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PubMed1990

Antinuclear antibody profiles in relation to specific disease manifestations of systemic lupus erythematosus.

UNLABELLED: The aim of the study was to investigate the prevalence of antinuclear and associated antibodies (anti-dsDNA, anticardiolipin, anti-RNP, anti-Sm, anti-SSA and anti-SSB) and/or combinations thereof in systemic lupus erythematosus (SLE) patients with respect to their diagnostic and pathogenetic significance. The prevalence of anti-dsDNA antibodies was strongly influenced by the selection criteria of the patient; the lowest prevalence was found in SLE patients with central nervous system (CNS) involvement; the highest prevalence in patients with nephritis. The results were also influenced by the different assays. Combinding different assays (Farr/PEG ratio) quantitative as well as qualitative differences could be shown between patients with nephritis (Farr/PEG ratio greater than or equal to 5) and with CNS involvement (Farr/PEG ratio less than 5). No difference in anticardiolipin antibody prevalence between the different SLE patient groups could be demonstrated. Regarding antibodies against RNP, Sm, SSA and SSB, the prevalence was found to be strongly influenced by the criteria used for patient selection. Only in CNS patients and association with anti-RNP and anti-SSB antibodies alone or in combination was found. In pleuropericarditis a weak association with RNP antibodies existed. IN CONCLUSION: studying the prevalence and possible pathogenetic significance of antibodies one should always consider patient selection criteria and the effect of the different assays used when analysing the results.

Antibodies, Antinuclear↗
PubMed1995

Antibodies to human immunodeficiency virus (HIV-1) in autoimmune diseases: primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis and autoimmune thyroid diseases.

The aetiology of autoimmune diseases remains unknown. The relationship between virus, and more recently retrovirus, has been suggested with this group of diseases. Immunoblotting is a useful method for determining the presence of proteins coded by different retrovirus genes. Since the prevalence of these types of proteins in patients with primary Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and autoimmune thyroid diseases has not been fully established, the aim of this work was to determine the prevalence of antibodies to immunodeficiency human virus type 1 (HIV-1) proteins in these diseases and their possible relationship with the presence of anti-nuclear, anti-DNA, anti-SSA (Ro) and anti-SSB (La) autoantibodies. Antibodies to human immunodeficiency virus (HIV-1) were studied in a group of 341 patients with autoimmune diseases (77 SS, 98 SLE, 75 RA, 91 autoimmune thyroid diseases) and 126 blood donors as a control group. A Western blot was used to detect antibodies to HIV-1, and a double polymerase chain reaction (PCR) using nested primers in the gag and pol gene of HIV-1. Antinuclear antibodies, anti-DNA, anti-SSA (Ro) and anti-SSB (La) were determined by enzyme-linked immunosorbent assays. At least one band was shown on immunoblotting in 26% of patients with autoimmune diseases and 35% of controls. The presence of antibodies to p55 or p68 proteins in patients with SS or SLE proved to be the only statistically significant difference between the other autoimmune diseases studied and the control group. These antibodies were not associated with autoantibodies ANA, DNA, SSA (Ro) or SSB (La).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1998

Glucose repression on RIM1, a gene encoding a mitochondrial single-stranded DNA-binding protein, in Saccharomyces cerevisiae: a possible regulation at pre-mRNA splicing.

The mitochondrial single-stranded DNA-binding protein (SSB) encoded by anuclear gene, RIM1, is a homolog of Escherichia coli SSB. The addition of glucose decreased the amount of RIM1-mRNA in cells growing in a glycerol medium, but increased the amount of the immature RIM1-mRNA. The changes in the amounts of both mature and immature RIM1-mRNAs were dependent on SRN1/REG1/HEX2, a gene relating to pre-mRNA-splicing and glucose repression. These observations suggest that the expression of the mitochondrial SSB is regulated, at least in part, by pre-mRNA splicing under the control of glucose repression.

Culture Media↗
PubMed1999

Quantitative modelling of DNA damage using Monte Carlo track structure method.

