Single-and Double-stranded DNA Research Articles

PH-dependent Activities and Structural Stability of Loop-2-anchoring Helix of RadA Recombinase from Methanococcus voltae

RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous double-stranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein’s ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA’s pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase-inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between single- and double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

Origin of Overstretching Transitions in Single-Stranded Nucleic Acids

We combined single-molecule force spectroscopy with nuclear magnetic resonance measurements and molecular mechanics simulations to examine overstretching transitions in single-stranded nucleic acids. In single-stranded DNA and single-stranded RNA there is a low-force transition that involves unwinding of the helical structure, along with base unstacking. We determined that the high-force transition that occurs in polydeoxyadenylic acid single-stranded DNA is caused by the cooperative forced flipping of the dihedral angle formed between four atoms, O5'-C5'-C4'-C3' (γ torsion), in the nucleic acid backbone within the canonical B-type helix. The γ torsion also flips under force in A-type helices, where the helix is shorter and wider as compared to the B-type helix, but this transition is less cooperative than in the B type and does not generate a high-force plateau in the force spectrums of A-type helices. We find that a similar high-force transition can be induced in polyadenylic acid single-stranded RNA by urea, presumably due to disrupting the intramolecular hydrogen bonding in the backbone. We hypothesize that a pronounced high-force transition observed for B-type helices of double stranded DNA also involves a cooperative flip of the γ torsion. These observations suggest new fundamental relationships between the canonical structures of single-and double-stranded DNA and the mechanism of their molecular elasticity.

Malignant Gastrocolic Fistula as a Late Complication of Radiation Therapy

Malignant gastrocolic fistula is a rare mode of presentation of gastric or colonic carcinoma [1–3]. A benign aetiology is more common, including peptic ulcer disease, particularly benign gastric ulcers which may relate to non-steroidal antiinflammatory use. Other benign causes include inflammatory bowel disease, post-PEG insertion, post-gastroenterostomy, pancreatitis, abdominal tuberculosis, trauma, perforated diverticular disease and infections including CMV, histoplasmosis, actinomycosis, amoebiasis, echinococcosis and lymphogranuloma venereum [2, 4–7]. A malignant gastrocolic fistula is most commonly found between the greater curvature of the stomach and the distal half of the transverse colon [8]. They are usually seen in the setting of large infiltrative tumours with a surrounding inflammatory reaction, and lymph node involvement is uncommon [8]. The colon rather than the stomach is the more usual site of origin. In a review of 1,500 cases of stomach cancer and 3,200 cases of colon cancer at the Lahey Clinic, 11 cases of gastrocolic fistula were reported, with 10 being of colonic origin [9]. Radiation therapy is associated with an increased risk of second cancers. Abdominal radiation therapy has been most frequently administered in the context of testicular cancer, where the overall cure rate is greater than 90 % [10]. The average age of presentation of testicular cancer is 35, and second neoplasms are uncommon but well-recognised complications where radiation therapy has been given [11–14]. Exposure to radiation may induce a broad variety of DNA damage including singleand double-stranded DNA breaks, cross-linking and damage to nucleotide bases [15]. Radiation-related genomic instability enhances the rate at which mutations and epigenetic changes are acquired in the descendants of that cell line after many generations of replication. In a cohort study of 40, 576 survivors of testicular cancer, Travis et al. reported on 2,285 second solid tumours observed where 1,619 new cancers were expected, demonstrating an elevated relative risk (ERR) as well as an excess absolute risk of developing a second neoplasm in comparison with the general population [16]. A further report of 5,142 patients treated with radiation therapy describes a 3.4fold increased risk of stomach cancer in survivors of testicular cancer and Hodgkin’s lymphoma [17]. This study found that the risk of second cancer begins approximately 10– 15 years after the original cancer diagnosis. We report herein a case of an adenocarcinoma presenting acutely as a J. Costelloe : C. O’Connell :O. Mc Cormack :N. Ravi : J. V. Reynolds Department of Surgery, St. James’s Hospital, Dublin, Ireland

