Photosensitization by imatinib. A photochemical and photobiological study of the drug and its substructures.

Imatinib (IMT) is a promising tyrosine kinase inhibitor used in the treatment of some types of human cancer. It constitutes a successful example of rational drug design based on the optimization of the chemical structure to reach an improved pharmacological activity. Cutaneous reactions, such as increased photosensitivity or pseudoporphyria, are among the most common nonhematological IMT side effects; however, the molecular bases of these clinical observations have not been determined. Thus, to gain insight into the IMT photosensitizing properties, we addressed its photobehavior together with that of its potentially photoactive anilino-pyrimidine and pyridyl-pyrimidine fragments. In this context, steady-state and time-resolved fluorescence as well as laser flash photolysis experiments have been conducted, and the DNA photosensitization potential has been investigated by means of single-strand break detection using agarose gel electrophoresis. The obtained results reveal that the drug itself and its anilino-pyrimidine fragment are not DNA photosensitizers. By contrast, the pyridyl-pyrimidine substructure displays a marked phototoxic potential, which has been associated with the generation of a long-lived triplet excited state. Interestingly, this reactive species is efficiently quenched by benzanilide, another molecular fragment of IMT. Clearly, integration of the photoactive pyridyl-pyrimidine moiety in a more complex structure strongly modifies its photobehavior, which in this case is fortunate as it leads to an improved toxicological profile. Thus, on the bases of the experimental results, direct in vivo photosensitization by IMT seems unlikely. Instead, the reported photosensitivity disorders could be related to indirect processes, such as the previously suggested impairment of melanogenesis or the accumulation of endogenous porphyrins.

Novel method for quantifying radiation-induced single-strand-break yields in plasmid DNA highlights 10-fold discrepancy.

The widely used agarose gel electrophoresis method for assessing radiation-induced single-strand-break (SSB) yield in plasmid DNA involves measurement of the fraction of relaxed-circular (C) form that migrates independently from the intact supercoiled (SC) form. We rationalized that this method may underestimate the SSB yield since the position of the relaxed-circular form is not altered when the number of SSB per DNA molecule is >1. To overcome this limitation, we have developed a novel method that directly probes and quantifies SSBs. Supercoiled (3)H-pUC19 plasmid samples were irradiated with γ-rays, alkali-denatured, dephosphorylated, and kinated with γ-[(32)P]ATP, and the DNA-incorporated (32)P activities were used to quantify the SSB yields per DNA molecule, employing a standard curve generated using DNA molecules containing a known number of SSBs. The same irradiated samples were analyzed by agarose gel and SSB yields were determined by conventional methods. Comparison of the data demonstrated that the mean SSB yield per plasmid DNA molecule of [21.2±0.59]×10(-2)Gy(-1) as measured by direct probing is ~10-fold higher than that obtained from conventional gel-based methods. These findings imply that the SSB yields inferred from agarose gels need reevaluation, especially when they were utilized in the determination of radiation risk.

Gold nanoparticles enhance DNA damage induced by anti-cancer drugs and radiation.

The chemotherapeutic agent cisplatin was chemically linked to pGEM-3Zf(-) plasmid DNA to produce a cisplatin-DNA complex, Gold nanoparticles, which bind electrostatically to pure DNA, could also be added to this complex. Dry films of pure plasmid DNA and DNA-cisplatin, DNA-gold nanoparticles and DNA-cisplatin-gold nanoparticles complexes were bombarded by 60 keV electrons. The yields of single- and double-strand breaks were measured as a function of exposure by electrophoresis. From a comparison of such yields from the different type of films, we found that the binding of only one gold nanoparticle to a plasmid-cisplatin complex containing 3197 base pairs increases by a factor of 3 the efficiency of the chemotherapeutic agent cisplatin to produce double-strand breaks in irradiated DNA. Furthermore, adding two cisplatin molecules and one gold nanoparticle to DNA enhances radiation-induced DSBs by a factor of 7.5. A number of phenomena could contribute to this huge enhancement, including the higher density of low-energy electrons and reactive species around the gold nanoparticles and the weakening of bonds adjacent to cisplatin in the DNA backbone. The addition of gold nanoparticles to cisplatin and other platinum agents may therefore provide interesting avenues of research to improve the treatment of cancer by concomitant chemoradiation.

