Polydeoxyribouridylic acid and its complexes with polyribo- and deoxyriboadenylic acids

J. 1MoZ.Biol. (1969) 46, 169-183

Polydeoxyribouridylic Acid and its Complexes with Polyriboand Deoxyriboadenylic Acids BARBARA~MUDZKA,

F.J. BOLLUMAND

D. SHUQAR

Department of Radiobiology, Institute of Oncology, Warsaw, Poland Department of Biochemistry, University of Kentucky, Lexington, U.S.A. and Institute of Biochemistry and Biophysics, Academy of Sciences Warsaw, Poland (Received 8 April 1969, and in revised form 24 July 1969) Polydeoxyribouridylic acid has been prepared (a) by polymerization of dUTP with the aid of terminal deoxynucleotidyl transferase, and (b) by deamination at, elevated temperatures of polydeoxyribocytidylic acid. The two procedures gave similar products. Polydeoxyribouridylic acid is a random coil, with only a low degree of base stacking over the experimentally accessible range of temperature and ionic strength. It is hydrolysed to monomers by phosphodiesterase I. The complexes of polydeoxyribouridylic acid with polyriboadenylic acid and polydeoxyriboadenylic acid, and the various transitions and rearrangements undergone by these as a function of temperature and ionic strength, have been studied in detail. The results make possible, for the first time, a comparison of the properties of all the possible complexes, and the transitions and rearrangements, of the ribose and deoxyribose analogues of polyuridylic acid (and polythymidylic acid) with polyadenylic acid. The results of such comparisons are presented in graphical form and discussed in relation to the structure of both the synthetic polynucleotide complexes and natural nucleic acids.

1. Introduction Among the various synthetic ribo- and deoxyribohomopolynucleotides containing residues of adenine or the complementary uracil or thymine, poly dUt has hitherto been conspicuous by its absence. Poly rA, poly rU and poly rT were initially obtained with the aid of polynucleotide phosphorylase. Subsequently poly dAT (Schachman et al., 1960), and then poly dA and poly dT were prepared by taking advantage of the ability of DNA polymerase to utilize poly rA: rU as a matrix for incorporation of dATPP and dTTP (Lee-Huang & Cavalieri, 1963). Finally, purified 7 Abbreviations used : poly dU, polydeoxyribouridylic acid ; poly dA, polydeoxyriboadenylic acid; poly dT, polythymidylic acid; poly d1, polydeoxyriboinosinio acid; poly dC, polydeoxyribocytidylio aoid; poly SMedC, poly 5-methyldeoxyribocytidylic acid; poly dA:dU (or simply dA: dU), twin-stranded complex of poly dA and poly dU; poly dA: 2dU (or dA: 2dU), triplestranded complex of poly dA and 2 poly dU, with similar connotations for other complexes. Replacement of the prefix “d” by “r” refers to the corresponding ribose polymer. The symbols “2 + 2” represent a transition from a triple- to a twin-stranded helix, with similar connotation for other types of transitions or rearrangements. 169

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deoxynucleotidyl terminal transferase (Yoneda & Bollum, 1965) made possible the synthesis of a variety of homodeoxyribopolynucleotides, including poly dA and poly dT, using only a short oligonucleotide initiator. The preparation of poly dU appears to have encountered considerable di&ulties. Notwithstanding that the replacement of dTTP by dUTP maintains DNA synthesis by Escherichia wli DNA polymerase with a yield of 54% (relative to that obtained with dTTP), and that poly dAU may be prepared with the aid of this enzyme system, it has not lent itself to the preparation of poly dU. Poly dU was ultimately prepared with the aid of the terminal transferase enzyme following extensive purification of commercially available dUTP. It also occurred to us that the readily available poly dC should be capable of providing poly dU by simple deamination, and initial trials immediately demonstrated the feasibility of such a procedure. The present communication describes the preparation and some of the properties of poly dU and its complexes with poly rA and poly dA. The data relating to the complexes are particularly relevant, since they fill the last important gap in our knowledge of the nature of the complementary helical complexes and hybrids formed between A and U (or T) ribo- and deoxyribo- residues. Poly rU and poly rA are known to form twin-stranded (Warner, 1957) and triplestranded (Felaenfeld & Rich, 1957) helices, and the conditions for formation of these two types of helices and the rearrangements which they may undergo have been extensively studied (Stevens & Felsenfeld, 1964; Blake, Massoulie & Fresco, 1967; Michelson, Massoulid t Guschlbauer, 1967). A detailed investigation of the reaction of poly rU with poly dA (Chamberlin, 1965; Riley, Maling t Chamberlin, 1966) demonstrated that these form uniquely the triple-stranded dA: 2rU; their failure to obtain the twin-stranded dA : rU was taken to indicate that the latter is less stable than the triple-stranded helix. An analogous situation prevails for the pair rA and rT, which form uniquely the triple-stranded rA: 2rT (Barszcz & Shugar, 1968). From a comparison with the corresponding complexes between poly dA and poly dT, this was postulated as being due to the lower degree of stacking of rU residues as compared to T in complexes of poly dA with poly dT (Riley et al., 1966) or poly rT (Barszcz & Shugar, 1968), and to the empirical observation that hybrid helical complexes containing deoxypurine and ribopyrimidine residues are less stable than other pairs in the same series.