Dimeric Fe-Co Phthalocyanine Complex as a Reagent for the Selective Damage of Nucleic Acids

The complexes of FeII and CoII with phthalocyanines are extremely good catalysts for oxidation of organic compounds with molecular oxygen and hydrogen peroxide. Their solubility and reactivity are increased by conjugation with oligonucleotides and by formation of dimeric complexes between negatively and positively charged phthalocyanines. In our work such complexes were formed directly on single-stranded DNA through interaction between negatively charged CoII phthalocyanine in conjugate and positively charged FeII phthalocyanine in solution. The resulting oppositely charged phthalocyanine complexes showed significant increase of catalytic activity compared with monomeric forms of phthalocyanines FeII. The site-directed modification of singleand double-stranded DNA by H2O2 in the presence of dimeric complexes of negatively charged CoII and positively charged FeII phthalocyanines was detected. These complexes catalyzed the DNA oxidation with high efficacy and led to direct DNA strand cleavage.

Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the lambda phage DNA with N6-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca2+/Mg2+- and Zn2+-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16–80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.

Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response: Figure 1.

The DNA damage signaling pathways mediated by the ataxia-telangiectasia mutated (ATM) and the ATM and Rad3-related (ATR) kinases play crucial roles in the maintenance of genomic integrity and may function as an anti-cancer barrier during early tumorigenesis. Although the ATM and ATR pathways share some of their downstream functions, the DNA damage that evoke these two pathways are distinct. While ATM plays a primary role in the response to double-stranded DNA breaks (DSBs), ATR controls the response to a much broader spectrum of DNA damage, including many that interfere with DNA replication. And, unlike ATM, ATR is crucial for maintaining genomic integrity during S phase of the cell cycle, and is indispensable for cell survival. Clearly, revealing the DNA structure that elicits the ATR pathway would be a critical step toward understanding the essential function of ATR and the genomic instability that it counters. The versatility of the ATR pathway in DNA damage response suggests that this pathway is likely able to sense a common signal generated by different types of DNA damage and genomic instability. Two simple structures commonly generated at sites of DNA repair and stressed DNA replication forks are single-stranded DNA coated with replication protein A (RPA-ssDNA) and junctions of singleand doublestranded DNA. Both of these structures have been implicated in the activation of ATR checkpoint by a number of studies using different model organisms. In this issue of Genes & Development, Cimprich and colleagues (MacDougall et al. 2007) report that circular singlestranded DNA (ssDNA) annealed with primers specifically triggers the ATR-mediated checkpoint responses in Xenopus egg extracts, revealing the first defined DNA structure sufficient to activate the ATR checkpoint pathway.

Some Aspects of Dna-Etbr Interaction Peculiarities.

On increasing the EtBr/DNA ratio, the temperature transition width in DNA melting curves decreased, passed through a minimum, and then slightly increased, indicating that EtBr binds to both singleand double-stranded DNA (Fig. 1). In addition, the melting curves showed a dependence on ionic strength, indicating that electrostatic interactions influence the different binding modes. Scatchard analysis of fluorescence binding data of EtBr with DNA detected only intercalation of EtBr into DNA (Fig. 2). However, Scatchard analysis of the absorbance binding data detected the intercalative interaction as well as additional binding modes (Fig. 2). Subtraction of the fluorescence titration data from the absorbance data showed that the additional modes of binding involve nonintercalative interactions between EtBr and both singleand double-stranded DNA (Fig. 2). These results are consistent with previous theoretical and experimental results.

