Effects of hyperoxia on DNA synthesis in cultured porcine aortic endothelial cells

The mode of action of hyperoxia on the inhibition of DNA synthesis from thymidine (dThd) was studied in primary cultures of porcine aortic endothelial cells (EC) at confluence. A significant effect of hyperoxia on dThd uptake was detected only after a 48-h exposure to 95% O2. On the other hand, decrease in dThd kinase activity was already observed after a 12-h exposure, and the time course of its reduction followed closely that of the inhibition of dThd incorporation into DNA. The incorporation of dThd triphosphate into DNA in permeabilized EC was unaffected by hyperoxia. Determination of DNA alpha- and beta-polymerase activities showed that hyperoxia reduced the activity of the alpha-polymerase and increased that of the beta-polymerase. We conclude that most of the O2 effects on DNA synthesis from dThd can be attributed to dThd kinase inhibition. The increased activity of DNA beta-polymerase, an enzyme involved in DNA repair, also supports the view that hyperoxia could damage DNA.     

Specific inhibition of DNA polymerase beta by its 14 kDa domain: role of single- and double-stranded DNA binding and 5'-phosphate recognition.

DNA polymerase beta (beta-polymerase) has been implicated in short-patch DNA synthesis in the DNA repair pathway known as base excision repair. The native 39 kDa enzyme is organized into four structurally and functionally distinct domains. In an effort to examine this enzyme as a potential therapeutic target, we analyzed the effect of various beta-polymerase domains on the activity of the enzyme in vitro. We show that the 14 kDa N-terminal segment of beta-polymerase, which binds to both single- and double-stranded DNA, but lacks DNA polymerase activity, inhibits beta-polymerase activity in vitro. Most importantly, the 8, 27 and 31 kDa domains of beta-polymerase do not inhibit beta-polymerase activity, demonstrating that the inhibition by the 14 kDa domain is specific. The inhibition of beta-polymerase activity in vitro is abolished by increasing the concentrations of both of the substrates (template-primer and deoxynucleoside triphosphate). In contrast, an in vitro base excision repair assay is inhibited in a domain specific manner by the 14 kDa domain even in the presence of saturating substrates. The inhibition of beta-polymerase activity by the 14 kDa domain appears specific to beta-polymerase as this domain does not inhibit either mammalian DNA polymerase alpha or Escherichia coli polymerase I (Klenow fragment). These data suggest that the 14 kDa domain could be used as a potential inhibitor of intracellular beta-polymerase and that it may provide a means for sensitizing cells to therapeutically relevant DNA damaging agents.     

DNA polymerase beta from brain neurons is a repair enzyme.

DNA polymerase beta was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian beta-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no alpha-polymerase, 99.2% beta-polymerase and only 0.8% gamma-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in vitro. Since beta-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the beta enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2',3'-dideoxyribosylthymine 5'-triphosphate (d2TTP), a potent beta-polymerase inhibitor. However, the presence of minute amounts of gamma-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase gamma in DNA repair. In view of the similarity of the neuronal DNA polymerase beta with all other mammalian beta-polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all beta-polymerases.     

Direct interaction between mammalian DNA polymerase beta and proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNA interacting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases beta (beta-pol) that resemble the consensus PIM were identified, and we show here that beta-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a beta-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in beta-polymerase appears to be a functional PIM and was required in the interaction between beta-polymerase and PCNA.     

Transfected human beta-polymerase promoter contains a ras-responsive element.

beta-Polymerase is a vertebrate cellular DNA polymerase involved in gap-filling synthesis during some types of genomic DNA repair. We report that a cloned human beta-polymerase promoter in a transient expression assay is activated by p21v-rasH expression in NIH 3T3 cells. A decanucleotide palindromic element, GTGACGTCAC, at positions -49 to -40 in the promoter is required for this ras-mediated stimulation.     

Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level.     

Inhibition of unscheduled DNA synthesis and repair of potentially lethal X-ray damage by 2-deoxy-D-glucose in yeast.

The effects of the glucose antimetabolite, 2-deoxy-D-glucose (2-DG), on DNA repair (assayed by unscheduled DNA synthesis) and on the repair of potentially-lethal damage (assayed by cell viability after irradiation) have been studied in X-irradiated respiratory-deficient yeast cells (auxotroph for 5'-thymidine-monophosphate). Experimental results show that: (a) both these phenomena can be inhibited by 2-DG; (b) the repair of potentially-lethal damage occurs after the unscheduled DNA synthesis is almost complete; and (c) the repair of potentially-lethal damage can be inhibited by 2-DG even after the completion of the unscheduled DNA synthesis.     

Protein phosphatase 5 is necessary for ATR-mediated DNA repair

Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.     

Chromatin dynamics after DNA damage: The legacy of the access–repair–restore model

Eukaryotic genomes are packaged into chromatin, which is the physiological substrate for all DNA transactions, including DNA damage and repair. Chromatin organization imposes major constraints on DNA damage repair and thus undergoes critical rearrangements during the repair process. These rearrangements have been integrated into the “access–repair–restore” (ARR) model, which provides a molecular framework for chromatin dynamics in response to DNA damage. Here, we take a historical perspective on the elaboration of this model and describe the molecular players involved in damaged chromatin reorganization in human cells. In particular, we present our current knowledge of chromatin assembly coupled to DNA damage repair, focusing on the role of histone variants and their dedicated chaperones. Finally, we discuss the impact of chromatin rearrangements after DNA damage on chromatin function and epigenome maintenance.     

New tricks for old drugs: the anticarcinogenic potential of DNA repair inhibitors

Defective or abortive repair of DNA lesions has been associated with carcinogenesis. Therefore it is imperative for a cell to accurately repair its DNA after damage if it is to return to a normal cellular phenotype. In certain circumstances, if DNA damage cannot be repaired completely and with high fidelity, it is more advantageous for an organism to have some of its more severely damaged cells die rather than survive as neoplastic transformants. A number of DNA repair inhibitors have the potential to act as anticarcinogenic compounds. These drugs are capable of modulating DNA repair, thus promoting cell death rather than repair of potentially carcinogenic DNA damage mediated by error-prone DNA repair processes. In theory, exposure to a DNA repair inhibitor during, or immediately after, carcinogenic exposure should decrease or prevent tumorigenesis. However, the ability of DNA repair inhibitors to prevent cancer development is difficult to interpret depending upon the system used and the type of genotoxic stress. Inhibitors may act on multiple aspects of DNA repair as well as the cellular signaling pathways activated in response to the initial damage. In this review, we summarize basic DNA repair mechanisms and explore the effects of a number of DNA repair inhibitors that not only potentiate DNA-damaging agents but also decrease carcinogenicity. In particular, we focus on a novel anti-tumor agent, β-lapachone, and its potential to block transformation by modulating poly(ADP-ribose) polymerase-1.     

Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells.

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is essential for both DNA replication and repair. PCNA is required in the resynthesis step of nucleotide excision repair (NER). After UV irradiation, PCNA translocates into an insoluble protein complex, most likely associated with the nuclear matrix. It has not previously been investigated in vivo whether PCNA complex formation also takes place after oxidative stress. In this study, we have examined the involvement of PCNA in the repair of oxidative DNA damage. PCNA complex formation was studied in normal human cells after treatment with hydrogen peroxide, which generates a variety of oxidative DNA lesions. PCNA was detected by two assays, immunofluorescence and western blot analyses. We observed that PCNA redistributes from a soluble to a DNA-bound form during the repair of oxidative DNA damage. PCNA complex formation was analyzed in two human natural mutant cell lines defective in DNA repair: xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). XP-A cells are defective in overall genome NER while CS-B cells are defective only in the preferential repair of active genes. Immunofluorescent detection of PCNA complex formation was similar in normal and XP-A cells, but was reduced in CS-B cells. Consistent with this observation, western blot analysis in CS-B cells showed a reduction in the ratio of PCNA relocated as compared to normal and XP-A cells. The efficient PCNA complex formation observed in XP-A cells following oxidative damage suggests that formation of PCNA-dependent repair foci may not require the XPA gene product. The reduced PCNA complex formation observed in CS-B cells suggests that these cells are defective in the processing of oxidative DNA damage.     

