Enhanced Excision Repair Research Articles

Enhanced excision repair and lack of PSII activity contribute to higher UV survival of Chlamydomonas reinhardtii cells in dark

Plant cells are known to differentiate their responses to stress depending up on the light conditions. We observed that UVC sensitive phenotype of light grown asynchronous Chlamydomonas reinhardtii culture (Light culture: LC) can be converted to relatively resistant form by transfer to dark condition (Dark culture: DC) before UVC exposure. The absence of photosystem II (PSII) function, by either atrazine treatment in wild type or in D1 (psbA) null mutant, conferred UV protection even in LC. We provide an indirect support for involvement of reactive oxygen species (ROS) signalling by showing higher UV survival on exposures to mild dose of H2O2 or Methyl Viologen. Circadian trained culture also showed a rhythmic variation in UV sensitivity in response to alternating light–dark (12 h:12 h) entrainment, with maximum UV survival at the end of 12 h dark and minimum at the end of 12 h light. This rhythm failed to maintain in “free running” conditions, making it a non-circadian phenotype. Moreover, atrazine strongly inhibited rhythmic UV sensitivity and conferred a constitutively high resistance, without affecting internal circadian rhythm marker expression. Dampening of UV sensitivity rhythm in Thymine-dimer excision repair mutant (cc-888) suggested the involvement of DNA repair in this phenomenon. DNA excision repair (ER) assays in cell-free extracts revealed that dark incubated cells exhibit higher ER compared to those growing in light, underscoring the role of ER in conferring differential UV sensitivity in dark versus light incubation. We suggest that multiple factors such as ROS changes triggered by differences in PSII activity, concomitant with differential ER efficiency collectively contribute to light–dark (12 h: 12 h) rhythmicity in C. reinhardtii UV sensitivity.

Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein

The XPA (Xeroderma pigmentosum A) protein is one of the six core factors of the human nucleotide excision repair system. In this study we show that XPA is a rate-limiting factor in all human cell lines tested, including a normal human fibroblast cell line. The level of XPA is controlled at the transcriptional level by the molecular circadian clock and at the post-translational level by a HECT domain family E3 ubiquitin ligase called HERC2. Stabilization of XPA by downregulation of HERC2 moderately enhances excision repair activity. Conversely, downregulation of XPA by siRNA reduces excision repair activity in proportion to the level of XPA. Ubiquitination and proteolysis of XPA are inhibited by DNA damage that promotes tight association of the protein with chromatin and its dissociation from the HERC2 E3 ligase. Finally, in agreement with a recent report, we find that XPA is post-translationally modified by acetylation. However, contrary to the previous claim, we find that in mouse liver only a small fraction of XPA is acetylated and that downregulation of SIRT1 deacetylase in two human cell lines does not affect the overall repair rate. Collectively, the data reveal that XPA is a limiting factor in excision repair and that its level is coordinately regulated by the circadian clock, the ubiquitin–proteasome system and DNA damage.

Parathyroid hormone suppresses osteoblast apoptosis by augmenting DNA repair

Daily injection of parathyroid hormone (PTH) is a clinically approved treatment for osteoporosis. It suppresses apoptosis of bone-forming osteoblasts although its exact anti-apoptotic mechanism(s) is incompletely understood. In this study, PTH treatment of cultured osteoblasts blocked the pro-apoptotic effects of serum withdrawal and nutrient deprivation; hydrogen peroxide induced oxidative stress, and UV irradiation. We hypothesized that PTH might suppress osteoblast apoptosis by enhancing DNA repair. Evidence is provided showing that post-confluent, non-proliferating osteoblasts treated with PTH exhibited a protein kinase A-mediated activation of two proteins that regulate DNA repair processes (proliferating cell nuclear antigen and forkhead box transcription factor 3a) as well as a suppression of the pro-apoptotic growth arrest and DNA damage protein 153. Additional proof of a connection between DNA damage and osteoblast apoptosis came from an unexpected finding whereby a majority of fixed PTH-treated osteoblasts scored weakly positive for Terminal Deoxynucleotidyl dUTP Nick-End Labeling (TUNEL), even though similar cultures were determined to be viable via a trypsin replating strategy. TUNEL identifies DNA excision repair, not just apoptotic DNA fragmentation, and the most likely explanation of these TUNEL results is that PTH's activation of DNA repair processes would permit nucleotide incorporation as a result of enhanced excision repair. This explanation was confirmed by an enhanced incorporation of bromodeoxyuridine in PTH-treated cells even though a majority of the cell population was determined to be non-replicating. An augmentation of DNA repair by PTH is an unreported finding, and provides an additional explanation for its anti-apoptotic mechanism(s).