2. Materials and Methods Poly dA and poly dC were products synthesized with the aid of the terminal transferase enzyme (Bollum, 1966). Their extinction coefficients in O-001 M-Tris buffer (pH 8-O) were 9.65 x IO3 and 5.30 x 103, respectively, and their sedimentation coefficients Sao,w were 5.04 for poly cL4 at neutral pH, and 4-80 for poly dC at pH 8.0. Poly rA was a Miles product with a molar extinction coefficient of 10.3 x 103, determined by alkaline hydrolysis to mononucleotides. All spectral measurements were carried out with a Unicam SP600 instrument, using Teflon-stoppered semi-micro IO-mm path-length cuvettes with an effective volume of 03 ml. For running of temperature profiles, a specially constructed cuvette carriage was employed, through which a water-glycerol mixture wa8 circulated by means of a Hoeppler ultrathermostat. Temperatures were measured by means of a thermistor located in a dummy cuvette. A Radiometer PHM22 instrument, with a semi-micro glass electrode, was used for pH measurements. Sediment&ion coefficients were determined on solutions of polymers

POLYDEOXYURIDYLIC

in O-01 M-buffer containing 10m3 M-EDTA model E ultracentrifuge.

ACID

171

(sodium salt) aud 0.16 an-Nacl, using a Beckman

3. Results and Discussion (a) Enzymic syn&esie of poly dU Poly dU was prepared by polymerization of dUTP with terminal deoxynucleotidyl traasferase in the presence of cobalt chloride. The preparation of dUTP of suitable purity, i.e. entirely free of dUMP and dUDP, is rather difficult to achieve (see, for example, Zmudzka, Bollum & Shugar, 1969). However, commercial sources of dUTP, and chemically deaminated dCTP, were found by trial to be suitable substrates for the terminal transferase enzyme. A typical polymerization mixture contained: 10 PM-d(pT),, 10 m&r-dUTP, 10 mu-cobalt chloride, 200 m-potassium cacodylate, 40 pg/ml. of terminal transferase protein (specific activity 35,000 units/mg), and an excess of pyrophosphatase. Incubation at 35°C for 48 hours resulted in polymerization of 60% of the substrate. This yield could probably be improved upon, but the matter was not pursued further since the poly dU preparations were fully satisfactory. The total reaction mixture, following incubation, was extracted with aqueous neutralized phenol, and the aqueous layer removed and precipitated with two volumes of 95% ethanol. The ethanol precipitate was redissolved and traces of substrate removed by passage through a Sephadex 650 column. The sedimentation coefticienta of the final products were usually S,, ,W= 3.7 s. (b) Deamindion

of poly dC to poly dU

Initial attempts to deaminate poly dC at room temperature were unsuccessful. The reaction was extremely slow and incomplete even after a period of ten days, despite the addition of nitrite at intervals. This is due to the very stable twin-stranded helix formed by poly dC in the acid medium (Inman, 1964; Zmudzka et al., 1969) ueed for deamination, resulting in the non-availability of the hydrogen-bonded amino groups. Poly dC (35 pmoles) was dissolved in 130 ~1. water and to this solution was added 48 mg aodium nitrite (700 pmoles) and 50 ~1. glacial acetic acid. Additionof the latter resulted in precipitation of the polymer. The reaction mixture was immersed in a water bath at 60°C for 10 to 15 minutes; this caused rapid clarification of the auspension. The solution was then brought to room temperature, and a 5+1. sample diluted with water to 0.5 ml. was dialyzed overnight against water. The resulting absorption spectrum, almost identical in 10m3az-hydrochloric acid and in buffered medium at pH 7.8, was similar to that of poly rU (Fig. l), indicating complete deamination. The entire reaction mixture was then exhaustively dialyzed at 3°C against three changes of water, once against EDTA and &ally several times against water; starch-iodide paper was used to control the removal of NOz- ions. The sedimentation coefhcient S,, ,w of the final, lyophilized product was 3.7 s, a8 compared to a value of S,,,, of 45 s for the poly dC sample from which it was prepared. This may conceivably point to some degree of chain sciasion during the deamination reaction. By contraat, the sedimentation boundary of the deamination product of poly dC w&8 a8 homogeneous as that of the poly dC sample from which it was prepared, and closely similar to that of enzymically prepared poly dU. This may be regarded aa evidence for the absence of any cross-linked products such aa are found in

