S1 Endonuclease Research Articles

Isolation and Characterization of Novel Phages Targeting Pathogenic Klebsiella pneumoniae.

Klebsiella pneumoniae is a dominant cause of community-acquired and nosocomial infections, specifically among immunocompromised individuals. The increasing occurrence of multidrug-resistant (MDR) isolates has significantly impacted the effectiveness of antimicrobial agents. As antibiotic resistance is becoming increasingly prevalent worldwide, the use of bacteriophages to treat pathogenic bacterial infections has recently gained attention. Elucidating the details of phage-bacteria interactions will provide insights into phage biology and the better development of phage therapy. In this study, a total of 22 K. pneumoniae isolates were assessed for their genetic and phenotypic relatedness by multi-locus sequence typing (MLST), endonuclease S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), and in vitro antibiotic susceptibility testing. In addition, the beta-lactamase gene (bla KPC) was characterized to determine the spread and outbreak of K. pneumoniae carbapenemase (KPC)-producing enterobacterial pathogens. Using these ST11 carbapenem-resistant K. pneumoniae isolates, three phages (NL_ZS_1, NL_ZS_2, and NL_ZS_3) from the family of Podoviridae were isolated and characterized to evaluate the application of lytic phages against the MDR K. pneumoniae isolates. In vitro inhibition assays with three phages and K. pneumoniae strain ZS15 demonstrated the strong lytic potential of the phages, however, followed by the rapid growth of phage-resistant and phage-sensitive mutants, suggesting several anti-phage mechanisms had developed in the host populations. Together, this data adds more comprehensive knowledge to known phage biology and further emphasizes their complexity and future challenges to overcome prior to using phages for controlling this important MDR bacterium.

Facile and sensitive S1 endonuclease activity and inhibition assay using positively-charged Ag nanorods

In this work, we designed a facile and promising strategy for fluorescence determination of S1 endonuclease activity and inhibition based on fluorescence resonance energy transfer (FRET) between positively Ag nanorods (AgNRs) and negatively-charged ROX-labeled sing-stranded DNA (ROX-ssDNA). In the absence of S1 endonuclease, the fluorescence signal of the ROX-ssDNA was efficiently quenched when the ROX-ssDNA was adsorbed on the surface of AgNRs via strong electrostatic interaction. However, upon addition of different concentration of S1 endonuclease, the fluorescence was gradually restored owing to the reduction of FRET efficiency caused by S1 endonuclease specific cleavage ROX-ssDNA into short fragments, which reduced the electrostatic interaction between AgNRs and short oligonucleotide fragment. This assay strategy exhibited a high sensitivity and excellent specificity for S1 endonuclease with a detection limit of 0.004 U/mL and a dynamic concentration range from 0.01 U/mL to 5.0 U/mL. In addition, the capabilities for screening of S1 endonuclease inhibitors and S1 endonuclease detection from complex biological matrixes were also verified.

An efficient and rapid method to detect and verify natural antisense transcripts of animal genes

High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lncRNA (long non-coding RNA), can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix (Xist antisense RNA), chicken LXN (latexin) and GFM1 (G elongation factor, mitochondrial 1), were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence.

Poly(propylene imine) dendrimers modified with maltose or maltotriose protect phosphorothioate oligodeoxynucleotides against nuclease activity

The antisense oligonucleotides are promising agents for application in anti-HIV therapies. The antiretroviral nucleoside analogues administrated into circulatory system are vulnerable to nuclease degradation and require a vehicle which would not only facilitate therapeutic nucleotides into host cells, but would also provide protection against enzymatic degradation. Such potential is exhibited by poly(propylene imine) dendrimers – the branched cationic polymers easily interacting with oligonucleotides to form complexes called “dendriplexes”. The aim of the present study was to evaluate the abilities of the fourth generation poly(propylene imine) dendrimers partially modified with maltose (PPI-Mal G4) or maltotriose (PPI-Mal-III G4) to protect anti-HIV antisense oligonucleotides (ODNs) from nucleolytic degradation. The ODNs (AT, GEM91, SREV) were complexed with dendrimers and subjected to cleavage by serum nucleases or endonuclease S1. The results showed that all examined dendrimers protected ODNs against nucleases contained in FBS. Both PPI-Mal G4 and PPI-Mal-III G4 dendrimers completely prevented ODNs digestion by nuclease S1 at neutral pH. The protective capabilities of investigated dendrimers were significantly weaker in acidic environment. The time stability assay showed that the dendriplexes formed by AT, GEM91, SREV and carbohydrate-modified PPI G4 dendrimers still existed after 12h incubation both in low and at neutral pH buffers. The conformational change of dendriplexes in acidic environment was proposed as possible phenomenon leading to exposition of ODNs to nuclease attack and significantly diminishing dendriplexes’ resistance to nucleolitic digestion.