This paper presents data on modelling of DNA damage induced by electrons, protons and alpha-particles to provide an insight into factors which determine the biological effectiveness of radiations of high and low linear energy transfer (LET). These data include the yield of single- and double-strand breaks (ssb, dsb) and base damage in a cellular environment. We obtain a ratio of 4-15 for ssb:dsb for solid and cellular DNA and a preliminary ratio of about 2 for base damage to strand breakage. Data are also given on specific characteristics of damage at the DNA level in the form of clustered damage of varying complexity, that challenge the repair processes and if not processed adequately could lead to the observed biological effects. It is shown that nearly 30% of dsb are of complex form for low-LET radiation, solely by virtue of additional breaks, rising to about 70% for high-LET radiation. Inclusion of base damage increases the complex proportion to about 60% and 90% for low- and high-LET radiation, respectively. The data show a twofold increase in frequencies of complex dsb from low-LET radiation when base damage is taken into account. It is shown that most ssb induced by high-LET radiation have associated base damages, and also a substantial proportion is induced by low-energy electrons.

DNA↗
PubMed2006

A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans.

We report the identification and characterization of the single-stranded DNA-binding protein (SSB) from the mesophile and highly radiation-resistant Deinococcus radiopugnans (DrpSSB). PCR-derived DNA fragment containing the complete structural gene for DrpSSB protein was cloned and expressed in Escherichia coli. The gene consisting of an open reading frame of 900 nucleotides encodes a protein of 300 amino acids with a calculated molecular weight of 32.45 kDa and pI 5.34. The amino acids sequence exhibits 43, 44, 79 and 18% identity with Thermus aquaticus, Thermus thermophilus, Deinococcus radiodurans and E. coli SSBs, respectively. The DrpSSB includes two OB folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT), DrpSSB bound 24-31 nt depending on the salt concentration, and fluorescence was quenched by about 80%. In a complementation assay in E. coli, DrpSSB took over the in vivo function of EcoSSB. The half-lives of DrpSSB were 120 min at 90 degrees C, 60 min at 95 degrees C and 30 min at 100 degrees C. These results were surprising in the context of half-life of SSB from thermophilic T. aquaticus, which has only 30 s of half-life at 95 degrees C. DrpSSB is the most thermostable SSB-like protein identified to date, offering an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and analytical purposes.

Amino Acid Sequence↗
PubMed2001

Soluble interleukin-2 receptor in Sjögren's syndrome: relation to main serum immunological and immunohistochemical parameters.

Our aim was to study sIL-2R relationship with main serum immunological and LSG immunohistochemical parameters, including surface antigen expression of immune activation, in 27 patients with primary SS. Serum sIL-2R levels were significantly higher in SS (p<0.00005), as well as in SLE (p<0.05) and RA (p< 0.000001) patients than in controls. In SS patients with abnormal slL-2R values (n = 7) we found higher levels of anti-SSB/La antibodies (p<0.05), IgM-RF (p<0.014) and CRP (p<0.003) with respect to those with normal sIL-2R values (n = 20). Moreover, sIL-2R levels correlated positively with those of anti-SSB/La antibodies (p<0.0037) and with CRP (p<0.008). The comparison of groups with (A) and without (B) abnormal slL-2R levels reveals a statistically different percentage of patients with foci number > 1 (86% vs 40%; p <0.047), and CD25 expression on lymphocytes (100% vs 40%; p < 0.008). The frequency (p < 0.025) of CD25 expression on lymphocytes was higher in group A than in group B. The frequency of CD25 expression on the infiltrates correlated not only with sIL-2R levels (p<0.047), but also with anti-SSB/La antibody values (p < 0.044), with Tarpley histological classes (p < 0.009) and with frequency of HLA-DR expression on lymphocytes (p<0.004) and on epithelial cells (p<0.002). The frequency of epithelial CD25 expression also correlated with that of epithelial HLA-DR (p<0.004). Our report suggests that slL-2R is linked to glandular involvement in primary SS.