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A(mp) FIG. 1. Absorption spectra in 10m3 M-HCl (-----) and at pH 7.8 (-) of (a) poly ru; (b) poly dU obtained by polymerization of dUTP with terminal deoxynucleotidyl transferase; (c) poly dU obtained by deaminat)on of poly dC as described in text; (d) poly dC; at pH 2.8 (-----), and at pH 10.0 (--.

deaminated DNA. The similarity of the product obtained by deamination of poly dC with that obtained by enzymic polymerization of dUTP is further supported by the absorption spectra as shown in Figure I(b) and (c) : X,,,, and A,,,, are identical for the two preparations; the ratios E,,,,,,/Q,,~,,.are 3.73 and 3.94, respectively; those of ~s~c/~sc~are 0.78 and 0.79; and those of •~s~/~~ccare 0.33 and 034. These data, while indicating that deamination was essentially complete, and that no cross-linked products had been formed, do not eliminate conclusively the possible formation of traces of side products, which would not be revealed either by the spectra or the sedimentation pattern. This is countered by the fact that the complexing ability, with poly rA and poly dA, of the product of deamination of poly dC was similar to that of enzymically prepared poly dU. Nonetheless, the results described below were all obtained with the enzymically prepared poly dU. Attempts are also under way to test further the validity of the deamination reaction in the range of 50 to 60°C. (0) Structure

of poly d U

Over a temperature range from 0°C to 35”C, in the presence of 0.01~ to 0.02~~ magnesium ions, there was no detectable change in the absorption spectrum of poly dU. A further increase in temperature to above 50°C was likewise without effect. In this respect, poly dU is somewhat similar to poly dT; it is, in fact, perhaps even less ordered, since poly dT has been shown to exhibit a small degree of temperature hyperchromicity, about two to three per cent at its absorption maximum over a temperature range of 0 to 50°C (Riley et al., 1966). Addition of various polyamines (Szer, 1966) to a solution of poly dU at pH 7.4 was without any detectable effect on the optical density as a function of temperature.

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These results are in agreement with the small extent of base stacking in this polymer as deduced from its absorption spectrum following hydrolysis to mononucleoticles with phosphocliesterase I (Fig. 2). It will be seen that the residual hyper-

FIQ. 2. Absorption spectrum of poly dU in aqueous medium at neutral pH (--) and following hydrolysis to monomers by phosphodiesterase I at pH 8.95, and neutralization of the solution (------). Conditions of enzymic hydrolysis: 0.01 M-Tris, ,O.Ol M-Mg2+, 6x 10-s M-poly dU, 5 pg phosphodiesterase I/ml. (Worthington); incubation at 37°C until there was no further change in optical density at 261 rnp at pH 8.95.

chromicity at the absorption maximum, 261 rnp, is only about 3%. At slightly longer wavelengths there is some hypochromicity. That the base residues in this polymer do exhibit some degree of base stacking is, on the other hand, shown by the red shift in Lax. accompanying enzymic hydrolysis, together with a small hypochromicity at about 290 and 235 rnp (Fig. 2). The absence of any helical structure in poly clU and poly dT as compared with poly rU and poly rT, in the experimentally accessible ranges of ionic strength and temperature, is very striking and illustrative of the important role of the 2’-hydroxyl in the formation of helical structures, at least in some specific instances (see conclusions below). The properties of poly dU are being further investigated by means of optical rotatory dispersion and circular clichroism (Rabczenko & Shugar, 1969). In particular, the CD spectrum of poly dU is strikingly similar to that of the disordered form of poly rU at room temperature, with a positive band at 271 rnp the intensity of which is only 20% below that for the corresponding band in poly rU at 272 ml*. This may be considered as evidence for some degree of base stacking in ploy

au.

(cl) Complexes of poly d U with poly dA Figure 3 shows the melting profiles of complexes of poly clU with poly dA in mixtures of dA : clU of 1: 1 and 1: 2 at neutral pH and at different ionic strengths, measured at various wavelengths. At low salt concentrations, less than O-1M-SOdkIm ions, both mixtures exhibit identical simple single-stage profiles (Fig. 3(a) and (cl)). Furthermore, the temperature hyperchromicities at 259 rnp for the 1: 1 mixture (46%) and

(a) ,.4 L

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40

40

60

80 1 -

100

Tempcmture K) FIQ. 3. Absorbance-temperature profiles, at wavelengths indicated, for mixtures of poly dA and poly dU at molar ratios of 1: 1 ((a), (b) and (0)) and 1:2 ( (d), (e) and (f)) in 0.02 aa-phosphate buffer pH 7.8 snd et the following Na+ oonoentrations: (a) and (d), 0.038 ra; (b) and (e), 0.434 M; (c), 2.04 M; (f), 1.93 M; -- 0 -- l -- (in (c) and (f)), 1.166 M.

the 1: 2 mixture (27%) demonstrate that, irrespective of the ratio of the homopolymers, the profiles represent the melting of a twin strand to its constituent components, as follows: dA:dU-dA or dA:dU+dU

+ dU -

dA + 2dU.

