Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Polγ against UV-induced inactivation.

Manganese superoxide dismutase is a nuclear encoded primary antioxidant enzyme localized exclusively in the mitochondrial matrix. Genotoxic agents, such as ultraviolet (UV) radiation, generates oxidative stress and cause mitochondrial DNA (mtDNA) damage. The mtDNA polymerase (Polγ), a major constituent of nucleoids, is responsible for the replication and repair of the mitochondrial genome. Recent studies suggest that the mitochondria contain fidelity proteins and MnSOD constitutes an integral part of the nucleoid complex. However, it is not known whether or how MnSOD participates in the mitochondrial repair processes. Using skin tissue from C57BL/6 mice exposed to UVB radiation, we demonstrate that MnSOD has a critical role in preventing mtDNA damage by protecting the function of Polγ. Quantitative-PCR analysis shows an increase in mtDNA damage after UVB exposure. Immunofluorescence and immunoblotting studies demonstrate p53 translocation to the mitochondria and interaction with Polγ after UVB exposure. The mtDNA immunoprecipitation assay with Polγ and p53 antibodies in p53(+/+) and p53(-/-) mice demonstrates an interaction between MnSOD, p53 and Polγ. The results suggest that these proteins form a complex for the repair of UVB-associated mtDNA damage. The data also demonstrate that UVB exposure injures the mtDNA D-loop in a p53-dependent manner. Using MnSOD-deficient mice we demonstrate that UVB-induced mtDNA damage is MnSOD dependent. Exposure to UVB results in nitration and inactivation of Polγ, which is prevented by addition of the MnSOD mimetic Mn(III)TE-2-PyP(5+). These results demonstrate for the first time that MnSOD is a fidelity protein that maintains the activity of Polγ by preventing UVB-induced nitration and inactivation of Polγ. The data also demonstrate that MnSOD has a role along with p53 to prevent mtDNA damage.

Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis.

Chicken B lymphocyte precursors and DT40 cells diversify their immunoglobulin-variable (IgV) genes through homologous recombination (HR)-mediated Ig gene conversion. To identify DNA polymerases that are involved in Ig gene conversion, we created DT40 clones deficient in DNA polymerase eta (poleta), which, in humans, is defective in the variant form of xeroderma pigmentosum (XP-V). Poleta is an error-prone translesion DNA synthesis polymerase that can bypass UV damage-induced lesions and is involved in IgV hypermutation. Like XP-V cells, poleta-disrupted (poleta) clones exhibited hypersensitivity to UV. Remarkably, poleta cells showed a significant decrease in the frequency of both Ig gene conversion and double-strand break-induced HR when compared to wild-type cells, and these defects were reversed by complementation with human poleta. Our findings identify a DNA polymerase that carries out DNA synthesis for physiological HR and provides evidence that a single DNA polymerase can play multiple cellular roles.

Altered DNA polymerase iota expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis.

The recently discovered human enzyme DNA polymerase iota (pol iota) has been shown to have an exceptionally high error rate on artificial DNA templates. Although there is a considerable body of in vitro evidence for a role for pol iota in DNA lesion bypass, there is no in vivo evidence to confirm this action. We report here that pol iota expression is elevated in breast cancer cells and correlates with a significant decrease in DNA replication fidelity. We also demonstrate that UV treatment of breast cancer cells additionally increases pol iota expression with a peak occurring between 30 min and 2 h after cellular insult. This implies that the change in pol iota expression is an early event after UV-mediated DNA damage. That pol iota may play a role in the higher mutation frequencies observed in breast cancer cells was suggested when a reduction in mutation frequency was found after pol iota was immunodepleted from nuclear extracts of the cells. Analysis of the UV-induced mutation spectra revealed that > 90% were point mutations. The analysis also demonstrated a decreased C --> T nucleotide transition and an increased C --> A transversion rate. Overall, our data strongly suggest that pol iota may be involved in the generation of both increased spontaneous and translesion mutations during DNA replication in breast cancer cells, thereby contributing to the accumulation of genetic damage.

Role of PSO genes in repair of DNA damage of Saccharomyces cerevisiae.