Introduction of OXA-48-producing Enterobacteriaceae to Israeli hospitals by medical tourism

The carbapenemase OXA-48 has been reported from different Mediterranean countries. It is mostly encoded on a single plasmid in various Enterobacteriaceae species. We characterized the epidemiological and molecular features of OXA-48-producing Enterobacteriaceae (OPE) in Israel. Epidemiological investigation was conducted by the National Center for Infection Control. Genotyping was performed using multilocus sequence typing. The bla(OXA-48)-carrying plasmids were investigated using S1 endonuclease and restriction fragment length polymorphism (RFLP). Conjugation efficiency of the bla(OXA-48)-carrying plasmids was studied in a filter mating experiment. Since 2007, four OPE-infected patients were identified, all non-Israeli (two Palestinian, one Jordanian and one Georgian). Three had prior hospitalization; two in Jordan and one in Georgia. The bla(OXA-48) gene was detected in three Escherichia coli strains belonging to different clonal complexes, one Klebsiella oxytoca and one Klebsiella pneumoniae sequence type 101, as previously reported from Tunisia and Spain. In all isolates, the bla(OXA-48) gene was located inside Tn1999.2 and was carried on a 60 kb plasmid with an identical RFLP pattern. The plasmid was able to conjugate from Klebsiella spp. to E. coli, and had a conjugation efficiency up to ~10000 times higher than that of pKpQIL. OPE, introduced mainly by medical tourism, are an emerging threat to patients from affected Mediterranean countries. The bla(OXA-48)-carrying plasmid demonstrated remarkable conjugation efficiency, which is probably important in the success of its dissemination.

Method for Improved Illumina Sequencing Library Preparation Using NuGEN Ovation RNA-Seq System

In this study, we tested the NuGEN Ovation RNA-Seq System for library preparation followed by next-generation sequencing on an Illumina GAIIx. The cDNA product of the NuGEN kit may have significant amounts of ssDNA with hairpin structures that are generated during the amplification process. These structures interfere with efficient downstream end repair, A-tailing, and adapter ligation, all standard steps in post-amplification sequencing library construction. We were able to increase the efficiency of sequencing library yields 4- to 6-fold or greater by treatment of NuGEN-amplified cDNA product with the single-strand endonuclease S1. These results suggest that this treatment effectively cleaves hairpin structures generated during amplification that are resistant to the standard enzyme cocktails used for the end-repair step.

ChemInform Abstract: Syntheses and Binding Studies of Oligonucleotides Containing N-Hydroxycarbamate Linkages: Potential DNA Cleaving Antisense Oligomers.

Abstract An efficient synthesis of N-hydroxycarbamate containing thymidine dimer 9 was accomplished and it was incorporated into automatic DNA syntheses to make thymidine 16mers T*-1, T*-2 and T*-3. Binding abilities of T*-1, T*-2 and T*-3 with poly (dA)-16mer were assayed by melting denaturation (Tm) studies of the corresponding duplexes. Iron binding ability of a thymidine oligomer with N-hydroxycarbamate linkages was studied by MALDI-MS. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides.

Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases

Antisense oligonucleotides with iron binding hydroxamate linkages are designed to act as sequence-selective cleaving agents of complementary nucleic acids through Fenton chemistry. Oligothymidylate analogs with hydroxamate linkages were efficiently synthesized from coupling of nucleoside intermediates, activated as p-nitrophenyl carbonates, with hydroxylamine derivatized nucleosides. Iron binding studies showed that hydroxamate linked oligonucleotides are effective iron chelators when there are three nonadjacent internucleosidic hydroxamate linkages available in the same oligonucleotide molecule. However, analysis of the CD spectra of an oligothymidylate 16mer, which contained complete substitution of all phosphates with hydroxamates, indicated that the hydroxamate linkage was too rigid to allow the analog to base pair with the complementary DNA d(A(16)). Syntheses of mix-linked thymidine oligomers with up to three hydroxamate linkages incorporated in the center of the sequence are also reported. Iron binding of the thymidine oligomer with hydroxamate linkages was confirmed by matrix assisted laser desorption mass spectrometry analysis. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. A thymidine 16mer with three hydroxamate linkages incorporated in the center of the sequence was shown to be able to bind with both iron and its complementary polyA strand. A small destablizing effect was observed when the phosphodiester linkage was changed to the hydroxamate linkage. Under Fenton chemistry conditions, this novel iron binding oligothymidylate analog cleaved the complementary DNA strand sequence-selectively.

Binding with Serum Components Favorably Affects Cellular Uptake of111In-Oligonucleotide in a Leukemia Cell Line

The influence of serum components on the intracellular uptake of an 111In-oligonucleotide (ODN) against mdr1 mRNA was investigated in the murine leukemia cell line, P388/S, and its mdr1-overexpressed P388/R. 111In-ODNs naked and vectorized with lipids were analyzed for binding with serum components using high-performance liquid chromatography (HPLC). 111In-ODN was incubated in albumin and transferrin solutions. 111In-DTPA and 111In-mononucleotide were incubated in serum. Degradation of naked 111In-ODN was detected in phosphate buffered saline (PBS) and serum containing endonuclease S1. Cellular uptakes of naked and vectorized 111In-ODN in the above cells were examined with and without fetal calf serum (FCS). Time-dependent binding of naked and vectorized 111In- ODN with serum components was observed throughout 24 hours. Transchelation of 111In to transferrin was not detected. HPLC profiles of 111In-DTPA and 111In-mononucleotide did not change in serum. Degradation of 111In-ODN by S1 was less remarkable in serum than in PBS. Specific accumulation of vectorized 111In-ODN in P388/R cells was achieved in culture with and without 10% FCS. This study verified the intense binding of ODN with serum components, leading to no inhibition on ODN intracellular specific uptake. Binding with serum components protects 111In-ODN from degradation by endonuclease and thus may facilitate ODN transmembrane delivery.

Higher order chromatin degradation in glial cells: the role of calcium

Higher order chromatin degradation (HOCD), i.e. the scission of nuclear chromatin loops at the matrix attachment regions (MARs), is a hallmark of programmed cell death. We have previously demonstrated that hydrogen peroxide (H 2O 2) induces rapid HOCD in cultured oligodendrocytes generating two subpopulations of DNA fragments of ≥400 and 50–200 kb. In the present study, we examined the involvement of calcium in this process. HOCD was induced in primary rat oligodendrocytes by exposure to 1 mM H 2O 2 and assessed by field inversion gel electrophoresis with and without S1 endonuclease digestion, to detect single and double stranded fragmentation, respectively. Chelating intracellular calcium with BAPTA/AM prior to H 2O 2 exposure inhibited HOCD in a dose-dependent manner. Complete inhibition of HOCD was attained with 50 μM BAPTA/AM. The pretreatment of cells with desferroxamine mesylate, which may lower intracellular calcium levels, also resulted in a profound inhibition of HOCD, but the initial chromatin digestion into ≥400 kb single stranded DNA fragments was unaffected. Neither removing extracellular calcium nor blocking calcium release from intracellular stores with TMB-8 affected HOCD. Moreover, increasing intracellular calcium with A23187 calcium ionophore did not induce HOCD. Subsequent study in nuclei purified from C6 glioma cells revealed that the endonuclease responsible for HOCD is calcium-independent, but is magnesium-dependent. Magnesium-induced HOCD was not affected by the removal of calcium from nuclei with EGTA, but was practically abrogated in nuclei prepared from BAPTA/AM-pretreated cells. These results indicate that although H 2O 2-induced HOCD is not directly mediated by an increase of intracellular calcium concentration, normal resting levels of intracellular calcium are required for the maintenance of MAR-associated endonuclease in an active form.

