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PubMed2005

Repeat-length-independent broad-spectrum shuffling, a novel method of generating a random chimera library in vivo.

We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL(+) gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb(+) background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained.

Amino Acid Sequence↗
PubMed1979

Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization.

A mutation in the Escherichia coli gene for single-strand binding protein results in temperature-sensitive deoxyribonucleic acid replication (R. R. Meyer, J. Glassberg, and A. Kornberg, Proc. Natl. Acad. Sci. U.S.A. 76:1702-1705, 1979). The mutant (ssb-1) is also more sensitive to ultraviolet irradiation and about one-fifth as active in recombination. Single-strand binding protein is thus implicated in repair and recombination as well as in replication. The mutation in ssb is located between uvrA and melA at 90.8 min on the genetic map. The ssb gene appears to be allelic with lexC, a gene with a proposed role in regulating inducible deoxyribonucleic acid repair.

Bacterial Proteins↗
PubMed1995

Vaccinia virus gene A18R encodes an essential DNA helicase.

The vaccinia virus A18R protein is a DNA-dependent ATPase that contains the canonical sequence motifs associated with the DEXH group of DNA and RNA helicases. Investigation of A18R protein function during infection indicated it functions in the early and late phases of vaccinia virus transcription. The A18R protein shares sequence similarity with the mammalian DNA helicase ERCC3. The ERCC3 protein has a dual function: it is a component of the transcription factor TFIIH and is an essential participant in the cellular nucleotide excision repair pathway. Here we present evidence that the A18R protein is a DNA helicase that unwinds duplex DNA in a 3'-to-5' direction. The A18R helicase was inactive on RNA-DNA and RNA-RNA hybrids. The A18R unwinding activity was most efficient on DNA substrates with lengths of 20 nucleotides or less, and its unwinding activity was not stimulated by the addition of Escherichia coli single-strand-binding protein (SSB), the bacteriophage T4 gene 32 SSB, or the vaccinia virus I3L protein, a putative SSB. We have used an electrophoretic gel mobility shift assay to show that the A18R protein forms a stable complex with single-stranded DNA, and to a lesser extent RNA, in a reaction that does not require ATP.

Adenosine Triphosphatases↗
PubMed2002

Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system.

We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5' end and displacing the other strand while progressing in a 3'-to-5' direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5' end of the nick in the process and providing a 3' OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO(4) oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase delta, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.

Animals↗
PubMed2004

Specific antinuclear antibodies are associated with clinical features in systemic lupus erythematosus.

OBJECTIVES: To study associations between antinuclear antibodies (ANA) and signs/symptoms in patients with systemic lupus erythematosus (SLE). METHODS: A consecutive cohort of 289 patients with SLE was included; 235 fulfilled ACR criteria for SLE and were further analysed. ANA profiles were determined by line immunoassay and by indirect immunofluorescence on Crithidia luciliae. An extensive list of signs/symptoms was evaluated. RESULTS: Five clusters of antibodies were defined by cluster analysis: 1-antibodies to SmB, SmD, RNP-A, RNP-C, and RNP-70k; 2-antibodies to Ro52, Ro60, and SSB; 3, 4, and 5-antibodies to ribosomal P, histones and dsDNA, respectively. Significant associations (p< or =0.01) were found between anti-RNP-70k, anti-RNP-A, anti-RNP-C and Raynaud's phenomenon, between anti-RNP-A, anti-RNP-70k and leucopenia, and between anti-RNP-A, anti-RNP-C and a lower prevalence of urine cellular casts. Anti-SSA, anti-SSB were associated with xerostomia, and anti-SSB with pericarditis. Antibodies to ribosomal P were associated with haemolytic anaemia, leucopenia, and alopecia. Patients with anti-dsDNA antibodies had a higher risk for cellular casts and a lower risk for photosensitivity. Antihistone antibodies were associated with arthritis. CONCLUSIONS: In a large and consecutive cohort of patients with SLE, clusters of antibodies were identified. Previously reported associations of antibodies with symptoms were confirmed and new associations found.

Adolescent↗
PubMed1980

Evaluation of colonic myoelectrical activity in health and functional disorders.