(1) (2)

For, if we accept that the 1: 1 mixture with 46% hyperchromicity is indeed twinstranded, then the calculated hyperchromicity for the 1: 2 mixture should be 27%, in ageement with that observed experimentally. Note that the numerical values for the corresponding hypochromicity et 283.5 rnp (Fig. 3(a) and (d)) are fully in accord with the foregoing conclusion. For a dA; dU mixture of 1: 1 at a salt concentration of about O-3 M, the melting profle (Fig. 3(b)) is essentially similar to that described above. The measured T,

POLYDEOXYURIDYLIC 100~ -

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Fm. 4. Dependence of T, values of oomplexes of poly dU with poly dA ((8) and (b)) and poly rA ((c) and (d)) on the Ne+ conoentration (including bulk cation) in phosphate buffered medium (pH 7.8). (--e--e--) Melting of twin-stranded helix to homopolymers; (--O--O--) melting of triple-&ended helix to homopolymers; (--A--A--) melting of triple-stranded helix to twinstranded; (--x -- x --) rearrengement of twin-stmnded helix to triple-stranded. T, values obteined with solutions containing A :dU 8t ratios of 1: 1 and 1: 2. Continuous lines (A to D) represent: (A) melting of twin strand to homopolymers; (B) melting of triple strand to homopolymers; (C) melting of triple to twin strand; (D) thermal rearrangement of twin to triple strand for: (a) poly rA 8nd poly rU (d8te from Michelson et al., 1967). (b) poly dA with poly dT d8ta from (c) poly dA with poly rU Riley et al. (d) poly rA with poly dT 1 (1966) Romen numerals I to IV define regions in which there exist: I, an admixture of the complexes dA : dU and dA : 2dU, depending on the reletive ratio of poly dA to poly dU in the range of 1: 1 to 1: 2; II, the twinstrended dA: dU, irrespective of the ratio of the oomponents; III, the triple-atrended dA:2dU regardless of the ratio of the components; IV, only the homopolymer oomponents poly dA and poly dU.

value under these conditions is located on the straight line which expresses the dependence of the T, values, for the twin-stranded helix, on the logarithm of the salt concentration at higher salt concentrations (Fig. 4). However, when the dA: dU ratio is increased to 1:2 under these conditions, the melting profile exhibits two stages (Fig. 3(e)). For the second of these two, the T, and the magnitude of the temperature hyperchromicity correspond to the melting of the twin-stranded helix

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(Fig. 3(e)) ; whereas the first, which occurs at a lower temperature, appears to represent the melting of a triple to a twin strand, as follows: dA:2dU

____t

dA:dU

+ dU.

(3)

The increase in total temperature hyperchromicity of a 1:2 mixture from 27% (Fig. 3(d)) to 52% (Fig. 3(e)) accompanying the two-stage melting process supports the assumption that two moles of poly dU are involved in a complex with one mole of poly dA at the lower temperature. With further increase in the salt concentration, the two-stage melting process for the 1: 2 mixture is replaced by a single helix-coil transition (Fig. 3(f)). But the temperature hyperchromicity remains above 50%, from which it may be concluded that at the lower temperature we have a triple-stranded helix and that the melting process represents a transition directly to homopolymers, as follows : dA: 2dU -

dA + 2dU.

(4)

For a 1: 1 mixture of the homopolymers at the same salt concentration, the melting process is two-step in nature, and is much clearer for measurements made at 280 rnp than at 259 rnp. If we recall the analogous data described by Stevens & Felsenfeld (1964) and Blake et al. (1967) for mixtures of poly rA and poly rU, as well as the appearance of the spectra for the sum of the two homopolymer components and their twin- and triple-stranded complexes (shown in Fig. 7, and discussed below), it seems logical to assume that the profile in this instance represents initially a rearrangement of a twin- to a triple-stranded helix, the latter of which then melts to its constituent homopolymer components, as follows : dA:dU ______t ,tdA:dU

+ +dA -

&iA:dU + 8 dA dU + dA.

