to the wild type. In addition, if a 2-h split-dose scheme was used with a treatment of 40 degrees C given before the first dose, between the dose fractions and after the second dose, the recovery from sublethal damage (SLD) was almost completely inhibited in the mutant cells. These observations implied that a pool of proteins was involved in the recovery from sublethal X-ray damage. However, if molecular repair was measured in the mutant cell by the alkaline-unwinding technique under the same time and temperature schemes as those demonstrating a reduction in the recovery from SLD, no difference in the kinetics of DNA strand rejoining was observed compared to similar measurements made under conditions permitting SLD recovery. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. Split-dose experiments were also done using cycloheximide to chemically inhibit protein synthesis. Under conditions which mimicked those used in the temperature-shift experiments both cell lines showed a reduction in the recovery from sublethal damage comparable in magnitude to that observed in the mutant cells when they were treated with 40 degrees C. Both the chemical and thermal inhibition of protein synthesis substantiate its necessity for the recovery from sublethal damage.     

The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast

The formation of RNA–DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.     

Killing of Escherichia coli K-12 by near-ultraviolet radiation in the presence of hydrogen peroxide: Role of double-strand DNA breaks in absence of recombinational repair

Near-ultraviolet (NUV) radiation killing of Escherichia coli K-12 can be enhanced by a sub-lethal concentration of hydrogen peroxide. This can be divided into a “RecA-dependent” and “RecA-independent” synergistic killing action. Stationary phase wild-type and 8 closely related repair-deficient mutants were examined for their NUV sensitivities in the presence and absence of H₂O₂. All exhibited the “RecA-independent” synergism; i.e., H₂O₂ enhanced NUV lethality when RecA repair was not operating. The “RecA-independent” synergism did not result from destruction of repair enzymes. Very few DNA—protein crosslinks could be detected following NUV plus H₂O₂ treatment. However, double-strand (DS) DNA breaks were produced, apparently by conversion of closely spaced single-strand (SS) breaks on opposite strands. The correlation between DS-break formation and lethality in wild-type and a polA mutant indicates that the RecA-independent synergistic killing results from the conversion of SS into lethal DS breaks.     

DNA damage and cytotoxicity of nitracrine in cultured HeLa cells

The effects of nitracrine (1-nitro-9-(3,3-N,N-dimethylaminopropylamino)acridine on DNA of cultured HeLa cells were studied. DNA strand breakage and interstrand cross-linking as well as DNA-protein cross-linking were measured by means of an alkaline elution technique and were compared with the cytotoxic effect of the drug. Interstrand cross-links were not detectable in the concentration range that inhibited cell growth up to 99%. DNA single-strand breaks were found when cells were treated with highly cytotoxic doses of the drug. DNA breakage was not reparable and exhibited a tendency to increase during incubation after drug removal. The only chromatin lesion induced by sublethal doses of nitracrine were DNA-protein cross-links which persisted for 24 h after drug treatment. It is concluded that DNA breaks represent degraded DNA from dying cells, whereas DNA-protein cross-links are specific cellular lesions, which may be responsible for the cell-killing effect of nitracrine.     

Mitotic inter-homologue junctions accumulate at damaged DNA replication forks in recQ mutants

Mitotic homologous recombination is utilised to repair DNA breaks using either sister chromatids or homologous chromosomes as templates. Because sister chromatids are identical, exchanges between sister chromatids have no consequences for the maintenance of genomic integrity unless they involve repetitive DNA sequences. Conversely, homologous chromosomes might differ in genetic content, and exchanges between homologues might lead to loss of heterozygosity and subsequent inactivation of functional genes. Genomic instability, caused by unscheduled recombination events between homologous chromosomes, is enhanced in the absence of RecQ DNA helicases, as observed in Bloom's cancer-prone syndrome. Here, we used two-dimensional gel electrophoresis to analyse budding yeast diploid cells that were modified to distinguish replication intermediates originating from each homologous chromosome. Therefore, these cells were suitable for analysing the formation of inter-homologue junctions. We found that Rad51-dependent DNA structures resembling inter-homologue junctions accumulate together with sister chromatid junctions at damaged DNA replication forks in recQ mutants, but not in the absence of Srs2 or Mph1 DNA recombination helicases. Inter-homologue joint molecules in recQ mutants are less abundant than sister chromatid junctions, but they accumulate with similar kinetics after origin firing under conditions of DNA damage. We propose that unscheduled accumulation of inter-homologue junctions during DNA replication might account for allelic recombination defects in recQ mutants.     