Nucleosides and Nucleotides. 204. Synthesis of Oligodeoxynucleotides Containing 6‘α-[N-(Aminoalkyl)carbamoyloxy]-carbocyclic-thymidines and the Thermal Stability of the Duplexes and Their Nuclease-Resistance Properties

To construct the nuclease-resistant oligodeoxynucleotides (ODNs) with natural phosphodiester linkages, we synthesized ODNs that contain 6'alpha-[N-(aminoalkyl)carbamoyloxy]-carbocyclic-thymidines (4, 5, and 6). The stability of these ODNs to nuclease hydrolysis was examined by using snake venom phosphodiesterase (3'-exonuclease) and nuclease S1 (endonuclease). It was found that the ODNs containing 4, 5, or 6 were more resistant to both the enzymes than the unmodified ODN. These nuclease-resistant properties are noteworthy, since they have natural phosphodiester linkages. Next, the thermal stabilities of duplexes consisting of these ODNs and either the complementary DNA or RNA were studied by thermal denaturation. The ODNs that contain 4 were found to enhance the thermal stability of the duplexes with the complementary DNA, while those containing 5 or 6 decreased the thermal stability of the ODN-DNA duplexes. On the other hand, all ODNs that contained 4, 5, or 6 decreased the thermal stability of the ODN-RNA duplexes.

Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H 2O 2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H 2O 2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H 2O 2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50–200 kb fragments. Chromatin digestion could be elicited with H 2O 2 concentrations as low as 50 μM. After the removal of H 2O 2, most >400 kb fragments were religated within 2 h; however, digestion into 50–200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H 2O 2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.

Syntheses and binding studies of oligonucleotides containing N-hydroxycarbamate linkages: potential DNA cleaving antisense oligomers

An efficient synthesis of N-hydroxycarbamate containing thymidine dimer 9 was accomplished and it was incorporated into automatic DNA syntheses to make thymidine 16mers T*-1, T*-2 and T*-3. Binding abilities of T*-1, T*-2 and T*-3 with poly (dA)-16mer were assayed by melting denaturation (Tm) studies of the corresponding duplexes. Iron binding ability of a thymidine oligomer with N-hydroxycarbamate linkages was studied by MALDI-MS. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides.

Human amiloride-sensitive epithelial Na+ channel γ subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation

The mRNA for the epithelial Na(+) channel gamma subunit (gammaENaC) is regulated developmentally in the lung, colon and distal nephron and in response to Na(+) deprivation and systemic corticosteroids in the distal colon. Because such regulation is likely to be at the level of gene transcription, we examined the function of the promoter and other 5' flanking elements of the human gammaENaC gene. The proximal 5' flanking region contains two GC boxes but does not contain a TATA box. A 450 bp human gammaENaC fragment (-459 to +40) directed the expression of luciferase in H441 cells and primer extension analysis in transfected cells confirmed the correct initiation of human gammaENaC-luciferase chimaeric transcripts. By deletional analysis, GC boxes at -21 and -52 were found to be critical for this promoter activity. To begin to identify transcription factors that bind to the core promoter, a double-stranded oligonucleotide that corresponded to this region was synthesized and tested in a gel mobility-shift assay. Incubation of this radiolabelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells resulted in the formation of four specific and distinct DNA-protein complexes. On the basis of antibody 'supershift' assays, one of these factors corresponds to Sp1, whereas the other three correspond to Sp3. Further upstream, an approx. 300 nt (-1143 to -839) polypurine-polypyrimidine tract (PPy tract) containing internal mirror repeats was identified. When contained in a supercoiled plasmid, the approx. 1200 nt 5' flanking region was sensitive to S1 endonuclease, which was consistent with the formation of an intramolecular triplex DNA ('H-DNA') structure with an unpaired single strand. High-resolution mapping with S1 endonuclease and sequencing of S1-generated clones confirmed that all S1-sensitive sites were within the PPy tract. Finally, a negative regulatory element was identified between -1525 and -1296 that functioned in lung, colon and collecting duct cell lines.