(6)

The various types of transitions described above between poly dA, poly dU and their complexes were examined over a range of salt concentrations. This made it possible to delineate, as Blake et al. (1967) h ave previously done for complexes of poly rA and poly rU, the conditions of salt concentration and temperature for the existence (see Fig. 4(a)) of: I, An admixture of the complexes dA: dU and dA: 2dU, depending on the relative ratio of poly dA to poly dU over the range of 1: 1 to 1: 2 ; II, the twin-stranded &A: dU, irrespective of the ratio of the components; III, the triple-stranded dA: 2dU regardless of the ratio of the components ; IV, only the homopolymer components poly dA and poly dU. Three series of T, values, characterizing the transitions of triple-stranded helices to twin strands, and twin-stranded and triple-stranded helices to homopolymers (Fig. 4), increase approximately linearly with a logarithmic increase in salt concentration. On the other hand, the T, values characteristic of the rearrangement of a twin-stranded to a triple-stranded helix, as in the case of complexes of poly rA with poly rU (Stevens t Felsenfeld, 1964; Blake et al., 1967) and poly 5MedC with poly d1 (Zmudzka et al., 1969), decrease with an increase in ionic strength. A comparison of the profiles presented in Figure 3(a) and (f), which represent respectively the melting of dA: dU and dA: 2dU, indicates that in both instances the transitions are fully co-operative, as for the complex of poly rA with poly rU. The

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I.8

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dA:dU

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PC)

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FIQ. 6. Melting profiles in phosphate-buffered medium, pH 7.8, for: dA: dU in 0.038 M-Na+ ; rA: rU in 0.1 M-Na+ (data from Riley et aZ., 1906); dA: 2dU in 1.93 M-N&+ ; rA: 2rU in 0.2 M-N&+ (d&t8 from Riley et al., 1968); dAU and rAU in 0.01 M-N&+ and citrate buffer, pH 7.5 (data from Chamberlin, Baldwin & Berg, 1963).

broadening of the initial portions of these profiles, at lower temperatures, is certainly not due to any other types of interactions (see discussion above) and is perhaps a consequence of the properties of the polymers, i.e. chain length or heterogeneity. In general, however, the similarity of the transitions, and of the temperature hyperchromicities (Fig. 5) for the complexes of poly dA with poly dU to those of poly rA with poly rU suggests that the base-pairing schemes in dA: dU and dA : 2dU are similar to those accepted for rA:rU and rA:2rU. We shall revert to this point in Conclusions below. (e) Complexes of poly dU with poly rA The formation and melting profiles of hybrid complexes between poly dU and poly rA were examined over a wide range of ionic strengths (see Fig. 6). The results obtained 1.6-

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60

40

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60

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FIQ. 6. Absorbance-temperature pro&s for mixtures of poly rA and poly dU at molar ratios of (a) 1: 1, and (b) 1:2, in 0.02 aa-phosphate buffer, pH 7.8, and Na+ concentrations of 0.038 M (-O--O-) and 0.434 M (-+---a-). 12

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were identical over a range of sodium ion concentrations from 0.02 M to O-9 M, that is, the hyperchromicity of the complex formed in s, 1: 1 mixture was about 52% in all instances, whereas in a 2 : 1 mixture the temperature hyperchromicity was approximately 26%. Both mixtures exhibited a single helix-coil transition with identical T, values. It follows that, irrespective of the ionic strength of the medium, or the ratio of the homopolymer components, the only complex formed is the twin-stranded rA : dU. Figure 4 shows the dependence of the T, for the twin-stranded rA: dU on sodium ion concentration. (f) Absorption spectra of complexes of poly dU with poly dA and poly rA From the analyses presented above for the melting profiles of the twin- and triplestranded complexes of poly dU with poly dA, two salt concentrations were selected (O-038 and 1.93 M for which, at WC, a 1 :l mixture of poly dA and poly dU gives only the twin-stranded dA : dU, while a 1: 2 mixture gives only the triple-stranded dA : 2dU. The absorption spectra of these two types of complexes are represented by the solid lines in Figure 7. The molar extinctions for these complexes at 260 rnp are 6.9 x lo3

(b) FIG. 7. Absorption spectra of poly dA, poly dU and their complexes in phosphate-buffered medium, pH 7.8. dA: dU in 0.038 M-Na+ at 18T; (. . . .) (a) Containing ratio of dA to dU of 1: 1; (-) 4 dA:dU + $ dA in 0.038 M-NE+ at 67°C; (-----) poly dA + poly dU in 2.04 M-N&+ at 88’C. ) dA: 2dU in 1.93 M-N&+ at 18°C; (. . . .) (b) Containing ratio of dA to dU of 1: 2 ; (--dA:dU + dU in 0.038 M-Na+ at 18’C; (-----) poly dA + 2 poly dU in 1.93 M-N&+ at 87°C.