Adult↗
PubMed1985

Differentiation of apurinic/apyrimidinic sites and single-strand breaks in DNA by formamide- and alkaline-sucrose gradient sedimentation.

The excision repair of DNA damaged by physical or chemical agents may produce either apurinic/apyrimidinic (AP) sites or single-strand breaks (SSB) in the DNA. Alkaline-sucrose gradient sedimentation and alkaline elution, techniques generally used for the study of DNA repair which depend upon high pH to denature the DNA, cannot differentiate between these possibilities. A simple method for the quantitative measurement of SSB in DNA which leaves any AP sites intact is presented. This method relies upon the separation by size of the fragments resulting from the denaturation of the DNA under neutral conditions by sedimentation through gradients of sucrose in formamide. By combining the use of both formamide- and alkaline-sucrose sedimentation methods, we can quantify both AP sites and SSB in DNA.

Apurinic Acid↗
PubMed1990

Structural and functional studies of the rat mitochondrial single strand DNA binding protein P16.

The rat mitochondrial single strand DNA binding protein (SSB) P16 was purified to apparent homogeneity by elution from single strand DNA agarose with ethidium bromide. Each monomer of P16 contains two tryptophan residues, and the intrinsic fluorescence from these residues is quenched upon binding to single strand polynucleotides. From fluorescence quench titrations of ligand to fixed amounts of DNA lattice, a binding site size of 8 or 9 nucleotides per P16 monomer was found. Measurement of the affinity of P16 for isolated sites by titration with either oligo(dT)8 or 5'-dephosphorylated oligo(dT)8 indicated values on the order of 10(7) M-1. P16 exhibited a binding preference for single strand DNA, poly(dT), and poly(dC) in comparison to double strand DNA, poly(U), or poly[d(A-T)]. Although it was not possible to show that P16 destabilizes double helical DNA or even poly[d(A-T)], binding of P16 does inhibit the process of renaturation as shown by inhibition of duplex formation between poly(dA) and poly(dT). The binding of saturating amounts of P16 to single strand poly(dT).oligo(dA)50 template-primers enhanced approximately 10-fold the activity of both the homologous mitochondrial DNA polymerase and the Escherichia coli DNA polymerase I Klenow fragment. However, the mitochondrial DNA primase was nearly completely inhibited by the saturation of the poly(dT) template with P16. Amino-terminal sequence analysis of P16 and a protease-insensitive, DNA binding domain (Mr approximately 6000) revealed that the DNA binding domain residues, at least in part, in the amino-terminal third of the P16 molecule. Furthermore, the amino-terminal sequence was found to be strikingly similar to that of the Xenopus laevis mtSSB-1 and to a lesser extent similar to E. coli SSB and E. coli F sex factor SSB.

Amino Acid Sequence↗
PubMed1988

Enhancement of DNA damage in mammalian cells upon bioreduction of the nitroimidazole-aziridines RSU-1069 and RSU-1131.

The induction of DNA double-(dsb) and single-(ssb) strand breaks by RSU-1069, RSU-1131 and misonidazole in V79 mammalian cells has been investigated using sedimentation in isokinetic sucrose gradients after incubation for various times (1-3 hr) at 310 K under both hypoxic and aerobic conditions. Double strand breaks are produced by RSU-1069 and RSU-1131 predominantly under hypoxic conditions. Comparison of the cellular DNA damage induced by these agents leads to the following facts: (1) the yield of ssb induced by these agents is substantially increased under hypoxia, (2) RSU-1069 and RSU-1131 are much more effective than misonidazole, on a concentration basis, at causing strand breakage both under hypoxic and aerobic conditions; and (3) RSU-1069 is more efficient on a concentration basis than RSU-1131 at inducing both ssb and dsb under both conditions. From these findings and molecular studies it is suggested that these 2-nitroimidazole aziridines act as monofunctional alkylating agents under aerobic conditions, a factor that governs their aerobic cytotoxicity. Under hypoxic conditions, it is suggested that the induction of dsb and crosslinks by these agents (bifunctional character) may play a major role in determining the ability of such agents to act as hypoxia-selective cytotoxins.