Frequency-modulation SFM imaging of DNA molecules in UHV conditions

Dynamic mode scanning force microscopy with frequency-shift detection has been employed for imaging single-and double-stranded DNA under ultrahigh vacuum conditions. For the wet processes used to prepare the samples, the strong adhesion force at the surface is observed even in ultrahigh vacuum conditions. The DNA images can only be obtained with large frequency shifts, but provide sufficient resolution to see the coil or higher-order structures of the DNA molecules in detail. Because of the high sensitivity of the tip-sample interaction, frequency-shift detection mode provides a higher quality grey-scale resolution than tapping mode. In addition, contrast artefacts, which might include information on the detailed structure, are often observed, depending upon the feedback conditions.

The largest (70 kda) product of the bacteriophage T4 DNA terminase gene 17 binds to single-stranded DNA segments and digests them towards junctions with double-stranded DNA

Bacteriophage terminases are oligomeric multifunctional proteins that bind to vegetative DNA, cut it and, together with portal proteins, translocate the DNA into preformed heads. Most terminases are encoded by two partially overlapping genes. In phage T4 they are genes 16 and 17. We have shown before that the larger of these, gene 17, can yield, in addition to a full-length 70 kDa product, several shorter peptides. At least two of these, gene product (gp) 17′ and gp17″, are initiated in the same reading frame as the 70 kDa gp17 from internal ribosome binding sites. Most of the shorter gp17 s contain predicted ATPase motifs, but only the largest (70 kDa) peptide has a predicted single-stranded DNA binding domain.Here we describe the DNA binding and cutting properties of the purified 70 kDa protein, expressed from two different clones containing gene 17 but no other T4 gene. Epitope-specific antibodies, which recognize several different gene 17 products in extracts of induced clones or of T4-infected cells, precipitate the purified 70 kDa gp17. When Mg2+ is chelated by EDTA this 70 kDa protein binds to single-stranded DNA, preferentially to junctions of single-and double-stranded DNA segments. It does not bind to blunt-ended double-stranded DNA. When Mg2+ is present the purified 70 kDa gp17 digests single-stranded segments preferentially up to junctions with double-stranded DNA. A 70 kDa gp17 from a P379L temperature sensitive (ts) mutant, which has lost the nuclease and ATPase activities, retains the single-stranded DNA binding activity. Taken together with earlier findings these results support a model for packaging of T4 DNA from single-stranded regions in recombinational or replicative intermediates, which occur at nearly random positions of the genome. This mechanism may be an alternative to site-specific initiation of packaging proposed by other investigators.

Newer approach of screening test for antinuclear antibodies: an enzyme-linked immunosorbent assay detecting antinuclear antibodies characteristic of connective tissue diseases

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of antinuclear antibodies (ANAs) previously established as diagnostic and/or prognostic marker ANAs for various connective tissue diseases. The antigen used in ELISA is a mixture of purified recombinant or natural antigens including single-and double-stranded DNA, RNP, Sm, SS-A/Ro, SS-B/La, centromere, topoisomerase I and Jo-1 antigens. Thirty hundred and fifty nine patients sera from a variety of connective tissue diseases and 113 normal human sera (NHS) were examined. ELISA ANAs were positive in 3.5% of NHS and 80.2% of patients sera at cut off index 11.5, whereas indirect immunofluorescent antinuclear antibodies (FANAs) using HEp-2 cells were positive in 9.7% of NHS and 92.5% of patients sera at 1:160 serum dilution. More than 80% of sera from systemic lupus erythematosus, mixed connective tissue disease and primary Sjögrens disease were ELISA ANAs positive. Mean value of ELISA ANAs was highest in sera of patients with MCTD. ELISA ANAs were positive in 92.5% of sera with marker ANAs for connective tissue diseases. Mean value of ELISA ANAs was higher in sera with more than two marker ANAs than in sera with a single ANA or in sera without marker ANAs. In contrast incidence and mean value of ELISA ANAs were low in sera positive for anti topoisomerase I antibody or anti Jo-1 antibody. Sensitivity, specificity and agreement (accuracy) for connective tissue diseases with marker ANAs were as follows: ELISA ANAs (at index 11.5): 92.5%, 88.3% and 90.9%: FANAs (at 1:160 serum dilution): 99.0%, 70.4% and 88.1%, respectively. ELISA ANAs, thus, are specific for connective tissue diseases when compared to FANAs and previous ELISA for the detection of total ANAs. Moreover, ELISA ANAs are able to measure precise ANAs titers and are much less labor intensive when screening a large number of clinical specimens.