and 6.4 x 103, respectively (at the A,,,. values the molar extinctions are 6.9 x lo3 and 6.6 x 103, respectively). A comparison of these extinctions with those for the sum of the homopolymer components (which for poly dA + poly dU is 11.36 x lo3 and for poly dA + 2 poly dU is 10.85 x 103) renders possible an evaluation of the hypochromicity accompanying complex formation. In calculating the arithmetic sum of the extinctions of the component homopolymers, the value for poly dA was taken to be 30% above fhat at 2O”C, i.e. corresponding to that et about 85°C (Barszcz

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$ Shugar, 196g)t. This is similar to the value noted by Riley et al. (1966) at a temperature of about 100°C on a sample of poly dA obtained with the aid of DNA polymerase and with an SzoeWvalue of 13.6 S. The calculated values at 260 rnp of the hypochromicities accompanying formation of poly dA: dU and poly dA: 2dU (i.e. c Ebomopolymers - Ecomplex)were approximately ~%OlllOPOlYlll~~S

39% and 41%. A similar value is obtained for the formation of poly rA: rU, for which the sum of the extinctions of the components is 11.4 x lo3 (at 258.5 rnp) and that for the complex is 7.0 x lo3 (at 257 mp), the resultant hypochromicity (at 257 rnp) being 39% (Riley et al., 1966). The spectra of dA: dU and dA: 2dU are shown in Figure 7. For comparison purposes the dotted lines represent poly dA: dU formed in a mixture containing two moles dU to one of dA; and poly dA: 2dU formed in a mixture containing dU to dA in a ratio of 1: 1. The dashed lines represent the spectra of two or three homopolymer components at a temperature above the melting point. It will be seen from Figure 7 that, as for the formation of complexes or hybrids between poly A and poly rU or poly T, complex formation is accompanied by the appearance of hyperchromicity in the neighbourhood of 280 mp. Since, however, the sum of the extinctions of the homopolymer constituents increases with increase in temperature, largely as a result of the non-co-operative melting of poly dA, the point of intersection of this additive spectrum with that of the complex (at about 282 rnp) will also shift with change in temperature. For this reason we did not attempt to

IO-

a-

FIQ. 8. Absorption spectrum of poly rA: dU. ) in 0.02 M-phosphate buffer, pH 7.8, and 0.038 M-N&+ et 18°C; (-----) (------melting to homopolymers at 57’C.

following

t Unlike poly rA which, although it “melts” out non-co-operatively, nonetheless attains a plateau at about W’C, poly dA continues to melt in a non-co-operative manner up to lOO”C, with no signs of levelling off (Riley et al., 1966; Barszcz & Shugar, 1908).

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delineate the so-called isochromic wavelengths (Blake et al., 1967) for the appropriate rearrangements between poly dA and poly dU on one hand, and their complexes on the other. Instead, appropriate wavelengths in the neighbourhood of 280 and 283.5 rnp were selected, as far removed from the intersection points as possible, which made it feasible (either by an increase or decrease in extinction) to differentiate between two related processes (such as a 3 --f 2, or a 2 + 1, transition). These measurements in the longer wavelength region complement those made in the neighbourhood of the absorption maxima, about 259 rnp (cf. Fig. 3). The absorption spectrum of the hybrid complex poly rA: dU presented in E’igure 8 has a value of cZ6,,of 7.1 x 103, as compared to 11.15 for the sum of the components; the latter value was deduced from the known ezGOfor poly dU, and the ~~~~for poly rA at 25°C and increased by 19Go/o to give the extinction of the fully melted form (Barszcz & Shugar, 1968). The resultant calculated hypochromicity at 260 rnp accompanying formation of poly rA: dU comes out to be 36*5%, which is only slightly less than that for formation of poly rA: rU or poly dA: dU (39%). The spectrum of poly rA : dU, like those for the complexes discussed above, exhibits, with respect to its components, hyperchromicity at wavelengths to the red of 283 mp.

4. Conclusions As already referred to in the Introduction, the availability of poly dU now makes feasible a comparison of the properties of all the possible helical complexes between A and U, and A and T. Poly dU is particularly valuable in this respect, in the light of the results obtained in this study, since for the ribo series, rA and rT form only a triple-stranded helix (Barszcz & Shugar, 1968), whereas rA and rU may complex to either twin or triple strands (Stevens & Felsenfeld, 1964). Furthermore, the twin-stranded dA: dU and dA: dT furnish a direct comparison of the influence of a pyrimidine 5-methyl substituent ; while d.A: dU and rA : rU demonstrate directly the differences due to a 2’-hydroxyl between DNA and RNA type twin-stranded helices. I

0

-2

-1 Log[Na+l

0

-2

-1

I

0

FIU. 9. Dependence on log [Ne+] of the T, values for the various transitions and rearrangements undergone by various pairs of ribose and/or deoxyribose polymers of A and U (or T). Data for dA, dT from Riley et al. (1966) for rA, rU from Michelson et al. (1967), and for rA, rT from Barszoz & Shugar (1968).

