The interaction of hydroxymercurybenzene sulfonate (HOHgBzS) with 2-thiouridine (sWrd) and 2-thiocytidine (s2Cyd) by means of absorption-difference spectroscopy and spectrophotometric titration was investigated. The absorption spectra of the reaction mixtures displayed isosbestic points indicating uniform reactions. Scatchard plots of the titration data revealed the formation of 1 : 1 complexes with a dissociation constant for sWrd -HOHgBzS and s2Cyd -HOHgBzS of K d = 0.55 fl and K d = 0.54 pi%, respectively. The interactions of poly(saU) and poly(s2c) with hydroxymercurybenzene sulfonate were established by their corresponding absorption difference spectra. The binding of hydroxymercurybenzoate (HOHgBz) to poly(s2C) and poly(s2U) was investigated by means of equilibrium dialysis. The association of HOHgBz to 2-thiocytosine bases in poly(s2C) follows a simple hyperbolic binding isotherm. The Scatchard plot of the binding data disclosed that only a fraction (n = 0.32) of the s2CMP residues in poly(s2C) was accessible to the interaction with HOHgBz. The dissociation constant of this interaction was found to beThe binding of HOHgBz to the helical poly(s2U) is a cooperative process as indicated by the sigmoidal binding cuve. Under saturating conditions all saUMP residues in poly(s2U) interact with the ligand.It was originally established by Lipsett [l] that s4UMP, a minor constituent of various tRNAs, reacted in a specific way with hydroxymercurybenzoate. Spectrophotometric titrations of s4Ups4U and s4UpU with hydroxymercurybenzoate [Z] dislocsed the extraordinary stability of the complexes formed. Since then, this reaction has been successfully applied to the analysis and modification of polynucleotides and tRNAs containing s4UMP residues [3,4]. Recently we succeeded in the enzymatic synthesis of poly (s2C) [3] and poly s2U [4]. Furthermore, preliminary evidence was obtained that s2UTP or s2CTP could be used as substrates in the transcription of synthetic DNA5 by RNA polymerase from Escherichia coli (K. H. Scheit, A. Frischauf and P. Faerber, unpublished results). We became interested in the question of whether or not 2-thiopyrimidine nucleotides interact with mercury derivatives. The interaction of sWrd, s2Cyd, poly(s2U) and poly(s2C) with hydroxymercuryAbbreviations. s2Urd, 2-thiouridine ; SWMP, 2-thiouridine 5'-phosphate; poly(s2U), poly(2-thiouridylic) acid; s2Cyd, 2-thiocytidine; s2CMP, 2-thiocytidine 5'-phosphate ; poly(s2C), poly(2-thiocytidylic acid); HOHgBzS, hydroxymercurybenzene sulfomte; HOIIgBz, hydroxymercurybenzoate ; these and other abbreviations for nucleotides and polynucleotides follow CBN rules, see Eur. J . Biochenz. 15, 203 (1970).36 Eur. J. Biochem., Vol.33 benzene sulfonate was investigated by means of absorption-difference spectroscopy, spectrophotometric titration and equilibrium dialysis. MATERIALS AND METHODS Chemicals Spectrophotometric MeasurementsUltraviolet absorption spectra were recorded with a Zeiss PMQ I1 Spectrophorometer equipped with a BO Phillips Optilab readout system or a ...