Resonance Raman spectroscopy of 4-thiothymidine and oligodeoxynucleotides containing this base both free in solution and bound to the restriction endonuclease EcoRV.

The resonance Raman spectra of 4-thiothymidine [4ST] have been recorded (a) in the free deoxynucleoside form, (b) when incorporated into the single stranded oligodeoxynucleotide d(AG[4ST]-TC), and (c) within the double-stranded self-complementary dodecamer d(GACGA[4ST]ATCGTC). Vibrational mode assignments of almost all the major Raman bands observed in each spectra have been made, mainly by comparison with the published assignments of related nucleosides and nucleotides. Differences between the spectra were observed, particularly when [4ST] and d(AG[4ST]TC) were compared to d(GACGA[4ST]ATCGTC). This is explained in terms of the variations in structure between single-and double-stranded DNA. Good quality spectra were obtained at nucleotide/oligonucleotide concentrations of between 100 and 500 microM and this coupled with an apparatus that uses small volumes (100 microL) allowed measurement of the spectrum of d(GACGA[4ST]ATCGTC) bound to the EcoRV endonuclease. This well characterised nuclease, for which crystal structures are available, recognizes d(GATAT) sequences. When this is replaced with d(GA[4ST]ATC), a poor substrate results but turnover can be prevented during data accumulation by omission of the essential cation Mg2+. Large shifts in several of the Raman bands were observed, and these have been related to the environment of the [4ST] base in the protein-bound oligonucleotide as deduced from the crystal structure. The wavenumber for the C = S stretch vibration in free d(GACGA[4ST]ATCGTC) has been used to calculate the strength of the Watson-Crick hydrogen bond between the sulphur atom in [4ST] and the 6-NH2 group on its partner dA. On binding to the enzyme, the shift in the wavenumber of the C = S stretch indicates this Watson-Crick hydrogen bond is weakened, in good agreement with X-ray structures. The advantage of using [4ST] as a resonance Raman probe is that it absorbs at 340 nm, a wavelength where other nucleic acid and protein absorbance is minimal. Thus the spectra obtained are very simple and consist of signals that arise predominantly from the thiobase alone, and this facilitates data interpretation.

Description of an IL-1-responsive kinase that phosphorylates the K protein. Enhancement of phosphorylation by selective DNA and RNA motifs.

The K protein was first identified in the heterogeneous ribonucleoprotein particle (hnRNP). Subsequently, K protein was shown to bind sequence-specific single-and double-stranded DNA, stimulate transcription, and bind Src, Fyn, Lyn, and Vav via SH3 interactions. The K protein also binds to the kappa B enhancer motif which stimulates its phosphorylation in vitro by an associated serine/threonine kinase. To gain more insight into this unique nucleic acid-dependent phosphorylation process, we set out to examine the regulation of this kinase. We demonstrate that the K protein exists in a complex with an IL-1-responsive kinase and that phosphorylation of the K protein by this kinase is augmented by either cognate DNA or RNA sequences. The IL-1-responsive kinase activity associated with the K protein is reduced by phosphatase treatment, suggesting that the K protein kinase activity is regulated by phosphorylation. The observation that phosphorylation of the K protein is DNA- or RNA-dependent and IL-1-responsive suggests that the function of the K protein is tightly regulated.