POLYDEOXYURIDYLIC

181

ACID

The differences between dA: dT and rA: rU are probably highly representative of the differences between DNA and RNA twin helices, including the effects both of the 2’-hydroxyl and a pyrimidine B-methyl. The T, of dA : dT is 20 deg. C higher than that of rA: rU. Addition of a third, rU, strand to rA: rU occurs much more readily (e.g. at lower ionic strengths) than in the case of dA: dT (Fig. 9). (a) Effect of 2’-hydroxyl The presence of a 2’-hydroxyl in the individual strands of a helical complex manifests itself in two ways: (a) elevation of the T, as compared to the corresponding deoxyribo- helix; (b) greater ease of formation of triple-stranded helices (Fig. 10).

20 ,'

-2

,

-1

0

-2

I

-1

0 -2

-1

o-2

-1

0

Log [No+] FIO. 10. Dependence on log [Na+] of the T, values for the various transitions and rearrangements undergone by various pairs of ribose and/or deoxyribose polymers of A and U (or T). Data for rA, rU from Miohelson et oZ. (1967), for dA, dT; dA, rU and rA, dT from Riley et d. (1900), and for rA, rT and dA, rT from Barszcz & Shugar (1968). --) represents: ((a) and (b)) dA, dU; ((0) and (d)) rA, dU; ((e) and (f)) dA, dT; ((g) and (h)o rA, dT; (-----) represents: ((a) and (c)) rA, rU; ((b) and (d)) dA, rU; ((e) and (g)) rA. rT; ((f) and (h)) dA, rT.

(a) Apart from the complexes already described in this investigation, other ribose helices, e.g. rG: rC, r1: rC, rI:BMerC, all exhibit higher T, values than the corresponding deoxyribo- twin-stranded helices. The higher values for the ribose helices vary from 5 deg. C for rA : rU as compared to dA : dU (Fig. lo), and 10 deg. C for the alternating twin-stranded rAU as compared to dAU, to 34 deg. C for rG : rC as compared to dG : dC. (b) For triple-stranded helices, e.g. rA: 2rU, the addition of a third rU strand occurs at a lower ionic strength than for dA : 2dU. A direct comparison of the T, values for the 3 -+ 2 transitions is rather difficult, since (as can be seen from Figs 4(a) and 10) this requires extrapolations of the T, verse ionic strength curves beyond the

182

B.

ZMUDZKA,

F.

J.

BOLLUM

AND

D.

SHUGAR

experimentally accessible limits, which may not always be valid. However, if we assume that such extrapolations are admissible, then the T, values for 3 -+ 2 transitions inribose helices come out to be about 50 deg. C higher than for the corresponding deoxyribo helices (Figs 4(a) and 10). By contrast, the values of T, for 3 + 1 transitions in ribose helices are barely 6 deg. C above those for the corresponding deoxyribo helices (Figs 4(a) and 10). Figure 10 presents in graphical form comparative data for selected pairs of ribose, deoxyribose and hybrid helical complexes of A and U(T). It will be seen that, for all the complexes exhibited, only a change in the carbohydrate component of the pyrimidine strand(s) from deoxyribose to ribose favours formation of a triple-stranded helix. It must be borne in mind that the foregoing are strictly comparisons of experimental data, and involve no assumptions as to the nature of the base pairing in the individual helices. But we cannot, in fact, exclude the possibility that at least some of the differences described above are the consequence of differences in types of base pairing between ribose and deoxyribose helices. The ultimate resolution of this problem would obviously require different techniques, such as X-ray diffraction on fibres of synthetic helices. It is perhaps fairly well illustrated by the fact that, whereas it was for some time assumed that in rA:ZrU the O* of both uracil residues formed hydrogen bonds to the individual hydrogens of the adenine amino group, Miles (1964) demonstrated by means of infrared studies in aqueous medium, that attachment of the first strand of rU to rA involves hydrogen bonding of the uracil O* to the adenine amino group, whereas the second rU strand adds via hydrogen bonding of O2 (to the second adenine amino hydrogen). (b) Effect of a pyrimidine

