Rearrangement of interstrand cross-links into intrastrand cross-links in cis-diamminedichloroplatinum(II)-modified DNA.

In the reaction of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, bifunctional intrastrand and interstrand cross-links are formed. In this work, we show that at 37 degrees C interstrand cross-links (ICL) are labile and rearrange into intrastrand cross-links. The ICL instability was first studied with a 10 base pairs (bp) double-stranded oligonucleotide containing a unique site-specific ICL resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(GC/GC) site by a cis-diammineplatinum(II) residue. The bonds between the platinum and the N7 of guanine residues within the interstrand adduct are cleaved. In 50 mM NaCl or NaClO4, this cleavage results in the formation of monofunctional adducts which subsequently form intrastrand cross-links. One cleavage reaction takes place per cross-linked duplex in either of both DNA strands. Whereas the starting cross-linked 10 bp duplex is hydrogen bonded, the two complementary DNA strands separate after the cleavage of the ICL. Under these conditions, the cleavage reaction is irreversible allowing its rate measurement (t1/2= 29+/-2 h) and closure of monofunctional adducts to intrastrand cross-links occurs within single-stranded DNA. Within a longer cross-linked oligonucleotide (20 bp), ICL are apparently more stable (t1/2= 120+/-12 h) as a consequense of monofunctional adducts closure back to ICL. We propose that the ICL cleavage is reversible in DNA and that these adducts rearrange finally into intrastrand cross-links. Our results could explain an 'ICL unhooking' in previously reported in vivo repair studies [Zhenet al. (1993)Carcinogenesis14, 919-924].     

Iron(II)-mimosine catalyzed cleavage of DNA

Mimosine, DNA breaKs, Free Radicals, Fenton Reaction Supercoiled plasmid DNA was treated in vitro with H2O2, DTT and either Fe (II), Fe (II)-EDTA or Fe (II)-mimosine. The rate of DNA break formation was followed by the conversion of the supercoiled form into relaxed-circular and linear forms. In the concentration interval of 0-4 microM Fe (II), Fe (II)-EDTA slowed-down the formation of DNA breaks, while Fe (II)-mimosine enhanced the rate of break formation up to several times. A conclusion is drawn that this enhancement is due to the increased affinity of the Fe (II)-mimosine complex to DNA.     

Effects of DNA intercalating agents on topoisomerase II induced DNA strand cleavage in isolated mammalian cell nuclei

Intercalator-induced DNA double-strand breaks (DSB) presumably represent topoisomerase II DNA cleavage sites in mammalian cells. Isolated L1210 cell nuclei were used to determine the saturability of this reaction at high drug concentrations. 4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) and 5-iminodaunorubicin (5-ID) both produced DSB in a concentration-dependent manner, and the production of these breaks leveled off above 10 microM. Addition of m-AMSA to 5-ID-treated nuclei did not raise the plateau level. Thus, both drugs seemed to interact similarly on identical targets. The ellipticine derivative 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+) had two effects on the production of DSB. Below 10 microM, 2-Me-9-OH-E+ produced DSB as did ellipticine, m-AMSA, or 5-ID. Above 10 microM, 2-Me-9-OH-E+ did not induce DSB and inhibited the DSB induced by m-AMSA, 5-ID, or ellipticine. 2-Me-9-OH-E+ and m-AMSA competed with each other to produce either double-strand break formation (m-AMSA-induced reaction) or double-strand break inhibition (2-Me-9-OH-E+-induced reaction at concentrations greater than 10 microM). Because these results were reproduced in experiments using DNA topoisomerase II isolated from L1210 nuclei, it is likely that the intercalator-induced protein-associated DNA breaks detected by alkaline elution in nuclei represent DNA topoisomerase II-DNA complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nature of inactivating lesions produced by platinum(II) complexes in phage lambda DNA