Expression profile of residual breast cancer after primary chemotherapy based on doxorubicin

639 Background: Expression profile of the remaining malignant cells in breast cancer after the last cycle of primary chemotherapy (PCT) based on doxorubicin (AC) might help to identify a resistant phenotype. Methods: To analyse changes in gene expression in samples of primary breast tumors before and after systemic chemotherapy, ten pt (median age 46.5 years, 3 post-menopausal) clinically staged as II (n=2) or III (n=8) breast cancer were submitted to PCT, receiving at least 90% of the planned doxorubicin dose in a median interval of 66.5 days. Samples were obtained by tumor biopsy before PCT and after 4 courses of AC, upon surgical intervention. Surgery was performed at least 28 days after AC last course. All pts presented a tumor reduction according to RECIST. Samples were hand-dissected to eliminate non-tumoral tissue. RNA was extracted, amplified and gene expression analysed using a cDNA microarray plataform having 4608 genes. Results: One hundred and seven genes were found differentially expressed by both a permuted Student t test at a False Discovery Rate (FDR) level ≤ 0.001 and p≤0.05 by paired Student t test. Unsupervised hierarchical clustering employing 34 differentially expressed genes at FDR level < 0.001 and Bayesian Error Rate < 50% allowed a proper discrimination of 100% post treatment samples. The discriminators included genes up-regulated such as SEPT1, a member of GTPase family, a growth factor receptor (PDGFRL) implicated in transcapillary transport in tumors and Pumilio homolog1 (PUM-1), an essential protein for stem cell maintenance and self-renewal. Genes involved in proliferation were repressed including IRS1 (insulin receptor substrate-1), PTPRC (a regulator of citokyne receptor signaling), CORO1C (coronin) and CSE1L (CAS) which depletion leads to cell cycle arrest. Notably HMG2L1 (high-mobility group protein 2-like1) was also down regulated reflecting enhanced excision repair mechanism. Conclusion: Genes differentially expressed in residual breast cancer after PCT may potentially represent a signature profile for doxorubicin reistance. Supported by FAPESP. No significant financial relationships to disclose.

Stress-Inducible DNA Repair in Saccharomyces Cerevisiae

Saccharomyces cerevisiae shows altered radiation response under various stress conditions, such as nutrition depletion, nitrogen starvation, osmotic shock, heat shock, and mild chemical treatments. In general, the cells show higher levels of UV or gamma radiation resistance under the stress. However, not all the stress conditions affect the repair system in the same manner. For example, depletion of nitrogen supply in the growth medium has been shown to enhance the repair of gamma ray-induced DNA damage without significantly affecting the UV response of the cells. On the other hand, a mild treatment with alkali or hydrogen peroxide improves the response to UV light but not to gamma radiation. It has further been shown that the effect of these stresses are not additive, e.g., the alkali and hydrogen peroxide treatments given simultaneously show the same effect as either of them alone. Low levels of gamma and UV radiation exposures are also treated as stress in the present context. Studies show that irradiation of low-dose gamma rays results in enhanced excision repair of UV-induced pyrimidine dimers. However, in all the wild-type strains tested, none showed any effect on gamma rays response. The exposure to low doses of UV light did not show any effect on either the gamma rays or the UV response. It is suggested that the stress-induced enhancement of DNA repair can be of two types: 1) A general response to stress, which prepares the organism to survive in adverse circumstances (some of the proteins produced during this response also take part in the DNA repair), and 2) a particular response involving DNA damage, such as that caused by gamma irradiation. In this case, the DNA damage may act as a signal for enhancement of the DNA repair.