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G₂ arrest after only one round of replication. In these G₂-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G₂. In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2–hMSH6 from hMLH1–hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.     

Induction and repair of psoralen cross-links in DNA of normal human and xeroderma pigmentosum fibroblasts

Skin fibroblasts from normal human subjects were exposed in vitro to long-wave ultraviolet radiation (UVA, 320–400 nm) alone, or in combination with 8-methoxypsoralen (8-MOP). DNA damage was analysed with the alkaline elution technique before and after post-treatment incubation of the cells at 37°C for various times.Cells treated with UVA at 1.1 J/cm² showed an increased DNA elution rate, which returned to the normal level within 30 min of post-treatment incubation. In cells treated with PUVA (8-MOP at 20 μg/ml plus UVA at 0.04 J/cm²), the alkaline elution rate was not different from untreated control cells, either before or after post-treatment incubation for times up to 7 days.When the PUVA treatment was followed first by a washing, to remove any unbound 8-MOP, and then by UVA (PUVA + UVA) at 1.1 J/cm², the alkaline elution rate decreased below the control level. During the post-treatment incubation of the PUVA + UVA-treated cells there was a gradual increase of the alkaline elution rate to a level significantly above that in control cells. This increase was observed after 30 min. It reached a miaximum after 24 h and remained after 7 days of post-treatment incubation. Cells from a patient with xeroderma pigmentosum of complementation group A, which were given the same PUVA + UVA treatment, did not show any change in the alkaline elution rate during the post-treatment incubation.If, as seems likely, an increased alkaline elution rate indicates an increase of DNA breaks, and a decreased alkaline elution rate indicates the sealing of breaks and/or the formation of cross-links, the results would suggest the following: (1) UVA irradiation in itself is capable of inducing DNA breaks, which are rapidly sealed during post-treatment incubation; (2) PUVA treatment induces mono-adducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation and can be activated to form DNA cross-links by a second dose of UVA; (3) DNA cross-links induced by PUVA + UVA can be recognized by a repair process that involves the formation of DNA breaks. This process is not observed in xeroderma pigmentosum cells of group A.     

Requirement for repair of DNA double-strand breaks by homologous recombination in split-dose recovery

Utsumi, H., Tano, K., Takata, M., Takeda, S. and Elkind, M. M. Requirement for Repair of DNA Double-Strand Breaks by Homologous Recombination in Split-Dose Recovery. Radiat. Res. 155, 680-686 (2001). Split-dose recovery has been observed under a variety of experimental conditions in many cell systems and is believed to be the result of the repair of sublethal damage. It is considered to be one of the most widespread and important cellular responses in clinical radiotherapy. To study the molecular mechanism(s) of this repair, we analyzed the knockout mutants KU70-/-, RAD54-/-, and KU70-/-/RAD54-/- of the chicken B-cell line, DT40. RAD54 participates in the recombinational repair of DNA double-strand breaks (DSBs), while members of the KU family of proteins are involved in nonhomologous end joining. Split-dose recovery was observed in the parent DT40 and the KU70-/- cells. Moreover, the split-dose survival enhancement had all of the characteristics demonstrated earlier for the repair of sublethal damage, e.g., the reappearance of the shoulder on the survival curve with dose fractionation; cyclic fluctuation in cell survival at 37 degrees C; repair and no cyclic fluctuation at 25 degrees C. These results strongly suggest that repair of sublethal damage is due to DSB repair mediated by homologous recombination, and that these DNA DSBs constitute sublethal damage.