Localization of UvrA and Effect of DNA Damage on the Chromosome of Bacillus subtilis

We found that the nucleotide excision repair protein UvrA, which is involved in DNA damage recognition, localizes to the entire chromosome both before and after damage in living Bacillus subtilis cells. We suggest that the UvrA(2)B damage recognition complex is constantly scanning the genome, searching for lesions in the DNA. We also found that DNA damage induces a dramatic reconfiguration of the chromosome such that it no longer fills the entire cell as it does during normal growth. This reconfiguration is reversible after low doses of damage and is dependent on the damage-induced SOS response. We suggest that this reconfiguration of the chromosome after damage may be either a reflection of ongoing DNA repair or an active mechanism to protect the cell's genome. Similar observations have been made in Escherichia coli, indicating that the alteration of chromosome structure after DNA damage may be a widespread phenomenon.     

Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.     

A molecular bar-coded DNA repair resource for pooled toxicogenomic screens

DNA damage from exogenous and endogenous sources can promote mutations and cell death. Fortunately, cells contain DNA repair and damage signaling pathways to reduce the mutagenic and cytotoxic effects of DNA damage. The identification of specific DNA repair proteins and the coordination of DNA repair pathways after damage has been a central theme to the field of genetic toxicology and we have developed a tool for use in this area. We have produced 99 molecular bar-coded Escherichia coli gene-deletion mutants specific to DNA repair and damage signaling pathways, and each bar-coded mutant can be tracked in pooled format using bar-code specific microarrays. Our design adapted bar-codes developed for the Saccharomyces cerevisiae gene-deletion project, which allowed us to utilize an available microarray product for pooled gene-exposure studies. Microarray-based screens were used for en masse identification of individual mutants sensitive to methyl methanesulfonate (MMS). As expected, gene-deletion mutants specific to direct, base excision, and recombinational DNA repair pathways were identified as MMS-sensitive in our pooled assay, thus validating our resource. We have demonstrated that molecular bar-codes designed for S. cerevisiae are transferable to E. coli, and that they can be used with pre-existing microarrays to perform competitive growth experiments. Further, when comparing microarray to traditional plate-based screens both overlapping and distinct results were obtained, which is a novel technical finding, with discrepancies between the two approaches explained by differences in output measurements (DNA content versus cell mass). The microarray-based classification of Deltatag and DeltadinG cells as depleted after MMS exposure, contrary to plate-based methods, led to the discovery that Deltatag and DeltadinG cells show a filamentation phenotype after MMS exposure, thus accounting for the discrepancy. A novel biological finding is the observation that while DeltadinG cells filament in response to MMS they exhibit wild-type sulA expression after exposure. This decoupling of filamentation from SulA levels suggests that DinG is associated with the SulA-independent filamentation pathway.     