Evaluation of the Selective Binding Ability of Oligodeoxynucleotides to Poly(9-vinyladenine) Using Capillary Affinity Gel Electrophoresis

During the past decade, technical advances in the synthesis of nucleic acid analogues have stimulated further research in the development of gene-targeted drugs.l Nucleic acid analogues, in which the entire phosphate-sugar backbone of DNA has been replaced with a polyvinyl or a polyamide chain2-4, have recently been synthesized as potential therapeutic agents and found to form extremely stable heteroduplexes with complementary singleand double-stranded DNA. Although the evaluation of the binding ability of nucleic acid analogue to DNA is helpful for designing sequenceselective gene-targeted drugs using nucleic acid analogues, very little is known about it, because of a lack of research tools for evaluating the selective binding ability, except for melting-temperature (Tm) measurements. We recently developed capillary affinity gel electrophoresis (CAGE)S_8 by combining the high resolving power of capillary gel electrophoresis and the high specificity of a bioaffinity ligand. CAGE is a powerful new tool for the specific-recognition of DNA through selective binding of DNA to the bioaffinity ligand and the characterization of DNA-drug binding properties. In this study, the CAGE technique was examined and found to be applicable to evaluating the selective binding ability of oligodeoxynucleotides to a nucleic acid analogue. In the CAGE system, one of the nucleic acid analogues, poly(9-vinyladenine) [PVAd], is used as an affinity ligand, which interacts with the target oligodeoxynucleotide, which is injected into capillary as a sample to form a heteroduplex via a complementary hydrogen bonding. The migration times for some oligodeoxynucleotides were measured by changing the total concentration of PVAd. For a reliable measurement of the binding ability we used cytidine 5'-monophosphate (CMP) as an internal standard, and calculated the relative migration

Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport

Plant viruses establish a systemic infection by moving through plasmodesmata, but little is known of the mechanism(s) involved. The roles of two movement-associated proteins of a single-stranded DNA virus were investigated in vivo, using functional proteins expressed in E. coli and microinjection into plant cells. We report here that the BL1 protein of bean dwarf mosaic geminivirus moves extensively from cell to cell, increases mesophyll plasmodesmal size exclusion limit, and potentiates the movement of double-stranded DNA from cell to cell. Movement of single-and double-stranded DNA out of the nucleus is mediated by the BR1 protein. These results provide direct experimental evidence for intercellular macromolecular transport in plants, and suggest that the BR1 and BL1 proteins coordinate the movement of viral DNA across both nuclear and plasmodesmal boundaries.

A symptom variant of beet curly top geminivirus produced by mutation of open reading frame C4

Two beet curly top virus (BCTV) mutants have been constructed in vitro that contain G-to-T transversions either at nucleotide 2682 or at nucleotide 2802 within the overlapping open reading frames (ORFs) C1 and C4. The mutations introduce termination codons in ORF C4 without affecting the amino acids encoded by ORF C1. When agroinoculated into Nicotiana benthamiana the mutants caused stunting and yellowing of the plant and downward leaf curl but not the vein swelling and upward leaf curl symptoms that are characteristic of wild-type BC1V infection in this host. Levels of viral single-and double-stranded DNA forms were similar in mutant and wild-type infections. Symptoms induced by one such mutant in Nicotiana clevelandii and Datura stramonium were less severe than those in wild-type infections and were again qualitatively distinct. The mutants caused symptomless infections in Beta vulgaris, contrasting with stunting, severe leaf curl, and vein swelling symptoms associated with wild-type infection of this host. The levels of mutant DNA in newly expanding asymptomatic leaves frequently reached those of wild-type virus in leaves showing severe symptoms. The results suggest that ORF C4 encodes a protein that is a major determinant of pathogenesis that might affect the hyperplastic response of the host to BCTV infection.

Atomic force microscopy of single- and double-stranded DNA.