Reversible photopadlocking on double-stranded DNA.

We describe a highly efficient method for reversible photocircularization of oligonucleotide (ODN) on a double-stranded DNA template: 5-carboxyvinyl-2'-deoxyuridine-containing ODN was reversibly circularized around the target sequence of the double-stranded plasmid DNA resulting in formation of a catenated plasmid.

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major (S. M. Beverley, J. A. Coderre, D. V. Santi, and R. T. Schimke, Cell 38:431-439, 1984). Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.

Factors influencing the removal of thymine glycol from DNA in gamma-irradiated human cells.

The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.

Fractionated ionizing radiation accelerates loss of amplified MDR1 genes harbored by extrachromosomal DNA in tumor cells.

In tumor specimens such as those from neuroblastoma, ovarian, and lung carcinoma patients, the prevalence of extrachromosomal circular DNA molecules harboring amplified genes has been well established. In some cases, the amplified genes have been identified as oncogenes, and their increased expression appears to contribute to the maintenance and progression of the malignancy. The aim of this study was to investigate the effect of fractionated radiation treatment, given in daily doses similar to those administered clinically, on the stability of extrachromosomal circular DNA molecules in cancer cells. Our studies were conducted with multidrug-resistant KB cells, which harbor extrachromosomal copies of the multidrug resistance gene (MDR1) almost exclusively on circular DNA molecules of approximately 750 and 1500 kb pairs. This size range is representative of extrachromosomal circular DNA molecules that have been shown to harbor amplified oncogenes in vivo. Exponentially growing MDR KB cells were exposed to 1400 and 2800 cGy ionizing radiation administered in 7 and 14 fractions, respectively, at 200 cGy per fraction/day. A statistically significant decrease in MDR1 extrachromosomal gene copy number was reproducibly detected in the irradiated cells compared with unirradiated cells passaged for the duration of the experiment in the absence of radiation treatment. This decrease was accompanied by a reduction in multidrug resistance and in P-glycoprotein levels, as determined by clonogenic dose-response assays and Western analyses, respectively. P-glycoprotein is a multidrug transporter encoded by the MDR1 gene. Fluorescence in situ hybridization studies further determined that extrachromosomal circular DNA loss correlated to the entrapment of these DNA molecules in radiation-induced micronuclei. These results indicate that radiation-induced loss of extrachromosomally amplified genes from tumor cells via their entrapment in micronuclei contributes to the improved therapeutic response observed for some cancers.

Characterization of a complex 125I-induced DNA double-strand break: implications for repair.

PURPOSE: To examine the role of radiation-induced DNA double-strand break (DSB) structural organization in DSB repair, and characterize the structural features of 125I-induced DSBs that may impact the repair process. METHODS: Plasmid DNA was linearized by sequence-specific targeting using an 125I-labeled triplex-forming oligonucleotide (TFO). Following isolation from agarose gels, base damage structures associated with the DSB ends in plasmids linearized by the 125I-TFO were characterized by probing with the E. coli DNA damage-specific endonuclease and DNA-glycosylases, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-glycosylase (Fpg). RESULTS: Plasmid DNA containing DSBs produced by the high-LET-like effects of 125I-TFO has been shown to support at least 2-fold lower end joining than gamma-ray linearized plasmid, and this may be a consequence of the highly complex structure expected near an 125I-induced DSB end. Therefore, to determine if a high density of base damage exists proximal to the DSBs produced by 125I-TFOs, short fragments of DNA recovered from the DSB end of 125I-TFO-linearized plasmid were enzymatically probed. Base damage and AP site clustering was demonstrated within 3 bases downstream and 7 bases upstream of the targeted base. Furthermore, the pattern and extent of base damage varied depending upon the presence or absence of 2 M DMSO during irradiation. CONCLUSIONS: 125I-TFO-induced DSBs exhibit a high degree of base damage clustering proximal to the DSB end. At least 60% of the nucleotides within 10 bp of the 125I decay site are sensitive to cleavage by endo IV, endo III, or Fpg following damage accumulation in the presence of DMSO, whereas > or = 80% are sensitive in the absence of DMSO. The high degree of base damage clustering associated with the 125I-TFO-induced DSB end may be a major factor leading to its negligible in vitro repair by the non-homologous end-joining pathway (NHEJ).