Photoactivated psoralens used in treatment of skin diseases like Psoriasis and Vitiligo cause DNA damage, the repair of which may lead to mutations and thus to higher risk to have skin cancer. The simple eukaryote Saccharomyces cerevisiae was chosen to investigate the cells' genetic endowment with repair mechanisms for this type of DNA damage and to study the genetic consequences of such repair. Genetic studies on yeast mutants sensitive to photoactivated psoralens, named pso mutants, showed their allocation to 10 distinct loci. Cloning and molecular characterization allowed their grouping into three functional classes: (I) the largest group comprises seven PSO genes that are either generally or specifically involved in error-prone DNA repair and thus affect induced mutability and recombination; (II) one PSO gene that represents error-free excision repair, and (III) two PSO genes encoding proteins not influencing DNA repair but physiological processes unrelated to nucleic acid metabolism. Of the seven DNA repair genes involved in induced mutagenesis three PSO loci [PSO1/REV3, PSO8/RAD6, PSO9/MEC3] were allelic to already known repair genes, whereas three, PSO2/SNM1, PSO3/RNR4, and PSO4/PRP19 represent new genes involved in DNA repair and nucleic acid metabolism in S. cerevisiae. Gene PSO2 encodes a protein indispensable for repair of interstrand cross-link (ICL) that are produced in DNA by a variety of bi- and polyfunctional mutagens and that appears to be important for a likewise repair function in humans as well. In silico analysis predicts a putative endonucleolytic activity for Pso2p/Snm1p in removing hairpins generated as repair intermediates. The absence of induced mutation in pso3/rnr4 mutants indicates an important role of this subunit of ribonucleotide reductase (RNR) in regulation of translesion polymerase zeta in error-prone repair. Prp19p/Pso4p influences efficiency of DNA repair via splicing of pre-mRNAs of intron-containing repair genes but also may function in the stability of the nuclear scaffold that might influence DNA repair capacity. The seventh gene, PSO10 which controls an unknown step in induced mutagenesis is not yet cloned. Two genes, PSO6/ERG3 and PSO7/COX11, are responsible for structural elements of the membrane and for a functional respiratory chain (RC), respectively, and their function thus indirectly influences sensitivity to photoactivated psoralens.

Analysis of the stimulation of DNA polymerase V of Escherichia coli by processivity proteins.

Bypass of replication-blocking lesions in Escherichia coli is carried out by DNA polymerase V (UmuC) in a reaction that requires UmuD', RecA, and single-strand DNA-binding protein (SSB). The activity of this four-component basic bypass system is a low-fidelity and low-processivity activity. Addition of the processivity subunits of pol III, the beta subunit sliding DNA clamp, and the five-subunit gamma complex clamp loader increased the rate of translesion replication approximately 3-fold. This stimulation was specific to the lesion bypass step, with no effect on the initiation of synthesis by pol V. The beta subunit and gamma complex increased the processivity of pol V from 3 to approximately 14-18 nucleotides, providing a mechanistic basis for their stimulatory effect. Stimulation of bypass was observed over a range of RecA and SSB concentrations. ATPgammaS, which strongly inhibits translesion replication by pol V, primarily via inhibition of the initiation stage, caused the same inhibition also in the presence of the processivity proteins. The in vivo role of the processivity proteins in translesion replication was examined by assaying UV mutagenesis. This was done in a strain carrying the dnaN59 allele, encoding a temperature-sensitive beta subunit. When assayed in an excision repair-defective background, the dnaN59 mutant exhibited a level of UV mutagenesis reduced up to 3-fold compared to that of the isogenic dnaN(+) strain. This suggests that like in the in vitro system, the beta subunit stimulates lesion bypass in vivo.

DNA polymerase eta undergoes alternative splicing, protects against UV sensitivity and apoptosis, and suppresses Mre11-dependent recombination.

