No abstract

I 'tiiuersity o j Cal$'ur,iia, Dacis, C'alij'ortiia 95616 SynopsisThe helictal stability of a variety of DNA samples, raiigiirg iii base composition from 0 to i 2 mole-')b GC, has been studied by heat deriatriration a t neutral pH iii increasing concentrations of LiCI, NaC1, KC1, ( X I , LiSSO,, and K2S0,. The variation of melting temperatiire with average bitbe compositioii, dT,,,/dScc, was found to decrease drastically in the colicelitrated salt media, e.g., from 41°C ill 0.006N LiCl t,o 2Y°C in 3.251 LiCl, and from 39°C: in 0.008J1 T,i+Wi to 18°C iii 1.6Jf I&SOJ. At the same time, the t,hermal trarisit ion is milch more cooperat.ive in the concentrated salt, solutioris than at, low ionic strength. Iiideed, at limiting salt coiicerit ratioiis, the transition breadth seems to reach a miiiimiim valiie irrespective of the compositioiial heterogeneity of the DNA samples. Attempts to correlate the observed decrease of dT,,,'tlXc:~; with predicted changes iii t,he eiii halpg of melt irig, dedriced from a simple theoretical treat,ment, experimental data oii the biiidiiig of cwiiiiterioris aird water t o IINA, and experimental data on thermal deiiaturatiori, were iiiisriccessful. However, the stroiigly reduced composition depeiideiice of the nieltiiig temperatiire can be iuiderstood iri terms of a dest,abilizirig effect of the coilcentrated salt media oii GC-base pairs. It is suggested, t.hough not proven, that the dest abilizcttioii involves t.he displacemelit, of water molecules from the DNA helix.

When DNA's of differing GC:AT base ratios, e.g. synthetic poly dAT, T4 DNA,calf thymus DNA, E. coli DNA, and M. lysodeikticus DNA, are heat-denatured at neutral pH in increasing concentrations of N(a)(2)SO(4) or C(s)(2)SO(4) as supporting electrolytes,the variation of melting temperature with average base composition, dT(m)/dX(G)(C), changes from 45°C (in 0.002M Na) to ll°C (in 4.5M Na) and from 42°C (in 0.002M Cs) to 3°C(in 4.5M Cs). The decrease of dT(m)/dX(G)(C) is a monotonic function of decreasing water activity in the salt solutions. We interpret this decreased composition dependence of the thermal stability of the various DNA's as being due to a destabilization of the GC base pairs relative to the AT base pairs by the concentrated salt media. A simple quantitative treatment shows that k = 8GC/SAT decreases from a value of 4.14 (in 0.01MN(a)) to 1.86 (in 3M Na) and from 4.18 (in 0.01M Cs) to 1.42 (in 3M Cs). SAT is the equilibrium constant for the formation of a hydrogen-bonded AT base pair from a pair of unbonded bases at the junction between a helical region and a denatured region and SGC is the like constant for the formation of a GC base pair. These results corroborate our previous findings of a strongly reduced composition dependence of the negative logarithm of the methylmercuric hydroxide concentration necessary to produce 50% denaturation when the helix-coil transition of DNA is studied in concentrated Cs(s)SO(4)(ultracentrifugation) instead of in dilute N(a)(2)SO(4) (ultraviolet spectrophotometry).

The pyridoxal-catalyzed elimination of hydrogen sulfide or methyl mercaptan from -amino acids such as L-cysteine, S-methyl-L-cysteine, and dlmethionine was studied quantitatively in ah atmosphere of nitrogen at 100°C and at pH 5.8 or 6.2 in the presence and absence of Al(III), Fe(III), Fe(II), Sn(IV), and Sn(II). The catalytic activity of the metal ions decreases in the order Al(III) » Fe(III) > Fe(II) » Sn(IV) « Sn(II) in the case of L-cysteine. With S-methyl-L-cysteine, the order is

Exposing native calf thymus DNA to increasing concentrations of Hg(ClO4)2 not only produces dramatic changes in its circular dichroism (CD) but results also in the decrease, and ultimate cessation, of endonucleolytic DNA cleavage by staphylococcal nuclease. Let r = [moles of added Hg(II)]/[mole of DNA base]: the conservative CD spectrum of the DNA B-form becomes nonconservative in appearance at 0.01 less than r less than 0.12 (resembling DNA in C-form geometry) and assumes the spectral characteristics of a left-handed DNA double helix at 0.12 less than r less than or equal to 1.0. DNA cleavage proceeds at or near the rates exhibited by untreated DNA at 0 less than r less than 0.08. At Hg(II) levels of 0.08 less than r less than 0.5, the rate of DNA hydrolysis decreases monotonically with increasing Hg(II) concentrations, and at r greater than 0.4, DNA cleavage ceases. Both the CD changes and the changes in the rate of DNA digestion are totally reversible upon the removal of Hg(II), at least up to r = 1.0, demonstrating that Hg(II) keeps all base pairs in register. For comparison purposes, native calf thymus DNA was also treated with methylmercury [CH3Hg(II)], an agent known to disrupt the secondary structure of DNA. The treatment yielded single-stranded methylmercurated DNA with preserved right-handed helix screwness. In addition, this DNA is digested by staphylococcal nuclease much more rapidly than double-stranded control DNA. Lastly, neither the CD nor the cleavage rate changes are reversible upon the removal of methylmercury.(ABSTRACT TRUNCATED AT 250 WORDS)