Electrical activity of the colon was investigated in six healthy volunteers and 22 patients presenting functional colonic disorders associated with pain, constipation, or diarrhoea. Myoelectrical activity during 10 hours, including two daily meals, was recorded from eight groups of nickel-chrome electrodes using a 1.5 m length probe introduced by the rectal route. Electromyograms of volunteers consisted of slow waves at two distinct rhythms, approximately 3 and 10 cycles per minute, during 16 to 28% of the recording time respectively for each site. Action potentials were also recorded as 11 to 80 short spike bursts (SSB) per hour lasting 1.5 to 3.5 s at any site and 20-26 long spike bursts (LSB) per hour, lasting 17 to 21 s, occurring in series of four to eight propagated bursts. Additional high-velocity propagated bursts were recorded during the three hour postprandial period. Three typical changes in spike activity patterns were detected: an increase by 170-420% of the number of SSB associated to a high spiking activity was recorded in 13 patients (group I), the absence of SSB, a low spiking activity level and only 3 to 8 LSB per hour (group II), in six patients; and the postprandial response was absent (group III) in three other investigated patients. Evidence for three groups of motor disturbances and their possible relation to clinical manifestations of functional disorders is presented.

Action Potentials↗
PubMed1987

Colonic myoelectrical activity in IBS painless diarrhoea.

Colonic myoelectrical activity was recorded during 24 hours in 23 patients with painless diarrhoea and compared with a control group of 10 healthy subjects without digestive functional disorders. Diurnal fasting activity showed no significant difference in the total long spike bursts activity (LSB lasting greater than 7 seconds), but short spike bursts activity (SSB, lasting less than 7 seconds) was significantly lower (p less than 0.05) in diarrhoeal patients. A striking difference was observed in colonic response to eating, with an increased number of migrating long spike bursts (MLSB: mass movements) during the first postprandial hour in diarrhoeal patients (p less than 0.001), while short spike bursts (segmental activity) were almost absent in the rectosigmoid area. A marked decrease in the retrograde LSB activity was also observed in eight patients. During the night (from 2200 h to 0600 h) the number of migrating long spike bursts was increased in the diarrhoea group, but almost absent in controls (p less than 0.001). This study shows that colonic motor activity was altered in painless diarrhoea. These disturbances were not limited to the decreased SSB activity in the sigmoid, but involved the whole colon, with lower SSB activity and abnormal increase of migrating long spike bursts activity (MLSB) in postprandial and nocturnal periods.

Action Potentials↗
PubMed2005

DNA-protein cross-links and replication-dependent histone H2AX phosphorylation induced by aminoflavone (NSC 686288), a novel anticancer agent active against human breast cancer cells.

Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one) (NSC 686288) is a candidate for possible advancement to phase I clinical trial. Aminoflavone has a unique activity profile in the NCI 60 cell lines (COMPARE analysis; http://www.dtp.nci.nih.gov/docs/dtp_search.html), and exhibits potent cellular and animal antitumor activity. To elucidate the mechanism of action of aminoflavone, we studied DNA damage in MCF-7 cells. Aminoflavone induced DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB). Aminoflavone induced high levels of DPC and much lower level of SSB than camptothecin, which induces equal levels of DPC and SSB due to the trapping topoisomerase I-DNA complexes. Accordingly, neither topoisomerase I nor topoisomerase II were detectable in the aminoflavone-induced DPC. Aminoflavone also induced dose- and time-dependent histone H2AX phosphorylation (gamma-H2AX). Gamma-H2AX foci occurred with DPC formation, and like DPC, persisted after aminoflavone removal. Aphidicolin prevented gamma-H2AX formation, suggesting that gamma-H2AX foci correspond to replication-associated DNA double-strand breaks. Accordingly, no gamma-H2AX foci were found in proliferating cell nuclear antigen-negative or in mitotic cells. Bromodeoxyuridine incorporation and fluorescence-activated cell sorting analyses showed DNA synthesis inhibition uniformly throughout the S phase after exposure to aminoflavone. Aminoflavone also induced RPA2 and p53 phosphorylation, and induced p21(Waf1/Cip1) and MDM2, demonstrating S-phase checkpoint activation. These studies suggest that aminoflavone produces replication-dependent DNA lesions and S-phase checkpoint activation following DPC formation. Gamma-H2AX may be a useful clinical marker for monitoring the efficacy of aminoflavone in tumor therapies.

Breast Neoplasms↗
PubMed1994

The effects of anti-TNF-alpha antibody and dexamethasone on TCDD-induced oxidative stress in mice.

Recent studies have implicated tumor necrosis factor alpha (TNF-alpha) in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since TNF-alpha sensitizes and activates phagocytic cells to agents that induce them to release reactive oxygen species, TNF-alpha may act as an amplifying loop in TCDD-induced oxidative stress (OS). Therefore, the effects of anti-TNF-alpha antibody (40 micrograms/mouse) and dexamethasone (2 mg/kg) treatment on TCDD-induced OS as measured by DNA single-strand breaks (SSB) in hepatic nuclei, lipid peroxidation in hepatic mitochondria and microsomes, and activation of peritoneal lavage cells (PLC) in C57BL/6J mice were studied. One day after treatment with 125 micrograms TCDD/kg, anti-TNF-alpha resulted in 70, 27, 33 and 21% decreases in DNA-SSB, mitochondrial and microsomal lipid peroxidation and PLC activation, respectively, relative to TCDD-treated mice. Dexamethasone produced 8, 32, 35 and 9% decreases in DNA-SSB, mitochondrial and microsomal lipid peroxidation and PLC activation, respectively, in TCDD-treated animals. The combination of anti-TNF-alpha and dexamethasone resulted in 67, 55, 61 and 25% decreases in the above parameters of OS, respectively. The results suggest that TNF-alpha release may play a role in sensitizing and activating phagocytic cells following treatment with TCDD, contributing to the overall OS of animals following exposure to TCDD.

Animals↗
PubMed1991

Repair of DNA single-strand breaks in lymphocytes from Alzheimer's disease patients.

We have studied DNA single-strand breaks (SSB) and their repair, after gamma-irradiation, in lymphocytes from patients with Alzheimer's disease (AD) and from normal, age-matched individuals, using alkaline sucrose gradient centrifugation. We have found that values for AD patients and normals are similar in each of the following: level of breaks in unirradiated cells, level in cells irradiated with 150 Gy and rate of repair. Also, preliminary results for young and old normal individuals showed no significant difference between their SSB levels in unirradiated cells, nor between levels in irradiated cells nor in repair rate. In all cases, post-irradiation repair appeared to be almost complete after 50 min incubation. We conclude that gamma-irradiation produces no gross difference in the initial number of SSB between lymphocytes from AD patients, old normals and young normals, and that their repair is equally proficient.

Aged↗
PubMed2004

Increased circulating levels and salivary gland expression of interleukin-18 in patients with Sjögren's syndrome: relationship with autoantibody production and lymphoid organization of the periductal inflammatory infiltrate.

IL-18, an immunoregulatory and proinflammatory cytokine, has been shown to play an important pathogenic role in Th1-driven autoimmune disorders. In this study, we evaluated the circulating levels and salivary-gland expression of IL-18 in patients with Sjögren's syndrome (SS), a mainly Th1-mediated disease. IL-18 serum levels were measured by ELISA in 37 patients with primary SS, 42 with rheumatoid arthritis, and 21 normal controls. We demonstrated high IL-18 serum levels in SS, similar to those in rheumatoid arthritis patients and significantly higher than in controls (P < 0.01). In addition, IL-18 serum concentrations were significantly higher in anti-SSA/Ro+ and anti-SSB/La+ than in anti-SSA/Ro- and anti-SSB/La- SS patients (respectively, P = 0.01, P < 0.01). Serum IL-18 correlated strongly with anti-SSA/Ro (P = 0.004) and anti-SSB/La (P = 0.01) titers. Salivary gland IL-18 expression was investigated by single/double immunohistochemistry in 13 patients with primary SS and in 10 with chronic sialoadenitis, used as controls. The expression of IL-18 was also examined in periductal inflammatory foci in relation to the acquisition of features of secondary lymphoid organs such as T-B compartmentalization, formation of follicular dendritic cell networks, and presence of germinal-center-like structures. IL-18 expression in SS salivary glands was detected in 28 of 32 periductal foci of mononuclear cells (87.5%), while no IL-18 production by infiltrating cells was detected in patients with chronic sialoadenitis. Within the inflammatory foci, IL-18 immunoreactivity co-localized almost exclusively with CD68+ macrophages. In addition, IL-18 was found in 15 of 19 foci (78.9%) with no evidence of T-B cell compartmentalization (nonsegregated) but in 100% of the segregated aggregates, both in T- and B-cell-rich areas. Strikingly, IL-18 was strongly expressed by CD68+ tingible body macrophages in germinal-centre-like structures both in SS salivary glands and in normal lymph nodes. IL-18 expression was observed in the ducts of all SS biopsies but in only 4 of 10 patients with nonspecific chronic sialoadenitis (P < 0.01). This study provides the first evidence of increased circulating levels and salivary gland expression of IL-18 in SS, suggesting an important contribution of this cytokine to the modulation of immune inflammatory pathways in this condition.

Adult↗
PubMed2000

Differences in clinical sensitivity of ELISA tests for autoantibodies with human and bovine extractable nuclear antigens.

Bovine antigens are routinely used in indirect ELISA tests to detect autoantibodies against extractable nuclear antigens (ENA). Here we investigate the difference in clinical sensitivity between ELISA tests prepared with native human and bovine antigens. SSA and SSB were obtained from spleen and nRNP/Sm complex from thymus. Each antigen was extracted with the same immunoaffinity column. ELISA tests with human and bovine antigens were set up under the same conditions of clinical specificity established on 50 blood bank donors. Of 109 random SLE and Sjogren's syndrome sera 49% and 35% were positive, respectively, for human and bovine SSA, 26% and 16% for SSB. Of 98 random SLE sera 52% and 41% were positive for human and bovine nRNP/Sm, respectively. A few specimens reacted only with bovine antigens, probably false positive reactions. The relative clinical sensitivity for all specimens identified as positive by human and/or bovine antigens was significantly higher with human than with bovine SSA, (93% vs 67%; P<0.001, chi2), SSB (93% vs 50%; P<0.001), and for nRNP/Sm (96% vs 75%; P<0.01). However, for values that exceeded 2.5-4 times the upper normal limit, the levels were similar for human and bovine antigens. We concluded that native human antigens offer clinical sensitivity superior to native bovine antigens for the measurement of anti-ENA antibodies by ELISA.

Animals↗
PubMed2002

Increased cellular carotenoid levels reduce the persistence of DNA single-strand breaks after oxidative challenge.

Dietary antioxidants, such as the carotenoids, may protect DNA from oxidative damage. This has been proposed to explain the epidemiological association between higher consumption of fruits and vegetables, which are rich in antioxidants, and lower incidence of cancer. However, this remains to be demonstrated conclusively. The effects of carotenoid supplementation on 1) baseline DNA damage, 2) susceptibility of cellular DNA to oxidative attack, and 3) DNA repair were measured in the human lymphocyte cell line Molt-17. Baseline DNA damage, susceptibility to oxidant attack (100 mumol/l H2O2 for 5 min at 4 degrees C), and disappearance of DNA single-strand breaks (SSB) after oxidative challenge were monitored by single-cell gel electrophoresis. DNA repair patch synthesis activity in cell extracts was determined using assays that measure nucleotide incorporation during repair of oxidative lesions in template DNA. Unlike single-cell gel electrophoresis, the parameters measured with these assays are not dependent on strand break religation. There was no evidence that beta-carotene, lutein, or beta-cryptoxanthin supplementation protected cellular DNA from oxidation under basal conditions or after oxidative challenge. However, only carotenoid-supplemented cells exhibited a significant decrease in numbers of SSB over a 2-h period after treatment with H2O2. Carotenoid supplementation did not provoke any detectable change in repair patch synthesis activity. We conclude that supplementation with carotenoids at 8 mumol/l does not provide significant antioxidant protection for DNA in Molt-17 lymphocytes but may enhance recovery of cells from oxidative challenge, as measured by loss of SSB. We argue that these data are most consistent with carotenoids acting to enhance DNA strand break repair.

Antioxidants↗
PubMed2000

Among friends? An examination of friendship and the self-serving bias.

Do friends bound each other's self-enhancement tendencies? Do friends display the self-serving bias (SSB; i.e. taking individual credit for success but blaming a partner for failure)? Dyads consisting of either friends or strangers engaged in an interdependent-outcomes creativity test, received bogus success or failure feedback at the dyadic level, and made responsibility attributions for the joint test performance. Strangers displayed the SSB. Friends, in contrast, refrained from the SSB: they shared responsibility for both successful and unsuccessful test outcomes. Friendship does place boundaries on self-enhancement.