Antineoplastic Agents↗
PubMed1989

Interrelationship between affinity for DNA, cytotoxicity and induction of DNA-breaks in cultured L1210 cells for two series of tricyclic intercalators. Simplified analogues of ellipticine derivatives.

The interrelationship between affinity for DNA, cytotoxicity and induction of single-strand DNA breaks in cultured L1210 cells was studied for 21 compounds belonging to two series of tricyclic intercalators: 1-amino-substituted 4-methyl-5H-pyrido[4,3-b]indoles (gamma CARB) and 1-amino-substituted 4-methyl-5H-pyrido[3',4':4,5]pyrrolo[2,3-c]pyridines (PPP), which are simplified analogues of Ellipticine derivatives obtained by deletion of one cycle. Adriamycin, m-AMSA (4'-(9-acridinylamino) methanesulfon-m-anisidide), PZE (10-[diethylaminopropyl amino]-6-methyl-5H-pyrido[3',4':4,5]-pyrrolo[2,3-g] isoquinoline and RTE [( 1-(3-diethylaminopropylamino)-9-methoxy ellipticine, bimaleate) are used as reference compounds. The intercalation of these compounds into DNA was strongly suggested by three experimental observations: (i) the competitive inhibition of ethidium bromide intercalation, (ii) bathochromic and hypochromic effects on absorption spectra induced by DNA, and (iii) drug-induced increase of the DNA length, measured by viscosimetry. PPP derivatives are generally less cytotoxic and induce DNA breaks less efficiently than the gamma CARB ones, both in terms of maximum breakage frequencies and required drug concentrations. The most active compounds induced SSB in the DNA of L1210 cells, in a bell-shaped manner: the SSB frequency increased, rose to a maximum and then decreased as the drug concentrations increased. The maximum SSB frequencies induced by the most active compounds are of the same order as those of reference compounds Adriamycin and PZE. The structurally important requirements are essentially the same for both DNA breakage activity and cytotoxicity: (i) a N-CH3 in the 5-position, (ii) a CH3 in the 4-position, (iii) a hydroxy in the 8-position and (iv) the presence of an (aminoalkyl)amino side chain with preferentially a 3 carbon unit. There is no direct relationship between DNA affinity in vitro and induction of DNA breaks in cells, although a relatively high affinity seemed to be a necessary condition, since the most active compounds have the highest affinities and compounds having a very low affinity are totally inactive. The close correlation between cytotoxicity and extent of induction of DNA breaks suggests that these breaks may be in fact the lethal lesions responsible for cell death and thereby for the antitumor properties of these tricyclic intercalators.

Alkaloids↗
PubMed1991

Interaction of cis-diamminedichloroplatinum (II) with single-stranded DNA in the presence or absence of Escherichia coli single-stranded binding protein.

We have compared the mode of fixation in vitro of the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) to single-stranded M13mp10 DNA either in the presence or absence of the Escherichia coli single-stranded binding protein (SSB). Platinum binding sites have been identified by taking advantage of their capacity to inhibit DNA replication of primed M13 DNA catalysed by E. coli DNA polymerase I large fragment. We report here that the presence of SSB increases the number of platinum-DNA lesions and alters their distribution. We also present evidence that SSB allows cis-DDP to bind to DNA sequences otherwise less accessible.

Bacterial Proteins↗
PubMed1991

The repair of DNA damage induced in V79 mammalian cells by the nitroimidazole-aziridine, RSU-1069. Implications for radiosensitization.