5methyl substituent

This appears, at first sight, to differ for ribose, as compared to deoxyribose he&es. Both dA and dT, and dA and dU, under similar conditions of ionic strength, form similar twin- and triple-stranded helices. The differences in values of T, (i.e. the increase due to the B-methyl) are 17 to 21 deg. C for a 2 --+ 1 transition?, 16 to 21 deg. C for a 3+ 2 transition, and 15 deg. C for a 3 -+ 1 transition (Figs 4(b) and 9). Likewise, in the case of the hybrids rA:dU and rA:dT, the influence of the pyrimidine 5-methyl manifests itself by an elevation of the T, value (Fig. 4(d)). For rA and rT, as compared to rA and rU, the effect of the B-methyl may be evaluated only for the 3 -+ 1 transition (this being the only one undergone by rA : 2rT, since rA and rT do not form the twin-stranded rA : rT in the accessible range of ionic strengths and temperature). The value of T, for rA:BrT is 15 deg. C higher than that of rA: 2rU, for the 3-+ 1 transition (Fig. 9). In view of what was said above regarding possible differences in base pairing between ribose and deoxyribose helices, it is perhaps not too surprising that the influence of a 5-methyl substituent in ribose helices differs from that in the deoxyribo analogues. One example in which the effect of a B-methyl is independent of the nature of the carbohydrate component is offered by the pairs r1: rC and r1: 5MerC, and d1: dC and d1 :BMedC. For both of these pairs the B-methyl results in an increase in T, of I6 to 17 deg. C. This is hardly coincidental, since there is only one way in which I can t Analogous

results

have

been noted

by Dr C. R. Cassani

(personal

communication,

1969).

POLYDEOXYURIDYLIC

ACID

183

base-pair with C (or 5MeC). In this case, where base pairing possibilities are unique, it would be of considerable interest to compare r1: dC with rI:BMedC, and dI:rC with d1: 5MerC. For the base pair A and U (T), the situation is considerably more complex, since there are four possible pairing schemes (Davies, 1967). This is best exemplified by the fact that mixed crystals of l-methylthymine and 9-methyladenine give the Hoogsteen type of base pairing, whereas in l-ethyluracil and 9-methyladenine the pyrimidine ring is rotated through 180”, so that O2 (instead of O*) is hydrogen-bonded to the adenine amino group. A few concluding remarks are called for with regard to the absence of any helical structure, over the experimentally accessible ranges of pH and ionic strength, in poly dU and poly dT, as compared to poly rU and poly rT. The latter two probably form twin-stranded helices in which the U (or T) residues are base-paired through the ring N, hydrogens and the O* carbonyls (Davies, 1967). It is not immediately apparent how the removal of the 2’-hydroxyls can lead to the collapse of such helical structures. There is considerable speculation on this point, including hydrogen-bonding of the 2’-hydroxyl to the O2 carbonyl of a pyrimidine, the N, of a purine, or to a neighbouring phosphate hydroxyl. These speculations are based largely on modelbuilding studies, and the different views put forward by various observers suggests that the conclusions in some instances may be subjective. For the moment, it appears that this controversy could best be resolved by an examination of the diffraction patterns for fibers of poly rU and poly rT. This investigation was supported by the Wellcome Trust, the World tion, the Agricultural Research Service, U.S. Department of Agriculture and to one of us, (F.J.B.), the National Cancer Institute (Public Health grant no. CA-08487).

Health Organiza(UR-E21-(32)-30) Service Research

REFERENCES Barszcz, D. & Shugar, D. (1968). Europ. J. Biochena. 5, 91. Blake, R. D., Massoulie, J. & Fresoo, J. R. (1967). J. Mol. Biol. 30, 291. Bollum, F, J. (1966). In Procedures in Nucleic Acid Research, ed. by G. L. Cantoni & D. R. Davies. New York: Harper & Row. Chamberlin, M. J. (1965). Fed. Proc. 24, 1446. Chamberlin, M., Baldwin, R. L. & Berg, P. (1963). J. Mol. Biol. 7, 334. Davies, D. R. (1967). Ann. Rev. Biochem. 36, 321. Felsenfeld, G. & Rich, A. (1957). Biochim. biophye. Acta, 26, 457. Inman, R. B. (1964). J. Mol. BioZ. 9, 624. Lee-Huang, S. t Cavalieri, L. F. (1963). Proc. Nat. Acud. Sk., Wash. 50, 1116. Michelson, A. M., Massoulie, J. & Guschlbauer, W. (1967). In Progress in Nucleic Acid Research und Molecular Biology, ed. by J. N. Davidson & W. E. Cohn, vol. p. 6,83. New York: Academic Press. Miles, H. T. (1964). Proc. Nat. Acud. Sk., Wash. 51, 1104. Riley, M., Maling, B. & Chamberlin, M. J. (1966). J. Mol. Biol. 20, 369. Schachman, H. K., Adler, J., Radding, C. M., Lehman, I. R. & Kornberg, A. (1960). J. Biol. Chem. 235, 3442. Stevens, C. & Felsenfeld, G. (1964). Biopolymrs, 2, 293. Szer, TV. (1966). J. MOE. BioZ. 16, 585. Warner, R. (1957). Ann. N.Y. Acad. Sci. 69, 314. Yoneda, M. & Bollum, F. J. (1965). J. BioZ. Chem. 240, 3385. Zmudzka, B., Bollum, F. J. & Shugar, D. (1969). Biochemistry, 8, 3049.