Poly(s2C) -poly(1) complex formation (where s2C = 2-thiocytidylate) was shown by spectrophotometric titration, absorption-difference spectroscopy and hydroxyl-ion titration. The absorption temperature profile of the complex does not display a transition between 10 and 100 "C. Cooperative melting with a transition midpoint at 77 "C was observed only after destabilisation by 30°/, ethyleneglycol. A stabilizing effect of s2CMP residues in helices is further shown by the observation that 2501, sWMP in poly(C,,s2C) * poly(1) significantly increased the thermal stability compared to poly(C) * poly(1). The ultraviolet absorption spectrum, first derivative absorption spectrum and circular dichroism spectrum suggest a weak n-n* transition at 320 nm in poly(s2C) * poly(1). Unlike other nucleoside diphosphates the polymerisation of s2CDP by polynucleotide phosphorylase from Escherichia coli occurred with it pronounced lag phase which disappeared after the addition of oligonucleotide primers.Recently we reported the enzymatic synthesis of a new poly(C) analog, poly(s2C) [l]. Poly(s2C) like poly(C) forms a helical structure a t acidic p H which is, however, much less stable than a poly(C) helix under corresponding conditions. We assumed that the low stability of the poly (s2C)helix in the acid pH range is the consequence of N-H . . . S hydrogen bonding. For a better understanding of the function of the rare nucleotide s2CMP in tRNA [2] knowledge of the behaviour of poly(s2C) under neutral conditions would be desirable. I n this paper we describe the interaction between poly(s2C) and poly(1) leading to the formation of the helical complex poly(s2C) -poly(1). Contrary to our original expectation, we find a stabilizing effect due to the presence of s2C in the helix. I n fact we found it necessary to extend our studies to copolymers of CMP and s2CMP to demonstrate the differences in the properties of CMP and s2CMP due to the introduction of the 2-thioketo group. Polymers with s2CMP only were so stable we could not detect helix-coil ~~ Abbreviations. sZCyd, 2-thiocytidine ; sZCMP, 2-thiocytidine-5'-phosphate ; s2CDP, 2-thiocytidine-(i'-diphosphate poly(s2C), poly-2-thiocytidylic acid; poly(C,,s2C), random copolymer of average composition CMP : s2CMP = 3 : 1 ; these and other abbreviations for nucleatides and polynucleotides follow CBN rules, see Eur. J . Biochenz. 15, (1970) 203. A,,, unit, amount of material which in 1 ml of solution gives an absorbance of 1 a t 250nm when measured in a cell of 1-cm path length. CD, circular dichroism.

Polymerisation of 2-thiocytidine-5'-diphosphate (s2CDP) by polynucleotide phosphorylase from Escherichia coli leads to the formation of poly(s2C) which has been characterized by ultracentrifugation, enzymatic hydrolysis and phosphorolysis. The ultraviolet absorption derivative, optical rotatory dispersion and circular dichroism spectra of 2-thiocytidine and poly(s2C) have been measured. Evidence for n-nd* transitions in the absorption spectra of these compounds is derived from derivatives of ultraviolet absorption and circular dichroism spectra. The absorption-temperature profiles of poly(s2C) in the acid pH range prove the existence of double-stranded structures which are much less stable than those of poly(C). From these observations it is concluded that hydrogen bonding predominantly controls the stability of the poly(C) helix under acidic conditions. Poly(C), like poly(A), forms double-stranded structures in the acid pH range [I-31. Hydrogen bonding between bases and their cation and electrostatic interactions are thought to determine the stability of these helices. However, evidence was presented recently that charge-transfer interactions between cytosine and its cation in stacked structures might take place as well [4]. I n a variety of rare bases transfer RNA of Echerichia coli has been found to contain 2-thiocytosine [5]. The presence of a 2-thioketo group in cytosine should reduce the ability to undergo horizontal hydrogen bonding in helical structures, but a t the same time should intensify vertical interactions of the hydrophobic or electronic type. The properties of poly(s2C) as compared to those of poly(C) should indicate which factors contribute to the stability of the poly(C) helix under acidic conditions. The attempt was therefore made to synthesize poly(s2C) via the polymerisation of s2CDP by polynucleotide phosphorylase of E. coli.

Die chemische Synthese von 2-Thio-desoxythymidin (6)*J, 2.4-Dithio-5-methyl-uridin (3), und 2.4-Dithio-desoxythymidin (7) wird beschrieben. Die Massenspektren, Ahsorptionsspektren iind pK-Werte dieser Verbindungen werden angegehen. The Chemical Synthesis of 2,4-Dithiothymine NucleosidesThe chemical synthesis of 2-thiodeoxythymidine (6) *), 2,4-dithio-5-methyluridine (3), and 2,4dithtodeoxythymidine (7) is reported. Mass spectra, absorption spectra, and pK-values of these compounds are given. *I 2-Thto-desoxythymidin bedeuted hier 2-Thio-2'-desoxy-ribosylthymin. 1' K . H . Scheit und E. Guertner, Biochim. biophysica Acta [Amsterdam] 182, 1 (1969). 2 ) K . H . Scheir und E. Guertner, Biochim. biophysica Acta [Amsterdam] 182, I0 (1 969). 3) A . G . Lezius und K . H. Scheit, Europ.