Photoreversal of UV-potentiated glutamine tRNA suppressor mutations in excision proficient Escherichia coli

UV-irradiated excision proficient Escherichia coli were exposed to light for photoenzymatic reversal (PR) of cyclobutane pyrimidine dimers (CPD) and assayed for reversion mutation (glutamine tRNA suppressor mutations) on semi-enriched medium or on the same medium containing acriflavine to inhibit excision repair. The initial mutation frequency without PR was relatively greater when assayed with acriflavine, and this difference increased as larger UV fluences were used. The PR kinetics were first order and about the same or slightly faster when cells were assayed with acriflavine (after 15, 30 or 45 J/m2, respectively). The results indicated mutation targeting by CPD in excision proficient cells. These results and conclusion contrast sharply with the original study of this type done several years ago. PR kinetics were considerably slower with assays containing acriflavine, sustaining the idea that PR causes repair of non-dimer targeting lesions by enhancing excision repair. To explain this contrast we devised a fluence-decrement rate for estimating the effectiveness of PR and measured PR-dependent excision repair (PER) as the difference in the fluence-decrement rate with excision proficient and deficient cells. PER was more evident when cells were prepared as in the original study but was still an insufficient factor. More importantly, the original study included a component of indirect photoreactivation or photoprotection (using unfiltered PR light) which accentuated the role of excision repair. Taking these factors into account, the original data also are consistent with the model that glutamine tRNA suppressor mutations produced by UV-mutagenesis in excision proficient E. coli result from targeting by CPD just as in excision defective cells. Thus, with regards to a common UV mutation assay, there does not appear to be two types of targeting lesion depending on excision proficiency.

Enhanced excision repair of DNA damage due to cis-diamminedichloroplatinum(II) in resistant cervix carcinoma HeLa cells

We have previously reported a cisplatin-resistant HeLa cell line which exhibits overproduction of nuclear proteins preferential for cisplatin-modified DNA (Chao et al., Cancer Res. 51: 601–605, 1991; Biochem. J. 277: 875–878, 1991). In this study, excision repair of cisplatin-DNA adducts in a resistant and a revertant cell lines was investigated using in situ detection of cisplatin-DNA adducts by an immunoassay and the measurement of repair-associated DNA strand breaks by a sensitive alkaline elution method. The resistant cells exhibited a 2-fold decrease in the accumulation of cisplatin-DNA adducts; whereas, the revertant cells showed a similar level of cisplatin-DNA adducts as the parental cells in the parallel experiment. Immediately following cisplatin treatment, the resistant and the revertant cells accumulated respectively ∼ 50% and 90% cisplatin-DNA adducts of the parental cells. However, the kinetic patterns of repair rate following peak accumulation of cisplatin-DNA adducts (which took ∼ 4 h) was the same in the three cell lines. This finding was supported by the measurement of repair-associated DNA strand breaks using alkaline elution which showed 1.6- and 1.5-fold increase in the resistant and the revertant cells respectively. In addition, following transfection with plasmid DNA carrying cisplatin damage, the resistant and the revertant cells displayed a 2.4- and 1.4-fold enhancement in host cell reactivation, respectively. Furthermore, the acquired resistance in HeLa cells was partially reversed by nontoxic aphidicolin, a DNA polymerase-α and DNA repair inhibitor. The results strongly suggest the improved excision repair of cisplatin-DNA adducts as a mechanism of phenotypic resistance of cells to cisplatin.

Heat-shock-increased survival to far-UV radiation in Escherichia coli is wavelength dependent

Heat-shock-induced resistance to far-UV (FUV) radiation was studied in Escherichia coli. The induction of FUV resistance was shown to be dependent on the products of the genes uvrA and polA in bacteria irradiated at 254 nm. Heat shock increased the resistance to 280 nm radiation in a uvrA6 recA13 mutant. Heat shock lowered the mutation frequency (reversion to tryptophan proficiency) in wild-type or uvrA strains irradiated at 254 nm. When these strains were irradiated at 280 nm, heat shock did not interfere with the mutation frequency in the wild-type strain, but greatly enhanced mutations in the uvrA mutant. After heat-shock treatment, the wild-type strain irradiated at 254 nm showed increased DNA degradation, indicating enhanced repair activity. However, heat shock did not stimulate SOS repair triggered by FUV. An increased survival of bacteriophages irradiated with FUV and inoculated into heat-shock-treated bacteria was not detected. The possibility that heat shock enhances excision repair activity in a wavelength-dependent manner is discussed.