The induction and repair of single (ssb) and double (dsb) strand breaks in DNA under aerobic or hypoxic conditions have been determined using sucrose sedimentation techniques following incubation of V79 mammalian cells with RSU-1069 or misonidazole, representative of a conventional 2-nitroimidazole radiosensitizer, for 1-1.5 hr at either 293 or 277 degrees K and subsequent irradiation at 277 degrees K. In all cases, the dose dependences for the induction of strand breaks are linear and consistent with an enhancement in the yield of DNA damage induced by the 2-nitroimidazoles under hypoxic conditions. With RSU-1069 at 293 degrees K, the dose dependence of ssb is displaced reflecting DNA damage induced during pre-incubation. From these dependences, it is evident that the enhanced radiosensitization by RSU-1069 may not be accounted for in terms of accumulation of the agent at DNA. From the repair studies, DNA breaks induced by RSU-1069 in the absence of radiation have been shown to persist for at least 3 hr. With a combination of RSU-1069 and radiation under hypoxic conditions, the repair timescale of the induced breaks is significantly longer and an increase in the residual yields of both ssb and dsb (at 2-3 hr) was observed when compared with the observation in the presence of misonidazole or oxygen. From these studies, it is inferred that the enhanced radiosensitization of RSU-1069 at 293 degrees K is a consequence of the formation of non-repairable DNA damage together with a modification of the repairability of the radiation-induced DNA breaks.

Animals↗
PubMed1992

Quinone-induced DNA single strand breaks in rat hepatocytes and human chronic myelogenous leukaemic K562 cells.

In rat hepatocytes exposed to the quinones menadione and 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) a decrease in NAD+ is observed. DNA damage and activation of poly(ADP-ribose)polymerase are often associated with a decrease in NAD+. Using rat hepatocytes and human myeloid leukaemic cells (K562), we examined the extent of DNA damage induced by these quinones at non-toxic concentrations, i.e. at concentrations at which the cells completely exclude the dye trypan blue. Both quinones caused significant DNA damage at very low concentrations (5-100 microM). With 2,3-diOME-1,4-NQ (15 microM) or menadione (15 microM) single strand breaks (SSB) were observed at very early time points (less than 5 min), reaching a maximum between 20 and 30 min. Most SSB were repaired within 45 min of the removal of the quinones. Whilst extensive repair was observed within 4 hr of the removal of 2,3-diOMe-1,4-NQ (15 microM), only partial repair was observed following exposure to menadione (15 microM). SSB induced by 2,3-diOMe-1,4-NQ (15 microM) were completely inhibited by the iron chelator 1,10-phenanthroline (25 microM), whereas in cells exposed to menadione (15 microM) they were only partially inhibited. Finally, although the membrane integrity of K562 cells was unaffected by exposure to high concentrations of both quinones (less than or equal to 400 microM), cytostasis was observed at much lower concentrations (50 microM). Our results demonstrate that at very low concentrations these quinones induce extensive DNA damage possibly caused by hydroxyl radicals. The DNA damage was accompanied by an early cytostasis but no loss of membrane integrity.

Animals↗
PubMed1993

Escherichia coli single-stranded DNA-binding protein alters the structure of intramolecular triplexes in plasmids.

The ability of the Escherichia coli single-stranded DNA-binding protein (SSB) to recognize structural features associated with intramolecular triplex formation in oligopurine.oligopyrimidine (pur.pyr) inserts in recombinant plasmids was evaluated. The SSB protein binds to supercoiled plasmids and causes a site-preferential increase in OsO4 reactivity of the pyrimidine strand involved in the formation of the Hy-3 isomer of the triplex structure. The E. coli RecA protein showed no reaction with triplexes in similar studies. This behavior is consistent with SSB-mediated unpairing of the H-DNA-forming region.

Base Sequence↗
PubMed1989

Negative co-operativity in Escherichia coli single strand binding protein-oligonucleotide interactions. II. Salt, temperature and oligonucleotide length effects.