DNA-repair reactions by purified HeLa DNA polymerases and exonucleases

PM2 duplex DNA substrates containing small gaps were utilized to study DNA repair reactions of extensively purified HeLa DNase V (a bidirectional double strand DNA exonuclease) and DNA polymerases beta, gamma (mitochondrial and extramitochondrial), and alpha holoenzyme, and delta as a function of ionic strength. At 50 mM NaCl, DNase V carried out extensive exonucleolytic degradation, and beta-polymerase exhibited strand displacement synthesis. However, at 150 mM NaCl, the DNase appeared only to remove damaged nucleotides from DNA termini while beta-polymerase catalyzed only gap-filling synthesis. When present in equimolar amounts, beta-polymerase and DNase V (which can be isolated as a 1:1 complex) catalyzed more degradation than synthesis at 50 mM NaCl; however, at 150 mM NaCl a coupled very limited nick translation reaction ensued. At physiological ionic strength DNA polymerase alpha holoenzyme was not active upon these substrates. In 15 mM KCl it could fill small gaps and carry out limited nick translation with undamaged DNA, but it could not create a ligatable substrate from UV-irradiated DNA incised with T4 UV endonuclease. Mitochondrial DNA polymerase gamma was more active at 150 mM NaCl than at lower ionic strengths. It readily filled small gaps but was only marginally capable of strand-displacement synthesis. The extramitochondrial form of gamma-polymerase, conversely, was less sensitive to ionic strength; it too easily filled small gaps but was not effective in catalyzing strand displacement synthesis. Finally, DNA polymerase delta was able to fill gaps of several to 20 nucleotides in 0.05 M NaCl, but at higher NaCl concentrations there was little activity. DNA polymerases delta did not demonstrate strand displacement synthesis. Therefore, at physiological ionic strength, it appears that either DNA polymerase beta or extramitochondrial DNA polymerase gamma might aid in short patch DNA repair of nuclear (or transfecting) DNAs, whereas mitochondrial gamma-polymerase might fill small gaps in mitochondrial DNA.     

Recovery from sublethal and potentially lethal damage in an X-ray-sensitive CHO cell

It has been suggested that DNA strand breaks are the molecular lesions responsible for radiation-induced lethality and that their repair is the basis for the recovery of irradiated cells from sublethal and potentially lethal damage. EM9 is a Chinese hamster ovary cell line that is hypersensitive to killing by X rays and has been reported to have a defect in the rate of rejoining of DNA single-strand breaks. To establish the importance of DNA strand-break repair in cellular recovery from sublethal and potentially lethal X-ray damage, those two parameters, recovery from sublethal and potentially lethal damage, were studied in EM9 cells as well as in EM9's parental repair-proficient strain, AA8. As previously reported, EM9 is the more radiosensitive cell line, having a D0 of 0.98 Gy compared to a D0 of 1.56 Gy for AA8 cells. DNA alkaline elution studies suggest that EM9 cells repair DNA single-strand breaks at a slower rate than AA8 cells. Neutral elution analysis suggests that EM9 cells also repair DNA double-strand breaks more slowly than AA8 cells. All of these data are consistent with the hypothesis that DNA strand-break ligation is defective in EM9 cells and that this defect accounts for increased radiosensitivity. The kinetics and magnitude of recovery from sublethal and potentially lethal damage, however, were similar for both EM9 and AA8 cells. Six-hour recovery ratios for sublethal damage repair were found to be 2.47 for AA8 cells and 1.31 for EM9 cells. Twenty-four-hour recovery ratios for potentially lethal damage repair were 3.2 for AA8 and 3.3 for EM9 cells. Both measurements were made at approximately equitoxic doses. Thus, the defect in EM9 cells that confers radiosensitivity and affects DNA strand-break rejoining does not affect sublethal damage repair or potentially lethal damage repair.     

DNA damage excision repair in microplate wells with chemiluminescence detection: development and perspectives

The development of in vitro repair assays with human cell-free extracts led to new insights on the mechanism of excision of DNA damage which consists of incision/excision and repair synthesis/ligation. We have adapted the repair synthesis reaction with cells extracts incubated with damaged plasmid DNA performed in liquid phase to solid phase by DNA adsorption into microplate wells. Since cells extracts are repair competent in base excision and nucleotide excision repair, all types of substrate DNA lesions were detected with chemiluminescence measurement after incorporation of biotin-deoxynucleotide during the repair synthesis step. Derivatives of our initial 3D-assay (DNA damage detection) have been set up to: i) screen antioxidative compounds and NER inhibitors; ii) capture genomic DNA (3D(Cell)-assay) that allows detection of alkylated base and consequently determines the kinetics of the cellular repair; and iii) immunodetect the repair proteins in an ELISA reaction (3D(Rec)-assay). The 3D derived assays are presented and discussed.