Alteration of irradiated shuttle vector processing by exposure of human lymphoblast host cells to single or split gamma-ray doses.

The repair of damaged DNA by mammalian cells exposed to single or split doses of radiation was probed with shuttle vector pZ189. Human lymphoblast hosts who received a single 120 cGy dose 2 h before transfection with 2500 cGy-damaged pZ189 yielded a two-fold higher frequency of progeny plasmids with mutations in their supF-tRNA target genes than did unirradiated host cells. Delaying transfection for 12 h, however, reduced the mutation frequency by half versus unirradiated controls. Plasmid survival was also affected by the time between host cell irradiation and transfection. Splitting doses of 50-500 cGy into two equal fractions separated by 4 h lowered mutation frequency and increased plasmid survival compared with equivalent acute doses; increasing the interval between dose fractions to 8 h, however, lowered plasmid survival compared with acute doses. Sequence analyses of the target gene in mutant plasmids revealed increased multiple-base substitution mutations among progenies recovered from irradiated hosts, indicating enhanced excision repair. These findings support modulation of mammalian cell DNA repair by ionizing radiation, disclose the transient nature of the effect of radiation on DNA repair, and demonstrate a quantitative difference in the effectiveness of single and split doses.

DNA excision repair as a component of adaptation to low doses of ionizing radiation in Escherichia coli.

In this study we examined whether or not DNA excision repair is a component of adaptation induced by very low-dose ionizing radiation in Escherichia coli, a well-characterized prokaryote, and investigated the relationship between enhanced excision repair and the SOS response. Competent E. coli cells were irradiated using low doses (0.1-10 Gy) of 137Cs gamma-rays, allowed to recover for 2 h and were then transformed using pUC18 DNA containing approximately 22 oxidized thymine residues (thymine glycols) per molecule. Successful transformants were identified by recovery of plasmid-borne ampicillin resistance and the resulting data (colony counts) were used to calculate ratios of plasmid recovery in irradiated to control cells. Results showed that cells irradiated with very low doses (0.1-0.5 Gy) were up to 30-40% more efficient at utilizing thymine glycol-containing pUC18 DNA. The enhanced excision repair by very low doses (< 0.5 Gy) of gamma-rays was shown to be independent of the recA-controlled SOS response in experiments using recA cells or cells carrying recA-lacZ gene fusions. The stimulating effect in AB1157 prototype cells were subsequently confirmed using a DNA precipitation assay in which DNA incision events were accumulated by inhibiting DNA ligation with ethidium bromide. Our data suggest that there seems to be narrow 'windows' of dose-effect for the induction of SOS-independent DNA excision repair. Being similar to mammalian cell studies, the dose range for this effect was about 200-fold less than D37 for radiation survival.

Enhanced Excision Repair Activity in Mammalian Cells After Ionizing Radiation

Monkey CV-1 cells which had received 5 Gy 12 h before harvesting lysates from their cell cultures contained approximately three times as much DNA excision repair enzyme activity as unirradiated cells. The activity was determined in crude cell lysates by the release of intermediate mobility DNA fragments and fragments with 3'-phosphoryl ends from 5'-32P-end labelled irradiated 95 bp alpha DNA. Different 3'-termini endow the fragments with differing mobilities, signifying steps in the processing of radiation damaged DNA. Similar results were obtained when Krebs II mouse tumour cells growing in mice as ascites received 5 Gy 12 h before harvest. The enzyme activities from CV-1 cells and from Krebs II cells were partially purified as 60-70 kDa proteins on Superose 12 or Ultrogel AcA-54 columns. Divalent cations were not required for enzyme activity. A 23 nucleotide long defined duplex oligodeoxynucleotide substrate containing a single 8-oxodG residue was also very actively cleaved by the partially purified cell enzymes. 8-oxoguanine is a major product of ionizing radiation's action on DNA and was recognized by the enzymes described here. The mechanism by which radiation increased excision repair activity of cellular enzymes is not understood.

DNA.RNA helicase activity of RAD3 protein of Saccharomyces cerevisiae.