Controlled Microwave Heating Accelerates Rolling Circle Amplification.

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

PicoGreen assay of circular DNA for radiation biodosimetry.

We developed a simple, rapid and quantitative assay using the fluorescent probe PicoGreen to measure the concentration of ionizing radiation-induced double-stranded DNA (dsDNA) in mouse plasma, and we correlated this concentration with the radiation dose. With 70 µl of blood obtained by fingerstick, this 30 min assay reduces protein interference without extending sample processing time. Plasma from nonirradiated mice (BALB/c and NIH Swiss) was pooled, diluted and spiked with dsDNA to establish sensitivity and reproducibility of the assay to quantify plasma dsDNA. The assay was then used to directly quantify dsDNA in plasma at 0-48 h after mice received 0-10 Gy total-body irradiation (TBI). There are three optimal conditions for this assay: 1:10 dilution of plasma in water; 1:200 dilution of PicoGreen reagent in water; and calibration of radiation-induced dsDNA concentration through a standard addition method using serial spiking of samples with genomic dsDNA. Using the internal standard calibration curve of the spiked samples method, the signal developed within 5 min, exhibiting a linear signal (r(2) = 0.997). The radiation-induced elevation of plasma DNA in mice started at 1-3 h, peaked at 9 h and gradually returned to baseline at 24 h after TBI (6 Gy). DNA levels in plasma collected from mice 9 h after 0-10 Gy TBI correlated strongly with dose (r(2) = 0.991 and 0.947 for BALB/c and NIH Swiss, respectively). Using the PicoGreen assay, we observed a radiation dose-dependent response in extracellular plasma DNA 9 h after irradiation with an assay time ≤ 30 min.

Two independent molecular pathways for recombinant adeno-associated virus genome conversion occur after UV-C and E4orf6 augmentation of transduction.

Numerous environmental influences have been demonstrated to enhance recombinant adeno-associated virus (rAAV) transduction. Such findings are the foundation of developing new and innovative strategies to improve the efficiency of rAAV as a gene therapy vector. Several of these environmental factors included genotoxic stresses such as UV and y irradiation as well as certain adenoviral gene products such as E4orf6. The mechanisms by which these environmental stimuli increase rAAV transduction are only partially understood but have been suggested to involve both endocytosis and uptake of virus to the nucleus, as well as conversion of single-stranded DNA viral genomes to double-stranded expressible forms. Two molecular intermediates of rAAV genomes, which have been demonstrated to correlate with transgene expression and/or the persistence of rAAV, include both replication form (Rf) monomers and dimers as well as circular intermediates. In the present study, we demonstrate that augmentation of rAAV transduction by UV irradiation and the adenoviral protein E4orf6 correlates with distinct increases in either circular or replication form intermediates, respectively. UV irradiation of primary fibroblasts at 15 J/m2 resulted in a 15-fold induction of head-to-tail circular intermediates, with minimal induction of replication form rAAV genomes. In contrast, E4orf6-augmented rAAV transduction was correlated with the formation of replication form intermediates, with no alteration in the abundance of circular intermediates. These findings demonstrate that rAAV transduction can occur through two independent molecular pathways that convert single-stranded AAV genomes to expressible forms of DNA.

Cloning of the phr gene and amplification of photolyase in Escherichia coli.

A new technique is developed for physically enriching recombinant DNA molecules in an in vitro recombination mixture. UV-irradiation of the donor DNA before recombination enables photoreactivating enzyme (PRE) (deoxyribodipyrimidine photolyase, EC 4.1.99.3) to attach to the donor segments in recombinant molecules. This attached protein causes retention of the recombinant molecules on a nitrocellulose filter, while molecules not containing donor DNA pass through. The bound DNA is repaired of its UV damage and released for insertion into cells by exposure to photoreactivating light in situ, yielding approx. 350-fold enrichment. Although applicable to any gene, this procedure has been used in cloning the Escherichia coli phr gene itself, permitting 100-fold amplification of the gene product in vivo.

DNA strand breaks photosensitized by benoxaprofen and other non steroidal antiinflammatory agents.

Benoxaprofen, a non steroidal antiinflammatory drug is known to be highly phototoxic. Upon irradiation at 300 nm, benoxaprofen is shown to enhance the cleavage of phi X 174 DNA in buffered aqueous solution (pH 7.4). A linear relationship between the number of single strand breaks and the irradiation time is found. In deaerated solutions, these breaks are three times greater in the presence than in the absence of benoxaprofen. In both cases the rate of cleavage decreases in the presence of air. The rate of DNA damage increases with the drug per base pair ratio up to approximatively 0.2 and then decreases at higher ratios. Other NSAIDs, naproxen, ketoprofen, diflunisal, sulindac and indomethacin have been tested as photocleavers of DNA by using the same experimental conditions. A comparison of the efficiency of cleavage of all these drugs (including BNP) was obtained at drug concentrations such that the light absorbance was the same. Benoxaprofen, naproxen, ketoprofen and diflunisal induce single strand breaks. Sulindac and indomethacin do not cause breaks, and they can in some conditions even act as screening agents. The most efficient of the series are naproxen and ketoprofen. In the presence of oxygen, at the same concentrations as above, the efficiency of benoxaprofen, ketoprofen and diflunisal is decreased while that of naproxen is increased. This suggests that all these compounds do not interact with DNA by the same mechanism. In the case of BNP, the mechanism of photoinduced DNA cleavage is discussed in detail. It is shown that the photoactive agent is the decarboxylated derivative of benoxaprofen, as the photodecarboxylation of benoxaprofen is much faster than the photocleavage of DNA.

Modifications of circular DNA by photoalkylation.

The effects of photoalkylation on superhelical PM2 DNA were examined. The chief product was 8-(2-hydroxy-2-propyl)guanine, formed exclusively in sequences of alternating purines and pyrimidines. Other purine damages included 8-(2-hydroxy-2-propyl)adenine and smaller quantities of two uncharacterized adenine products. DNA strand breaks were formed with increasing irradiation. A small quantity of thymine-containing photodimers was formed. Photoalkylation of poly(dG-dC):poly(dG-dC) reduced the concentration of salt required to effect inversion of the circular dichroic spectrum. This suggests that photoalkylation induces the transition of poly(dG-dC):poly(dG-dC) from the right-handed B form of DNA to the left-handed Z form.

Radiosensitivity of DNA minicircles.

DNA minicircles of 207 bp were constructed by the ligation of linear restriction fragments in the presence of various concentrations of ethidium bromide. Three topoisomers characterized by linking numbers (Lk) of 20, 19 and 18, and with helical repeats of 10.35, 10.9 and 11.5 bp/turn respectively, were obtained. They are called, respectively, relaxed minicircle or topoisomer 0, topoisomer -1 and topoisomer -2. Owing to the limited flexibility of such small circles, the stress created by the lack of 1 or 2 turns cannot be eliminated by a spatial circle-axis writhing (supercoiling) of the circular molecules. These two undertwisted, stressed topoisomers have to adopt a flat, non-crossed shape, similar to that of the relaxed minicircle. The three minicircles were irradiated with gamma-rays or fast neutrons. The same yields of single-strand breaks, double-strand breaks and alkali-induced single-strand breaks were observed for the three topoisomers showing that their base and sugar moieties are attacked equally by gamma photon- or fast neutron-induced radicals. We conclude that untwisting of a B helix does not modify the radiosensitivity of DNA.

Mechanism of protection against radiation-induced DNA damage in plasmid pBR322 by caffeine.