Polymerase eta (pol eta) is a low-fidelity DNA polymerase that is the product of the gene, POLH, associated with the human XP variant disorder in which there is an extremely high level of solar-induced skin carcinogenesis. The complete human genomic sequence spans about 40 kb containing 10 coding exons and a cDNA of 2.14 kb; exon I is untranslated and is 6 kb upstream from the first coding exon. Using bacterial artificial chromosomes (BACs), the gene was mapped to human chromosome band 6p21 and mouse band 17D. The gene is expressed in most tissues, except for very low or undetectable levels in peripheral lymphocytes, fetal spleen, and adult muscle; exon II, however, is frequently spliced out in normal cells and in almost half the transcripts in the testis and fetal liver. Expression of POLH in a multicopy episomal vector proved nonviable, suggesting that overexpression is toxic. Expression from chromosomally integrated linear copies using either an EF1-alpha or CMV promoter was functional, resulting in cell lines with low or high levels of pol eta protein, respectively. Point mutations in the center of the gene and in a C-terminal cysteine and deletion of exon II resulted in inactivation, but addition of a terminal 3 amino acid C-terminal tag, or an N- or C-terminal green fluorescent protein, had no effect on function. A low level of expression of pol eta eliminated hMre11 recombination and partially restored UV survival, but did not prevent UV-induced apoptosis, which required higher levels of expression. Polymerase eta is therefore involved in S-phase checkpoint and signal transduction pathways that lead to arrest in S, apoptosis, and recombination. In normal cells, the predominant mechanism of replication of UV damage involves pol eta-dependent bypass, and Mre11-dependent recombination that acts is a secondary, backup mechanism when cells are severely depleted of pol eta.

Effects of high ELF magnetic fields on enzyme-catalyzed DNA and RNA synthesis in vitro and on a cell-free DNA mismatch repair.

Environmental electromagnetic fields have been implicated in human cancers. We examined whether high extremely low frequency (ELF) AC magnetic fields could affect DNA synthesis, transcription or repair, using in vitro model systems with defined sequences. The rate and fidelity of DNA polymerase catalyzed DNA synthesis, as well as of RNA polymerase catalyzed RNA synthesis, were not statistically significantly affected by 60 Hz 0.25-0.5 Tesla magnetic fields. The efficiency of mutS dependent mismatch repair with human cell extracts was also not affected by the magnetic field exposure. The results suggest that the core processes related to the transmission of genetic information are stable under high ELF magnetic fields.

Engagement of DNA polymerases during apoptosis.

DNA replicative and repair machinery was investigated by means of different techniques, including in vitro nuclear enzymatic assays, immunoelectron microscopy and confocal microscopy, in apoptotic cell lines such as HL-60 treated with methotrexate, P815 and K562 exposed to low temperatures and Friend cells exposed to ionizing radiation. The results showed a shift of DNA polymerase alpha and beta activities. DNA polymerase alpha, which in controls was found to be the principal replicative enzyme driving DNA synthesis, underwent, upon apoptosis, a large decrease of its activity being replaced by DNA polymerase beta which is believed to be associated with DNA repair. Such a modulation was concomitant with a topographical redistribution of both DNA polymerase alpha and the incorporation of BrdUrd throughout the nucleus. Taken together, these results indicate the occurrence of a dramatic response of the DNA machinery, through a possible common or at least similar behaviour when different cell lines are triggered to apoptosis. Although this possibility requires further investigation, these findings suggest an extreme attempt of the cell undergoing apoptosis to preserve its nuclear environment by switching on a repair/defence mechanism during fragmentation and chromatin margination.

[Effect of gamma radiation on the components of hepatitis B virus and the delta antigen]. / Vliianie gamma-izlucheniia na komponenty virusa gepatitia B i del'ta-antigen.

The influence of ionizing irradiation on the serological activity of purified HBsAg, receptors to polymerized albumin, DNA-polymerase activity, delta agent, and delta antigen was studied. Different radiosensitivity of hepatitis B virus components and delta-system was demonstrated. The possibility of sterilization by irradiation of purified HBsAg preparations suitable for construction of a vaccine against hepatitis B was shown.

Inhibition of mammalian DNA polymerases by hematoporphyrin derivative and photoradiation.