We have examined the salt and temperature dependences of the equilibrium binding of the Escherichia coli single strand binding (SSB) tetramer to a series of oligodeoxythymidylates, dT(pT)N-1, with N = 16, 28, 35, 56 and 70. Absolute binding isotherms were obtained, based on the quenching of the intrinsic protein fluorescence upon formation of the complexes. The shorter oligonucleotides, with N = 16, 28 and 35, bind to multiple sites on the SSB tetramer and negative co-operativity is observed among these binding sites. We have quantitatively analyzed these isotherms, using a statistical thermodynamic ("square") model to obtain the intrinsic binding constant KN, and the negative co-operativity constant, sigma N. For all oligonucleotides, we find that KN decreases significantly with increasing concentration of monovalent salt, indicating a large electrostatic component to the free energy of the interaction (e.g. delta log KN/delta log [NaBr] = -2.7, -4.6 and -7.1 for N = 16, 35 and 70, respectively), with contributions from both cations and anions. For oligonucleotides that span two or more subunits, there is a significant unfavorable contribution to the binding free energy for each intersubunit crossing, with an accompanying uptake of anions. Therefore, the extent of anion uptake increases as the number of intersubunit crossings increase. There is a strong temperature dependence for the intrinsic binding of dT(pT)15, such that delta Ho = -26(+/- 3) kcal/mol dT(pT)15. Negative co-operativity exists under all solution conditions tested, i.e. sigma N less than 1, and this is independent of anion concentration and type. However, the negative co-operativity constant does decrease with decreasing concentration of cation. The dependence of sigma 16 on Na+ concentration indicates that an average of one sodium ion is taken up as a result of the negative co-operativity between two dT(pT)15 binding sites. These data and the lack of a temperature dependence for sigma 16 suggest that the molecular basis for the negative co-operativity is predominantly electrostatic and may be due to the repulsion of regions of single-stranded DNA that are required to bind in close proximity on an individual SSB tetramer.

Bacterial Proteins↗
PubMed1992

Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein.

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.

DNA, Single-Stranded↗
PubMed1982

Visualization of recA protein and its association with DNA: a priming effect of single-strand-binding protein.

A stoichiometric interaction of RecA protein with single-stranded DNA promotes homologous pairing of the single strand with duplex DNA and subsequent polar formation of a heteroduplex joint. Escherichia coli single-strand-binding (SSB) protein augments these reactions. Electron microscopic observations suggest structural bases for these interactions. Without triphosphates or DNA, RecA protein forms short linear filaments. With added circular single-stranded DNA, it forms extended circular filaments as well as collapsed and aggregated complexes of protein and DNA. The extended circular filaments are stiff and regular in appearance, contrasting with the convoluted structure formed by SSB protein and single-stranded DNA. Together, these two proteins form mixed filaments, which mostly resemble the extended structures containing RecA protein; moreover, SSB protein accelerates formation of extended filaments more than 50-fold, increasing the yield of these structures at the expense of heterogeneous aggregates. Other observations further define the interactions of RecA protein with partially single-stranded DNA, and the effects of ATP gamma S on the tendency of RecA protein to form polymeric structures even in the absence of DNA.

Adenosine Triphosphatases↗
PubMedDate not supplied

Molecular dosimetry of DNA damage caused by alkylation. II. The induction and repair of different classes of single-strand breaks in cultured mammalian cells treated with ethylating agents.

Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (SSB) or alkali-labile sites were measured by elution through membrane filters at pH 12.0 and pH 12.6, and by centrifugation in alkaline sucrose gradients after 1 h and 21 h lysis in alkali. Two agents with different tendencies to ethylate preferentially either at N or O atoms were compared, namely N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and diethyl sulphate (DES). The compounds differed greatly in their potency to induce lesions, but the ratios of SSB, measured with different methods after a treatment for 30 min, did not differ significantly. This suggested that the spectrum of lesions induced by the two compounds is very similar. However, when both agents were studied with alkaline elution at pH 12.0 after a short treatment time (5 min) only ENNG was found to induce rapidly-repairable SSB. Most of these were rejoined already within 5 min after treatment. These results suggest that rapidly-repairable lesions occurring in DNA after treatment of mammalian cells with ethylating agents are due mainly to alkylation at O-atoms.

Alkylation↗