Lambda DNA loses transfectivity and acquires interstrand cross-links after treatment with either trans-Pt(II) or cis-Pt(II). With trans-Pt(II) there is close to an equivalence between the fraction of lambda DNA cross-linked and the fraction inactivated. In contrast, with cis-Pt(II) there are approx. 5 inactivating lesions for each lambda DNA interstrand cross-link. These results suggested that trans-PT(II) does not introduce intrastrand inactivating lesions into lambda DNA while cis-Pt(II) does so. To verify this conclusion, the cross-linked and uncross-linked fractions of lambda DNA treated with trans-PT(II) or cis-Pt(II) were separated on alkaline sucrose gradients. After trans-Pt(II) treatment, the uncross-linked fraction of lambda DNA was transfective when renaturated. However after cis-Pt(II) treatment the uncross-linked fraction of lambda DNA was not transfective when renatured. Thiourea treatment restored transfectivity to all inactivated fractions, showing that these lesions are reversible. We conclude that trans-Pt(II) inactivates lambda DNA primarily by introducing interstrand cross-links but that cis-Pt(II), although it also introduces interstrand cross-links, inactivates lambda DNA primarily by introducing intrastrand lesions.     

Interstrand cross-links and sequence specificity in the reaction of cis-dichloro(ethylenediamine)platinum(II) with DNA

Characterization of the adducts produced in DNA by the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) and a radiolabeled analogue, [3H]-cis-dichloro(ethylenediamine)platinum(II) ([3H]-cis-DEP) was recently reported [Eastman, A. (1983) Biochemistry 22, 3927]. Both drugs reacted at identical sites in DNA, most of which produced intrastrand cross-links. DNA-interstrand cross-links, which represent less than 1% of total platination, have now been characterized. DNA containing interstrand cross-links was enriched for on the basis of its renaturability after boiling. This DNA was digested to deoxyribonucleosides, and the adducts were separated by high-pressure liquid chromatography. A cross-link between two deoxyguanosines was observed to be the most prominent adduct. It is proposed that the major sequence in which this cross-link occurs is 5'-CG-3'. DNA that was incubated with [3H]-cis-DEP for 1 h showed low levels of interstrand cross-links. After removal of unreacted drug, their frequency increased significantly over 6 h with a maximum occurring at about 12 h. A similar phenomenon was seen in the case of intrastrand cross-links that contained adenine, in particular when the cross-link was between the terminal bases in an ANG trinucleotide sequence (N is any nucleotide). The primary site of reaction is at guanine, with a slow subsequent cross-link to the adenine. A model is presented that is consistent with the observation that adenine is always at the 5' terminus of these adducts. The proportion of adducts at ANG sequences also increased at elevated temperatures. This is discussed with regard to potential significance during hyperthermia treatment of patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of nucleic acids with cis-diamminedichloroplatinum(II): interstrand cross-links

In the reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with double-helical (dC-dG)4.(dC-dG)4 or (dC-dG)5.(dC-dG)5, intrastrand and interstrand cross-links between two guanine residues are formed. This is shown by gel electrophoresis in denaturing conditions of the reaction products and by high-performance liquid chromatography (HPLC) analysis of the products digested with nuclease P1. In the reaction of cis-DDP and poly(dG-dC).poly(dG-dC), at relatively low levels of platination, it is mainly interstrand cross-links between two guanine residues that are formed. This is shown by HPLC analysis of the nuclease P1 digest and by gel electrophoresis in denaturing and nondenaturing conditions of the platinated polymer after cleavage with the restriction enzyme HhaI. Moreover, the antibodies to platinated poly(dG-dC).poly(dG-dC) cross-react with the interstrand cross-linked (dC-dG)4 or (dC-dG)5 but not with the intrastrand cross-linked (dC-dG)4 or (dC-dG)5. These antibodies cross-react with platinated natural DNA. The amount of interstrand cross-links deduced from radioimmunoassays (0.5% of the total bound platinum) is lower than that (2%) deduced by gel electrophoresis in denaturing conditions of a platinated DNA restriction fragment. By gel electrophoresis, it is also shown that in vitro the isomer trans-DDP is more efficient in forming interstrand cross-links than cis-DDP.     

Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin

Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage. The nucleotide sequence analysis exhibited considerably random DNA cleavage. The DNA strand scission by the camptothecin-Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction. The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy.     