A method has been developed for imaging single-stranded DNA with the atomic force microscope (AFM). phi X174 single-stranded DNA in formaldehyde on mica can be imaged in the AFM under propanol or butanol or in air. Measured lengths of most molecules are on the order of 1 mu, although occasionally more extended molecules with lengths of 1.7 to 1.9 mu are seen. Single-stranded DNA in the AFM generally appears lumpier than double-stranded DNA, even when extended. Images of double-stranded lambda DNA in the AFM show more sharp kinks and bends than are typically observed in the electron microscope. Dense, aggregated fields of double-stranded plasmids can be converted by gentle rinsing with hot water to well spread fields.

Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase.

Bacteriophage T7 DNA polymerase, the product of gene 5 of the phage, has both polymerase and single-and double-stranded DNA 3'-to 5'-exonuclease activities. The exonuclease activities can be inactivated selectively by an oxidation reaction that requires molecular oxygen, a reducing agent, and iron at a concentration less than or equimolar to that of the gene 5 protein. Both exonuclease activities can be diminished by several thousandfold, with only a small decline in the polymerase activity. Escherichia coli thioredoxin, an accessory protein that binds tightly to the gene 5 protein and increases the processivity of the polymerization reaction, has no effect on the rate of oxidation. We propose that iron binds specifically to the exonuclease domain and, in the presence of molecular oxygen and a reducing agent, generates reactive oxygen species that selectively modify amino acid residues essential for the exonuclease activities.

Estradiol-dependent trans-acting factor binds preferentially to a dyad-symmetry structure within the third intron of the avian vitellogenin gene.

The secondary activation of the avian vitellogenin II gene in isolated liver nuclei by cytoplasmatic liver extracts of estradiol-treated chicks is accompanied by the binding of a protein from the extract to the structural part of the cloned gene. Both the DNA-binding and gene-stimulatory activities, which cochromatograph on heparin-Sepharose, are apparently present only in the cytoplasmatic liver extracts of estradiol-treated roosters and in the oviduct extracts of egg-laying hens. DNA-binding competition assays combined with exonuclease III footprinting showed that the factor binds to the imperfect dyad-symmetry structure 5'GTCTTGTTCCAAAC3' within the third intron of the gene. The factor is sequence specific and binds equally well to both single-and double-stranded DNA with an estimated dissociation constant of 3.5 X 10(-10) M.

Lifetime of peroxyl radicals of poly(U), poly(A) and single-and double-stranded DNA and the rate of their reaction with thiols.

Peroxyl radicals of poly(U), poly(A), and single- and double-stranded DNA have been produced by photolysing H2O2 in oxygenated aqueous solution in presence of the substrates. The peroxyl radicals are formed by the reaction of OH radicals with the polynucleotides followed by addition of oxygen. The lifetime of the peroxyl radicals and the rate constant of their reactions with the thiols cysteamine, glutathione and dithiothreithol have been measured by time-resolved e.s.r. spectroscopy. The unusually long lifetimes range from 0.2 to 3.3 s. The activation energy for the decay for all four substrates is 10.3 +/- 1 kcal/mol (43 kJ mol-1). The reaction rate constants with the thiols range from k = 0.8 X 10(4) to 1.3 X 10(5) dm3 mol-1 s-1. The reactions of the thiols with the peroxyl radical of poly(U) are known to prevent strand break formation. This shows that the peroxyl radicals of poly(U) observed by e.s.r. are intermediates in the pathway leading to strand break formation.

Improved rapid methodology for the isolation of nucleic acids from agarose gels.

Improved methodology is presented with which DNA may be rapidly isolated from agarose gels. Hydroxyapatite is used to bind the nucleic acid after agarose solubilization and a sodium citrate buffer is used to elute the nucleic acid free of agarose. Rapid concentration of the sample may then be effected by ethanol precipitation. Purified oyster glycogen may be used as carrier in this regard and does not inhibit restriction endonucleases nor T4 DNA ligase in the concentrations used. This methodology is useful for the isolation of single-and double-stranded DNA, supercoil plasmid DNA, and mRNA.