Genomic and phenotypic characterization of the bacterium causing blight of kiwifruit in New Zealand

Strains of the pathogen causing bacterial blight of kiwifruit in New Zealand, previously identified as Pseudomonas viridiflava, were examined using phenotypic and genotypic methods. Percentage DNA–DNA reassociation values for strains of the pathogen, with the type strains representing P. viridiflava and P. savastanoi, and representative strains within P. syringae, were obtained using the S1 endonuclease method. Strains of the pathogen were most similar to the type strain of P. savastanoi. This similarity was supported by examination of the ΔTm between representative strains. It is concluded that the pathogen can be considered as a member of the P. savastanoi genomic species. The pathogen from kiwifruit in New Zealand was also differentiated in genomic terms from P. syringae pv. actinidiae. Strains of the kiwifruit pathogen compared using the Biomerieux API Biotype 100 system exhibited consistent determinative tests which distinguished the pathogen from P. viridiflava and P. syringae pv. actinidiae. The origins of the pathogen in New Zealand are discussed.

Investigation of some properties of oligodeoxynucleotides containing 4'-thio-2'-deoxynucleotides: duplex hybridization and nuclease sensitivity.

The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.

Different types of DNA cleavage involved in osteosarcoma cell death

To induce cell death in osteosarcoma, a murine osteosarcoma clone, DOS C14 was exposed to: i) topoisomerase II-reactive epipodophyllotoxin, etoposide (ETO); ii) glucocorticoid analogue, dexamethasone (DEX); and iii) ultraviolet light (UV) irradiation. In MTT assay, fifty micromolar ETO, 100 mu M DEX and UV irradiation for 90 min reduced the cell number to 20% of that of the control. The cytotoxic effects of ETO and DEX were dose-dependent, while those of UV irradiation were time-dependent. Endonuclease cleavage of DNA into internucleosomal fragments was not recognized on DOS C14 osteosarcoma treated with 50 mu M ETO, 100 mu M DEX or UV irradiation. Aurintricarboxylic acid (ATA) also failed to inhibit the reduction in the number of viable cells. However, DNA from DOS C14 osteosarcoma cells exposed to 1 h UV irradiation showed smearing DNA fragments after treatment with S1 endonuclease, while such single strand modification was not detected in DNA extracted from cells exposed to ETO or DEX. On the other hand, pulse field gel electrophoresis revealed that cleavage of DNA into high molecular weight fragments estimated as 50-150 kilobase pairs (kbp) with a peak of 100 kbp was found in DOS C14 osteosarcoma cells exposed to 50 mu M ETO and 100 mu M DEX. The cytotoxicity of ETO and DEX was reduced by okadaic acid, while UV-induced cytotoxicity was not reduced by okadaic acid. Furthermore, okadaic acid inhibited the formation of high molecular weight DNA fragments in a dose-dependent manner. These results suggest that two types of DNA degradation exist in osteosarcoma death; one is random breakage of DNA, and the other is large DNA fragmentation, which may be produced by an activation of putative ATA-insensitive and okadaic acid-sensitive endonuclease.

Inter-strand cross-linking of Vibrio cholerae DNA induced by furazolidone: a quantitative assay by four simple methods

Four simple methods, i.e., (i) UV absorption spectrophotometry, (ii) hydroxyapatite chromatography, (iii) fluorescence analysis of ethidium bromide bound to DNA and (iv) assay of S1 endonuclease action, were used in parallel for the estimation of furazolidone-induced inter-strand cross-links in Vibrio cholerae DNA. The data produced by the four methods were in reasonable agreement with each other and provided similar linear dose-response relations, the correlation (between dose and response) coefficient being in any case numerically greater than 0.98. When the data obtained by four independent methods were plotted in a single graph, the resulting dose-response relation could be described by the equation log NR = 1.41 − 0.54 log D, where NR is the % non-reversible DNA remaining in the cells treated by furazolidone at dose D μg/ml × h. The correlation coefficient in this plot was −0.98 and significant to a level better than 0.1%. This study thus brings out that any one of these four methods can be used with reasonable confidence for the diagnosis and assay of inter-strand cross-links in DNA.

Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.

Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved.