Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles

The effect of physiological concentrations of KCl and MgCl₂ on the chemical stability of double-stranded and single-stranded DNA has been studied at temperatures typical for hyperthermophiles. These two salts protect both double and single-stranded DNA against heat-induced cleavage by inhibiting depurination. High KCl concentrations also protect DNA cleavage at apurinic sites, while high MgCl₂ concentrations stimulate this cleavage. It has been previously proposed that salt protects double-stranded DNA against depurination by stabilizing the double helix. However, the inhibition of the depurination of single-stranded DNA by KCl and MgCl₂ indicates that this effect is more probably due to a direct interaction of salts with purine nucleotides. These results suggest that the number and nature of heat-induced DNA lesions which have to be repaired might be quite different from one hyperthermophile to another, depending on their intracellular salt concentration. High salt concentrations might be also useful to protect DNA in long polymerase chain reaction (PCR) experiments and for long-term preservation. Received: October 12, 1997 / Accepted: January 29, 1998     

Interaction between the adenovirus DNA-binding protein and double-stranded DNA.

DNA-binding protein was characterized by previous investigators as a single-stranded DNA-binding protein analogous to the gene 32 protein of phage T4 (Van der Vliet &; Levine, 1973; Sugawara et al., 1977). In the studies presented here the interactions between natural and synthetic polynucleotides and the DNA-binding protein of adenovirus 2-infected HeLa cells have been examined. Polynucleotide melting techniques revealed a tight yet dissociable binding to the helix structure of double-stranded DNA. In addition, binding and filter binding competition experiments at high DNA to protein ratios revealed a specific binding to double-stranded DNA termini with a dissociation constant of 1 × 10^?9 to 2 × 10^?9m. The ability of DNA-binding protein to bind to heat-denatured viral DNA was confirmed but the binding to double-stranded DNA termini was more specific on a molar basis. DNA-binding protein can recognize both flush and staggered ends of double-stranded DNA molecules.     

Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.     

Analysis of processivity of mungbean dideoxynucleotide-sensitive DNA polymerase and detection of the activity and expression of the enzyme in the meristematic and meiotic tissues and following DNA damaging agent

Analysis of the processivity of mungbean ddNTP-sensitive DNA polymerase showed the incorporation of ∼35-40 nucleotides per binding event in the replication assays involving M13 ss DNA template with 5′-labeled 17-mer primer. Optimal processivity was obtained with 100-150 mM KCl and 6-8 mM Mg²⁺ at pH 7.5. The enzyme showed preference for Mg²⁺ over Mn²⁺ as the metal activator for processivity. 2′, 3′ dideoxythymidine 5′ triphosphate (ddTTP) and rat DNA pol β antibody strongly influenced distributive synthesis. Considerable enhancement in processivity was noticed at 1 mM ATP and 2-4 mM spermidine while higher concentrations of spermidine caused distributive synthesis. The enzyme was found to be active in both meristematic and meiotic tissues and distinctly induced by EMS treatment. DNA-binding assays revealed distinct binding ability of the enzyme to template/primer and damaged DNA substrate. Together these observations illustrate the probable involvement of the enzyme in replication and repair machinery in higher plants.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.     

ATP-dependent unwinding of the double helix and extensive supercoiling by Escherichia coli recA protein in the presence of topoisomerase

recA protein, which is essential for genetic recombination in Escherichia coli, causes extensive unwinding of the double helix by an ATP-dependent reaction and accumulation of positive supercoiling in closed circular double-stranded DNA. Initiation of the extensive unwinding was largely dependent on homologous single-stranded DNA. Therefore, it is likely that the extensive unwinding is initiated mainly at the site of D-loops. "Nascent D-loops" in which the two DNA molecules did not interwind were also good initiation sites of extensive unwinding. When the concentration of Mg2+ was decreased from the standard conditions for D-loop formation (13 mM MgCl2; the higher Mg2+ condition) to the lower Mg2+ condition (1 to 2 mM MgCl2), extensive unwinding by recA protein was initiated very quickly in the absence of single-stranded DNA. Results showed that this single-stranded DNA-independent initiation of extensive unwinding (i) requires negative superhelicity of the double-stranded DNA and (ii) is a first order reaction with respect to the DNA. These observations suggest that, under the lower Mg2+ condition, the extensive unwinding starts at a transiently denatured site in the negative superhelical DNA. Once initiated, the unwinding by recA protein is propagated extensively, even under conditions that do not allow its initiation. Therefore, the propagation of unwinding is a processive reaction ("processive unwinding"). Previous studies indicated that recA protein promotes "distributive unwinding" of double helix which depends on single-stranded DNA. Therefore, recA protein promotes unwinding of the double helix by either of two distinct pathways. Stress caused by the processive unwinding could explain the dissociation of D-loops and reversible inactivation of the double-stranded DNA in a D-loop cycle.     

Affinity and Conformation of the Estrogen Receptor When Bound to Single-Stranded vs. Double-Stranded Nonspecific DNA

Abstract

The affinity of the hormone-bound estrogen receptor for single-stranded and double-stranded DNA was compared using isocratic elution chromatography. The receptor bound single-stranded DNA with a two-fold higher affinity than double-stranded DNA (17.9 × 10⁴ M^?1 vs. 9.1 × 10⁴ M^?1) at 0.2 M KCl. The same number of ions were released when the receptor bound either single-stranded or double-stranded DNA (11.8 vs. 10.6, respectively). These results indicate the hormone-bound estrogen receptor has no strong preference for single-stranded vs. double-stranded nonspecific DNA, and has a similar conformation when bound to either form of DNA at physiological salt concentrations.     

Modification of Escherichia coli RNA polymerase by diethylpyrocarbonate. II. Binding and unwinding of double-stranded DNA

E. coli DNA dependent RNA polymerase was modified by diethylpyrocarbonate. Binding to a double-stranded DNA and unwinding of the DNA at the enzyme binding site by the modified enzyme were examined. It was found that RNA polymerase reversibly lost the ability to unwind DNA helix as well as the RNA synthetic activity when 9 to 11 histidyl residues of the enzyme were modified. In addition ot modification of the most reactive sulfhydryl or amino groups of the enzyme accompanying histidyl residues modification results in irreversible decrease of the salt concentration which is necessary to remove the enzyme from DNA cellulose column. Further modification of the less reactive sulfhydryl or amino groups leads to irreversible loss of the DNA binding ability and to the enzyme structure alteration.     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Nonhistone proteins HMG1 and HMG2 unwind DNA double helix.

In a previous communication we have shown that both HMG1 and HMG2 nonhistone proteins change the DNA helical structure and the binding of HMG1 and HMG2 to DNA induces a net unwinding equivalent of DNA double helix (Javaherian, K., Liu, L. F. and Wang, J. C. (1978) Science, 199, 1345-1346). Employing melting absorption technique, we now show that in the presence of salt HMG1 and HMG2 destabilize DNA whereas in the absence of salt, they both stabilize DNA molecules. Consequently the folded structure of HMG must play an important role in melting DNA. Furthermore, by measuring topological winding number using competition unwinding experiments, we conclude that HMG1 has a higher affinity for a single-stranded DNA relative to double-stranded DNA. These results together suggest that HMG1 and HMG2 unwind DNA double helix by local denaturation of the DNA base pairs. The net unwinding angles have been measured to be 22 degrees and 26 degrees per molecule of HMG1 and HMG2 respectively.     

Mode of Action of Antibiotic U-20,661

Antibiotic U-20,661 was shown to inhibit predominantly deoxyribonucleic acid (DNA)-directed ribonucleic acid (RNA) synthesis by binding to the double-stranded DNA template. Specific binding to DNA was verified by difference spectroscopy, reversal of the RNA polymerase inhibitory effect by increasing concentrations of DNA template, and by moderately increasing the melting temperature of double-stranded DNA in the presence of the antibiotic. The RNA polymerase reaction primed with synthetic poly dAT was inhibited considerably, but not completely even with high concentrations of antibiotic. Thus, the agent might bind to adenine or thymidine or both bases in the double-stranded DNA helix.     

Purification and characterization of an endo-exonuclease from adult flies of Drosophila melanogaster.

An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45°C in the absence and presence of Mg²⁺. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. ΔH⁰ and ΔS⁰ for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, ΔH⁰ ≈ 15–16 kcal/mol (63–67 kJ/mol) and ΔS⁰ ≈ 50–57 cal/K per mol (209–239 J/K per mol). The presence of Mg²⁺ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg²⁺, the ΔH⁰ and ΔS⁰ values are larger and temperature-dependent, with ΔH⁰ ≈ 22 kcal/mol (92 kJ/mol) and ΔS⁰ ≈ 72 cal/K per mol (approx. 300 J/K per mol) at 25°C, and ΔC_p⁰ 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA.

A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNA-binding proteins that retain a biological function from Rauscher murine leukemia virus. By consecutive purification on oligo (dT)- cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein. The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH. After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800. This protein binds about as well as to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA. The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E. coli or calf thymus and by 70S murine or feline viral RNA. Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition. This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA.     

Chimeric Phi29 DNA polymerase with helix–hairpin–helix motifs shows enhanced salt tolerance and replication performance

Phi29 DNA polymerase (Phi29 Pol) has been successfully applied in DNA nanoball-based sequencing, real-time DNA sequencing from single polymerase molecules and nanopore sequencing employing the sequencing by synthesis (SBS) method. Among these, polymerase-assisted nanopore sequencing technology analyses nucleotide sequences as a function of changes in electrical current. This ionic, current-based sequencing technology requires polymerases to perform replication at high salt concentrations, for example 0.3 M KCl. Nonetheless, the salt tolerance of wild-type Phi29 Pol is relatively low. Here, we fused helix–hairpin–helix (HhH)₂ domains E-L (eight repeats in total) of topoisomerase V (Topo V) from the hyperthermophile Methanopyrus kandleri to the Phi29 Pol COOH terminus, designated Phi29EL DNA polymerase (Phi29EL Pol). Domain fusion increased the overall enzyme replication efficiency by fourfold. Phi29EL Pol catalysed rolling circle replication in a broader range of salt concentrations than did Phi29 Pol, extending the KCl concentration range for activity up to 0.3 M. In addition, the mutation of Glu³⁷⁵ to Ser or Gln increased Phi29EL Pol activity in the presence of KCl. In this work, we produced a salt-tolerant Phi29 Pol derivative by means of (HhH)₂ domain insertion. The multiple advantages of this insertion make it a good substitute for Phi29 Pol, especially for use in nanopore sequencing or other circumstances that require high salt concentrations.     

Cloning, expression, and purification of gene 3 endonuclease from bacteriophage T7

The structural gene for the single-stranded endonuclease coded for by gene 3 of bacteriophage T7 has been cloned in pGW7, a derivative of the plasmid pBR322, which contains the lambda PL promoter and the gene for the temperature-sensitive lambda repressor, cI857. The complete gene 3 DNA sequence has been placed downstream of the PL promoter, and the endonuclease is overproduced by temperature induction at mid-log phase of Escherichia coli carrying the recombinant plasmid pTP2. Despite the fact that cell growth rapidly declines due to toxic effects of the excess endonuclease, significant amounts of the enzyme can be isolated in nearly homogeneous form from the induced cells. An assay of nuclease activity has been devised using gel electrophoresis of the product DNA fragments from DNA substrates. These assays show the enzyme to have an absolute requirement for Mg(II) (10 mM), a broad pH optimum near pH 7, but significant activity from pH 3 to pH 9, and a 10-100-fold preference for single-stranded DNA (ssDNA). The enzyme is readily inactivated by ethylenediaminetetraacetic acid or high salt. The differential activity in favor of ssDNA can be exploited to map small single-stranded regions in double-stranded DNAs as shown by cleavage of the melted region of an open complex of T7 RNA polymerase and its promoter.     

DNA translocation activity of the multifunctional replication protein ORF904 from the archaeal plasmid pRN1

The replication protein ORF904 from the plasmid pRN1 is a multifunctional enzyme with ATPase-, primase- and DNA polymerase activity. Sequence analysis suggests the presence of at least two conserved domains: an N-terminal prim/pol domain with primase and DNA polymerase activities and a C-terminal superfamily 3 helicase domain with a strong double-stranded DNA dependant ATPase activity. The exact molecular function of the helicase domain in the process of plasmid replication remains unclear. Potentially this motor protein is involved in duplex remodelling and/or origin opening at the plasmid replication origin. In support of this we found that the monomeric replication protein ORF904 forms a hexameric ring in the presence of DNA. It is able to translocate along single-stranded DNA in 3′–5′ direction as well as on double-stranded DNA. Critical residues important for ATPase activity and DNA translocation activity were identified and are in agreement with a homology model of the helicase domain. In addition we propose that a winged helix DNA-binding domain at the C-terminus of the helicase domain could assist the binding of the replication protein specifically to the replication origin.     

Saltatory thermal denaturation of double-stranded viral RNAs.

The double-stranded RNAs from bacteriophage phi6 and the replicative form of mengovirus denature upon heating in a series of abrupt steps which resemble the subtransitions (thermalites) observed within the high resolution profiles of small, naturally occurring DNA molecules. Such RNA thermalites are approximately an order of magnitude narrower than typical thermal subtransitions of nominally single-stranded RNA. We conclude that the same features of nucleotide sequence that give rise to cooperative denaturation in DNA genomes are to be found also in RNA genomes. Thus, high resolution thermal denaturation profiles are useful for characterizing double-stranded RNA molecules as well as native DNA in the size range of common viruses. A medium containing dimethylsulfoxide was required to lower the Tm of the RNA samples to a satisfactory temperature range. For double-stranded RNA in 50% dimethylsulfoxide, the dependence of Tm on G . C composition was greater than that of DNA in the same medium and also greater than that of double-stranded RNA in an aqueous medium. The fact that RNA thermalites are broader than DNA thermalites and that the melting temperature of double-stranded RNA has a greater dependence on base composition than that of DNA, indicates that at least one of the thermodynamic parameters for double helix formation in RNA is different from that in DNA.