Hematoporphyrin derivative (HPD) plus photoradiation caused the inactivation of DNA polymerases from calf thymus and R3230AC rat mammary tumor. Photosensitization of purified DNA polymerase-alpha as well as two forms of DNA polymerase-delta (I and II) from calf thymus were evaluated. Although all polymerase enzyme forms were inactivated at 70 micrograms HPD/ml, DNA polymerase-delta II was the most sensitive, displaying a 90% inactivation under conditions that did not cause significant inactivation of the other polymerase forms. Unlike DNA polymerase-alpha, the delta-forms have an associated 3'- to 5'-exonuclease activity. The exonuclease associated with DNA polymerase-delta II was uniquely sensitive to a low level of HPD and light exposure. DNA polymerase-delta II can be distinguished from other polymerase forms in cell extracts by its relative insensitivity to the polymerase inhibitor N2-(p-n-butylphenyl)deoxyadenosine 5'-triphosphate. In cytosols prepared from calf thymus and R3230AC rat mammary tumors, DNA polymerase-delta II was preferentially inhibited by HPD plus light. Furthermore, in experiments in which tumor-bearing rats were administered HPD prior to preparation of tumor cytosols, DNA polymerase-delta II was specifically inactivated by exposure to light. These results are discussed in view of their possible role in cancer therapy, and the potential use of HPD as a specific inhibitory agent of DNA polymerase-delta II is suggested.

Properties of a DNA repair endonuclease from mouse plasmacytoma cells.

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.

Strand break repair, DNA polymerase activity and heat radiosensitization in thermotolerant cells.

HeLa S3 cells were made thermotolerant by 'chronic' (5 h at 42 degrees C) or 'acute' (15 min at 44 degrees C followed by 5 h at 37 degrees C) heat treatments. Cell survival, repair of radiation-induced DNA strand breaks, alpha and beta DNA polymerase activity and radiation sensitivity following hyperthermia were all measured in both control and thermotolerant cells. The ability to repair DNA strand breaks correlated well with cell survival following hyperthermia. Hyperthermic inhibition of strand break repair was reduced in thermotolerant relative to control cells, although the thermal tolerance ratios for repair were less than for hyperthermic cell killing. Both radiosensitization and DNA polymerase inactivation by hyperthermia were only slightly reduced in thermotolerant relative to control cells. Hence a poor correlation was found between these two parameters and hyperthermic cell survival. For all heat treatments applied, alpha and beta DNA polymerase activity correlated well with the extent of hyperthermic radiosensitization.

Sequence specificities in the interactions of chemicals and radiations with DNA.

Sequence specificities in the interactions of chemicals and radiations with DNA are reviewed. Emphasis is placed on information which has been obtained by adapting DNA sequencing techniques to the detection of DNA damage and modifications. The actions of anti-tumor drugs, non-covalent DNA binders and UV irradiation are discussed in terms of both modifications induced in DNA and the subsequent response of DNA polymerase and repair enzymes. Particular attention is paid to the evidence for 'sequence-specific' interactions of these agents with DNA. It is concluded that while most agents exhibit 'warm' or even 'hot' spots in their interactions with DNA there is not, as yet, compelling evidence for extreme selectivity down to say the gene level in their actions. There does, however, appear to be some affinity, particularly in the case of non-covalent binders, for certain tertiary structures rather than primary sequences per se. In addition, both misincorporation opposite DNA damage and the bypass of damage by polymerases are important phenomena which, to some extent, exhibit sequence specificities. The idea is advanced that DNA structures such as hairpins or cruciforms maybe important in vivo targets for many agents giving rise to specific biological effects.

DNA polymerase alpha and beta activity in gamma-irradiated HeLa S3 cells.

The acute effects (less than 2 hours) of gamma-irradiation on DNA polymerase alpha and beta activity in HeLa S3 cells were studied. The enzyme activities were measured in sonicates of the irradiated cells, using an exogenous DNA as template. Both DNA alpha- and beta-polymerase activities decreased following irradiation of the cells. Doses as low as 100 rad significantly reduced the activities of the enzymes. While the activities of both DNA polymerases decreased as the dose received by the cells increased, the major reduction in enzyme activity occurred with doses of 100--200 rad. The reduction in DNA alpha- and beta-polymerase activities was maximal by 30 min post-irradiation and recovered to control values by 2 hours post-irradiation.