In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA-protein cross-links

Lead is present in the natural and occupational environment and is reported to interact with DNA, but the mechanism of this interaction is not fully understood. Using the alkaline comet assay we showed that lead acetate at 1-100 microM induced DNA damage in isolated human lymphocytes measured the change in the comet tail length. At 1 and 10 microM we observed an increase in the tail length, whereas at 100 microM a decrease was seen. The former effect could follow from the induction of DNA strand breaks and/or alkali-labile sites (ALS), the latter from the formation of DNA-DNA and/or DNA-protein cross-links. No difference was observed between tail length for the alkaline and pH 12.1 versions of the assay, which indicates that strand breaks and not ALS are responsible for the tail length increase induced by lead. The neutral version of the test revealed that lead acetate induced DNA double-strand breaks at all concentrations tested. The presence of spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) did not influence the level of DNA damage induced by lead. Post-treatment of the lead-damaged DNA (at 100 microM treatment concentration) by endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II, an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage. Proteinase K caused an increase in comet tail length, suggesting that lead acetate might cross-link DNA with nuclear proteins. Vitamin A, E, C, calcium chloride and zinc chloride acted synergistically on DNA damage evoked by lead. The results obtained suggest that lead acetate may induce single-strand breaks (SSB) and double-strand breaks (DSB) in DNA as well as DNA-protein cross-links. The participation of free radicals in DNA-damaging potential of lead is not important and it concerns other reactive species than could be trapped by DMPO or PBN.     

DNA bending and unwinding due to the major 1,2-GG intrastrand cross-link formed by antitumor cis-diamminedichloroplatinum(II) are flanking-base independent

Antitumor cisplatin [cis-diamminedichloroplatinum(II)] forms on DNA predominantly intrastrand cross-links between neighboring purine residues. Several discoveries suggested that the toxicity of cisplatin originated from these lesions. The formation of 1,2-GG intrastrand cross-link of cisplatin leads to marked conformational alterations in DNA including a directional, rigid bend toward the major groove and local unwinding. These altered structures attract various cellular proteins. This phenomenon has been postulated to mediate antitumor properties of cisplatin. Importantly, the binding affinity of several proteins that specifically recognize 1,2-GG intrastrand cross-link to platinated DNA is modulated by the nature of the base pairs that immediately flank the platinated d(GpG) site. However, the influence of sequence context on DNA bending and unwinding due to the formation of the 1,2-GG intrastrand cross-link has not been extensively investigated. In the present study we have employed electrophoretic retardation (phasing) assay to analyze bending and unwinding induced by the single, site-specific 1,2-GG intrastrand cross-link immediately flanked by various bases formed by cisplatin in nine oligodeoxyribonucleotide duplexes. The results indicate that bending and unwinding of DNA as a consequence of the formation of the major adduct of cisplatin is, in the first approximation, independent of the base pairs flanking the platinated d(GpG) site.     

Naturally occurring cross-links in yeast chromosomal DNA.

Chromosome-size yeast DNA molecules with a number average molecular weight (Mn) of 3-4 X 10(8) were isolated from sucrose gradients after sedimentation of lysed yeast spheroplasts. Resedimentation showed that the molecules were isolated without introducing appreciable single-strand or double-strand breaks. The presence of cross-links in these molecules was suggested by the observation that the apparent Mn in alkali was greater than expected for separated single strands. Since cross-linked molecules would have strands which fail to separate upon denaturation, this was tested more directly. Neutralization of alkaline denaturing conditions resulted in up to 70% of the intact molecules rapidly reforming duplex structures, as shown by equilibrium banding in CsCI. Experiments with larger E. coli DNA molecules (Mn = 5.2 X 10(8)) indicated that the conditions used were sufficient to denature completely molecules of this size. Results of enzyme treatments suggest that the cross-links are not RNA or protein. Experiments with density-labeled yeast DNA molecules showed that the rapid reformation of duplex DNA is not the consequence either of a bimolecular reaction between separated DNA strands or of intrastrand renaturation. The data indicate that when the yeast DNA molecules are completely denatured, the strands fail to separate. Hence they must be cross-linked. Experiments with sheared DNA show that there are small number of cross-links, one to four, permolecule.     

A comparison of cobalt(II) and iron(II) hydroxyl and superoxide free radical formation

We have employed the electron spin resonance spin-trapping technique to study the reaction of Co(II) with hydrogen peroxide in a chemical system and in a microsomal system. In both cases, we employed the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and were able to detect the formation of DMPO/.OH and DMPO/.OOH. DMPO/.OOH was the predominant radical adduct formed in the chemical system, while the two adducts were of similar concentrations in the microsomal system. The formation of both of these adducts in either reaction system was inhibited by the addition of superoxide dismutase or catalase, and by chelating the cobalt with either ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The incorporation of the hydroxyl radical scavengers ethanol, formate, benzoate, or mannitol inhibited the formation of DMPO/.OH in both systems. We also repeated the study using Fe(II) in place of Co(II). In contrast to the Co(II) results, Fe(II) reacted with hydrogen peroxide to yield only DMPO/.OH, and this adduct formation was relatively insensitive to the presence of added superoxide dismutase. In addition, Fe(II)-mediated DMPO/.OH formation increased when the iron was chelated to either EDTA or DTPA rather than being inhibited as for Co(II). Thus, we propose that Co(II) does not react with hydrogen peroxide by the classical Fenton reaction at physiological pH values.     

Enhanced DNA repair as a mechanism of resistance to cis-diamminedichloroplatinum(II)

Murine leukemia L1210 cells, either sensitive or resistant to the toxic action of the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II), have been studied for potential differences in the formation and repair of drug-induced DNA damage. The sensitivity for these experiments was obtained by using the radiolabeled analogue [3H]-cis-dichloro(ethylenediamine)platinum(II). The resistant cells demonstrated a 40% reduction in drug accumulation but a qualitatively similar profile of DNA-bound adducts. These adducts resembled those previously characterized in pure DNA and represented intrastrand cross-links at GG, AG, and GNG (N is any nucleotide) sequences in DNA. Repair of these cross-links occurred in a biphasic manner: rapid for the first 6 h and then much slower. The resistant cells removed up to 4 times as many adducts during the rapid phase of repair. The extent of this repair did not directly correlate with the degree of resistance in that cells with 100-fold resistance were only slightly more effective at repair than cells with 20-fold resistance. Therefore, although enhanced DNA repair is thought to contribute markedly to drug resistance, other mechanisms for tolerance of DNA damage may also occur in these cells.     

Influence of copper(II) and magnesium(II) ions on the ciprofloxacin binding to DNA

The influence of magnesium(II) and copper(II) ions on the binding of ciprofloxacin to double stranded calf thymus DNA was studied by fluorescence emission spectroscopy, ultraviolet- and circular dichroism (CD) spectroscopy. The interaction of ciprofloxacin and copper(II) ions was followed by strong fluorescence quenching which was almost unaffected by the presence of DNA. On the other hand, only a slight decrease in fluorescence emission intensity, which was enhanced in the presence of DNA, was observed for ciprofloxacin interaction with magnesium(II) ions. Furthermore, magnesium(II) ions increase the thermal stability of the DNA, while, in the presence of ciprofloxacin, the degree of stabilisation is smaller. In contrast, copper(II) ions destabilise double helical DNA to heat, while ciprofloxacin slightly affects only the second transition of the biphasic melting curve of calf thymus DNA. Magnesium(II) ions at 25 degrees C induce conformational transitions of DNA at concentrations of 1.5 mM and 2.5 M, as monitored by CD. On the other hand copper(II) ions induce only one conformational transition, at a concentration of 12.7 microM. At higher concentrations of copper(II) ions (c>700 microM) DNA starts to precipitate. Significant changes in the CD spectra of DNA were observed after addition of ciprofloxacin to a solution containing DNA and copper(II) ions, but not to DNA and magnesium(II) ions. Based on our spectroscopic results, we propose that copper(II) ions are not directly involved into ciprofloxacin binding to DNA via phosphate groups as it has been suggested for magnesium(II) ions.     

Strand breaks in whole plasmid dna produced by the decay of (125)I in a triplex-forming oligonucleotide.

DNA strand breaks produced by the decay of (125)I positioned against a specific site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucleotide resolution and in the whole plasmid by measuring the percentages of supercoiled, open-circular and linear forms. The localized breaks are distributed within 10 bp in each direction from the decay site with maxima in both strands just opposite the (125)I-dC residue in the triplex-forming oligonucleotide. The distributions of breaks in the two DNA strands are almost symmetrical, in agreement with the geometry of the pyrimidine motif triplex. We found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequence. The ratio of single- to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triplex-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of (125)I was 0.46 for bound triplex-forming oligonucleotide and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, in addition to DNA breaks that are confined to a helical turn around the (125)I atom, the decay can produce breaks hundreds of base pairs away in the plasmid molecule. This linear plasmid molecule containing radiation-induced damage at a specific DNA site should be useful in studies of the molecular mechanisms of DNA repair.     

Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.     

The effect of cis-diamminedichloroplatinum (II) on the formation of DNA-Pt-DNA cross-links.

The effect of cis-diamminedichloroplatinum (II) (cis-DDP) on the formation of interstrand cross-links in DNA and in DNA and chromatin complex from leukocytes was studied. Following the use of cis-DDP the number of DNA-DNA interstrand cross-links was elevated with the increase of cis-DDP concentration and elongation of reaction time. It was also found that nucleic proteins reduce the quantity of the cis-DDP induced DNA-DNA interstrand cross-links in the DNA in nucleoprotein complex when compared with the links in the isolated DNA.     

Topoisomerase II cleavage in chromatin

We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.     

An invitro characterization of interstrand cross-links in DNA exposed to the antitumor drug cis-dichlorodiammineplatinum(II)

The $\text{cis}$-isomer of the antitumor drug dichlorodiammineplatinum(II) [$\text{cis}$-Pt(II)] was tested for its abilty to introduce nicks (single-strand breaks) into supercoiled PM2 DNA. Whereas incubations up to 24 h show no indication of $\text{cis}$-Pt(II)-treated DNA having single-strand breaks, DNA interstrand cross-links were detected in the first 15 min of incubation. Furthermore, the formation of DNA interstrand cross-links was $\text{both}$ inhibited and fully reversed after incubation with 2 mM thiourea.     

Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage

DNA topoisomerase II is believed to be the enzyme that produces the protein-associated DNA strand breaks observed in mammalian cell nuclei treated with various intercalating agents. Two intercalators--4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA, amsacrine) and 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+)--differ in their effects on protein-associated double-strand breaks in isolated nuclei. m-AMSA stimulates their production at all concentrations, whereas 2-Me-9-OH-E+ stimulates at low concentrations and inhibits at high concentrations. We have reproduced these differential effects in experiments carried out in vitro with purified L1210 DNA topoisomerase II, and we have found that concentrations of 2-Me-9-OH-E+ above 5 microM prevent the trapping of DNA-topoisomerase II cleavable complexes irrespective of the presence of m-AMSA. It also stimulated topoisomerase II mediated DNA strand passage, again with or without inhibitory amounts of m-AMSA (this result suggests that extensive intercalation by 2-Me-9-OH-E+ destabilized the cleavable complexes). From these data, it is concluded that intercalator-induced protein-associated DNA strand breaks observed in intact eukaryotic cells and isolated nuclei are generated by DNA topoisomerase II and that intercalators can affect mammalian DNA topoisomerase II in more than one way. They can trap cleavable complexes and inhibit DNA topoisomerase II mediated DNA relaxation (m-AMSA and low concentrations of 2-Me-9-OH-E+) or destabilize cleavable complexes and stimulate DNA relaxation (high concentrations of 2-Me-9-OH-E+).     

Mechanism of action of deoxyribonuclease II from human lymphoblasts.

Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE-cellulose and heparin-agarose chromatography followed by fractionation on Mono-S, Mono-Q and Superose-12 FPLC columns. In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single-strand nicks relative to double-strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3' termini to hydrolysis by this enzyme. Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 microM Fe3+. Likewise we confirmed the presence of 3'-phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzed by Escherichia coli DNA polymerase I.