PURPOSE: Caffeine (1,3,7-trimethyl xanthine), a dietary component, has been shown to have widely varying effects on DNA damage induced by UV and ionizing radiation, depending upon pre- or post-irradiation administration and its concentration. Caffeine administered post-UV irradiation is known to inhibit enzymatic repair of DNA lesions, leading to potentiation of damage, whereas its presence before or during irradiation elicits protection in a wide range of test systems: bacteria, cultured human cells, plant seeds and mouse. The purpose of this study is to test whether caffeine present during gamma-irradiation of plasmid DNA, a system devoid of replication and repair, could elicit protection by scavenging free radicals. MATERIALS AND METHODS: Plasmid pBR322 DNA was exposed to gamma-radiation in the presence or absence of caffeine at a dose-rate of 1.20 Gy min(-1) and damage measured as single-strand breaks. To understand the mechanisms of the observed protection, especially under oxic conditions, reaction of caffeine with superoxide radical (O(2)(-)), hydrogen peroxide (H(2)O(2)) and the deoxyribose peroxyl radical (ROO(*)) were studied. RESULTS: Irradiation of pBR322 was observed to induce a dose-dependent increase in single-strand breaks. Caffeine itself did not induce strand breaks but reduced radiation-induced strand breaks at micromolar to millimolar concentrations. Caffeine has been shown to react with the radiation-derived oxidants. The reaction rate constants observed were 7.5x10(1) M(-1) s(-1) with O(2)(-) 1.05x10(8) M(-1) s(-1) with ROO(*) and 8.8x10(1) M(-1) s(-1) with H(2)O(2). CONCLUSIONS: Caffeine effectively protects DNA against ionizing radiation in a system devoid of repair and replication machinery. Thus, DNA protection shown by caffeine is possibly due to the scavenging of radiation-derived primary as well as secondary reactive oxygen species, and this physicochemical protective pathway possibly pre-empts any subsequent inhibitory effect of caffeine on the enzymatic repair of DNA.

UV-A induced DNA nicking activities of skin photosensitive drugs: phenothiazines, benzothiadiazines and afloqualone.

Plasmid pBR 322 was subjected to UV-A irradiation in the presence of photosensitive drugs, i.e., phenothiazines [chlorpromazine hydrochloride (CPZ), promethazine hydrochloride (PMZ) and mequitazine (MQZ)], benzothiadiazines [penflutizide (PFZ), hydrochlorothiazide (HCT) and methyclothiazide (MCT)] and afloqualone (AQ). The distribution of the closed-circular and the open-circular form of the plasmid DNA was analyzed by means of neutral agarose gel electrophoresis. All the drugs used induced more or less DNA nicking to yield the open-circular form. The nicking activities of the phenothiazines were in the order: CPZ greater than PMZ greater than MQZ. CPZ elicited extensive degradation of the DNA by photosensitization. The nicking activities of the benzothiadiazines and AQ were much weaker than CPZ and PMZ.

Effects of AQ4N and its reduction product on radiation-mediated DNA strand breakage.

Supercoiled plasmid pBR322 DNA was irradiated in phosphate buffer by 60Co gamma-rays at a dose rate 19.26 Gy/min and total dose of 10 Gy in the presence of a bioreductive antitumour prodrug namely 1,4-bis [¿2-(dimethylamino-N-oxide)ethyl¿ amino] 5, 8-dihydroxyanthracene-9,10-dione (AQ4N) and its DNA affinic reduction product 1,4-bis[¿2(dimethylamino)ethyl¿ amino] 5,8-dihydroxyanthracene-9,10-dione (AQ4) under air and nitrogen. AQ4N and AQ4 were found to protect against radiation-induced plasmid single and double strand breakage as assessed by agarose gel electrophoresis. The differences between the two agents, and between atmospheres of air or nitrogen were negligible. It was also found that the protection efficiencies of the compounds were pH dependent and showed maximum protection at pH 6. These results indicate that protection of DNA by AQ4 and AQ4N against radiation damage is an indirect effect since both agents are equally effective despite major differences in their DNA affinity. It is likely that radiation-induced phosphate buffer radicals are intercepted by AQ4 and AQ4N in a pH-dependent process.

AP endonuclease and uracil DNA glycosylase activities in Deinococcus radiodurans.

An endonuclease specific for apurinic/apyrimidinic (AP) sites was identified and purified from extracts of Deinococcus radiodurans. The enzyme is 34.5 kD, has no activity towards normal, alkylated, uracil-containing, or UV-irradiated DNA, and is active in the presence of EDTA. The addition of up to 10 mM Mg2+ or Mn2+ did not affect activity, but higher concentrations were inhibitory. There is no associated exonuclease activity, either in the presence or absence of divalent cation. Optimal reaction conditions were 150 mM NaCl and pH 7.5. A uracil DNA glycosylase was also detected, active in the presence of EDTA, selectively removing uracil from DNA without generating other byproducts. The optimal reaction conditions were 50 mM NaCl and pH 7.5. Implications for base excision repair in D. radiodurans are discussed.