Adolescent↗
PubMed2006

Fes1p acts as a nucleotide exchange factor for the ribosome-associated molecular chaperone Ssb1p.

The HspBP1 homolog Fes1p was recently identified as a nucleotide exchange factor (NEF) of Ssa1p, a canonical Hsp70 molecular chaperone in the cytosol of Saccharomyces cerevisiae. Besides the Ssa-type Hsp70s, the yeast cytosol contains three additional classes of Hsp70, termed Ssb, Sse and Ssz. Here, we show that Fes1p also functions as NEF for the ribosome-bound Ssb Hsp70s. Sequence analysis indicated that residues important for interaction with Fes1p are highly conserved in Ssa1p and Ssb1p, but not in Sse1p and Ssz1p. Indeed, Fes1p interacts with Ssa1p and Ssb1p with similar affinity, but does not form a complex with Sse1p. Functional analysis showed that Fes1p accelerates the release of the nucleotide analog MABA-ADP from Ssb1p by a factor of 35. In contrast to the interaction between mammalian HspBP1 and Hsp70, however, addition of ATP only moderately decreases the affinity of Fes1p for Ssb1p. Point mutations in Fes1p abolishing complex formation with Ssa1p also prevent the interaction with Ssb1p. The ATPase activity of Ssb1p is stimulated by the ribosome-associated complex of Zuotin and Ssz1p (RAC). Interestingly, Fes1p inhibits the stimulation of Ssb1p ATPase by RAC, suggesting a complex regulatory role of Fes1p in modulating the function of Ssb Hsp70s in co-translational protein folding.

Amino Acid Sequence↗
PubMedDate not supplied

Induction of single- and double-strand breaks in plasmid DNA by monoenergetic alpha-particles with energies below the Bragg-maximum.

The yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alpha-particles at the end of their track in DNA-films was determined experimentally. Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target. The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo. For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined. The energy loss of the particles in the DNA-layer was calculated. DNA samples were separated into the three conformational isomers using agarose gel electrophoresis. After fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics. Linear dose effect correlations were found for ssb and dsb per plasmid molecule. In the case of 344 keV-alpha-particles the yield of dsb was (8.6 +/- 0.9) x 10(-11) breaks/Gy x dalton. The ratio of ssb/dsb was 0.5 +/- 0.2. This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations. Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.

Alpha Particles↗
PubMed2005

Influence of polymorphisms in DNA repair genes XPD, XRCC1 and MGMT on DNA damage induced by gamma radiation and its repair in lymphocytes in vitro.

DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.

Adult↗
PubMed2006

Mechanisms underlying production of double-strand breaks in plasmid DNA after decay of 125I-Hoechst.

Previously, the kinetics of strand break production by (125)I-labeled m-iodo-p-ethoxyHoechst 33342 ((125)IEH) in supercoiled (SC) plasmid DNA had demonstrated that approximately 1 DSB is produced per (125)I decay both in the presence and absence of the hydroxyl radical scavenger DMSO. In these experiments, an (125)IEH:DNA molar ratio of 42:1 was used. We now hypothesize that this DSB yield (but not the SSB yield) may be an overestimate due to subsequent decays occurring in any of the 41 (125)IEH molecules still bound to nicked (N) DNA. To test our hypothesis, (125)IEH was incubated with SC pUC19 plasmids ((125)IEH:DNA ratio of approximately 3:1) and the SSB and DSB yields were quantified after the decay of (125)I. As predicted, the number of DSBs produced per (125)I decay is one-half that reported previously ( approximately 0.5 compared to approximately 1, +/- DMSO) whereas the number of SSBs ( approximately 3/(125)I decay) is similar to that obtained previously ( approximately 90% are generated by OH radicals). Direct visualization by atomic force microscopy confirms formation of L and N DNA after (125)IEH decays in SC DNA and supports the strand break yields reported. These findings indicate that although SSB production is independent of the number of (125)IEH bound to DNA, the DSB yield can be augmented erroneously by (125)I decays occurring in N DNA. Further analysis indicates that 17% of SSBs and 100% of DSBs take place within the plasmid molecule in which an (125)IEH molecule decays, whereas 83% of SSBs are formed in neighboring plasmid DNA molecules.

Benzimidazoles↗