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability. The RAD3 protein exhibits a remarkable degree of sequence homology to the human excision repair protein ERCC2. The RAD3 protein is a single-stranded DNA-dependent ATPase and a DNA helicase capable of denaturing long regions of duplex DNA. Here, we demonstrate that RAD3 also possesses a potent DNA.RNA helicase activity similar in efficiency to its DNA helicase activity. The rad3 Arg-48 mutant protein, which binds but does not hydrolyze ATP, lacks the DNA.RNA unwinding activity, indicating a dependence on ATP hydrolysis. RAD3 does not show any RNA-dependent NTPase activity and, as expected, does not unwind duplex RNA. This observation suggests that RAD3 translocates on DNA in unwinding DNA.RNA duplexes. That the rad3 Arg-48 mutation inactivates the DNA and DNA.RNA helicase activities and confers a substantial reduction in the incision of UV-damaged DNA suggests a role for these activities in incision. We discuss how RAD3 helicase activities could function in tracking of DNA in search of damage sites and effect enhanced excision repair of actively transcribed genes.

Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.

Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.

Microcell-mediated transfer of a single human chromosome complements xeroderma pigmentosum group A fibroblasts.

Chromosomes from an immortalized aneuploid human fibroblast cell line were randomly tagged with the selectable marker neo by transfection with the plasmid pSV2neo. Somatic cell fusions between transfected human cells and mouse A9 cells generated pools of G418-resistant human-mouse hybrid clones containing various numbers of human chromosomes. Microcell-mediated chromosome transfer from the hybrid pools to xeroderma pigmentosum complementation group A (XP-A) cells in culture and selection for G418-resistant colonies resulted in the identification of XP cells with enhanced resistance to ultraviolet radiation. Screening of subclones from selected pools of human-mouse hybrids facilitated the identification of hybrids containing a single neo-tagged human chromosome. Transfer of this chromosome to XP-A cells (but not to XP-F or XP-C cells) results in enhanced resistance to ultraviolet light and enhanced excision repair capacity. The identification of a single human chromosome that complements the phenotype of XP-A cells in culture provides the potential for genetic mapping of the complementing gene and for its isolation by molecular cloning.

Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis.

The lexA41 mutant of E. coli is a UV-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. lexA41 carried an additional mutation which changed amino acid 132 in the LexA protein from Ala to Thr. The resultant protein was unstable and was degraded both before and after an inducing treatment. This instability was greater at 42 degrees than at 30 degrees. The protein was more stable in Lon- mutants at both temperatures. lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistant with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the UV-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA- mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis.

Liquid-holding effects in U.V.-irradiated phage T3.

Abstract— Holding complexes of u.v.‐irradiated (254 nm) T3 phage in E. coli B/r cells for several hours at 37°C in buffer, or broth with chloramphenicol, affects the phage survival in at least two different ways: (1) by enhancing excision repair, resulting under certain conditions in liquid‐holding recovery (LHR), and (2) by destroying the phage (holding inactivation). LHR is most apparent in buffer containing 20 μg ml‐1 chloramphenicol (CAP). It is expressed by as much as a 10–fold increase in the fraction of complexes that display host‐cell reactivation (resulting from excision repair), but the percentage of u.v. lesions repaired within repair‐proficient complexes is slightly decreased. LHR is not observed if T3 infects the repair‐deficient strain Bs‐1.Holding inactivation is readily observed with unirradiated phage complexes in broth containing CAP. The response of irradiated‐phage complexes to liquid‐holding conditions is more complex: holding inactivation is less effective for irradiated than for unirradiated phage DNA (i.e. the irradiated DNA is to some extent ‘protected’), and processes leading to LHR are superimposed. Thus under certain holding conditions one observes the paradoxical phenomenon that the viable titer of irradiated phage is several times higher than that of unirradiated phage.The nature of holding inactivation is not known, nor is the mechanism by which irradiated DNA is partially protected against it. Holding inactivation does not require protein synthesis; it is rather enhanced at high CAP concentration and seems to be favored by otherwise active cell metabolism. At high CAP concentrations (200–400 μg ml‐1, as compared to 20 μg ml‐1) irradiated‐phage complexes show neither LHR nor protection against holding inactivation. Likewise they fail to undergo some step by which the phage DNA becomes insensitive to repair inhibition by caffeine.