16 NUCLEIC ACIDS Part (i) By G. Michael Blackburn (Department of Chemistry University of Sheffield Sheffield S3 7HF) “THATwas Molecular Biology that was.” Gunther Stent’s essay’ provides the backcloth against which two of the year’s highlights can be seen in perspec- tive. The Nobel Prize awarded2 to Holley Khorana and Nirenberg signifies again the experimental success of the ‘Academic Phase’ of nucleic acid studies. Watson’s ‘The Double Helix’3 records the termination of the ‘Romantic Phase’ both with the solution of the structure of DNA4 and by the exposition of the Central Dogma; it also implies in diverse ways that there can be occupa- tions more profitable than bench experimentation. The book3 has provoked reviews,’ counter reviews,6 and reviews of reviews,’ none of which appears to have noted that its dust jacket depicts a left-handed golden helix! The year’s achievements have included Khorana’s synthesis of a DNA duplex of 30 nucleotide residues the increase to eleven of the number of known t-RNA sequences and the crystallisation of both homogeneous and hetero- geneous t-RNA’s.Topics reviewed elsewhere include RNA from animal cells,8 sense and non- sense in the Gmetic Code,g and the molecular biology of viruses.” In uiuo codon assignments have been summarised” and the physical chemistry of polynucleotides has been the subject of a group of papers. l2This and doubtless other literature searches has been assisted by SCAN a computer-controlled search profile for nucleic acids published by the Chemical Society.Prebiotic Chemistry.-Many components of the genetic system can be made from simple precursors under mild conditions. Among the amino-acids glycine alanine serine and aspartic acid are most easily produced and ribose is formed G. S. Stent Science 1968 160 390. ’ Chum iii Britain 1968,4 557; Cheni. adEng. Ne1i.q 1968 46,NO. 46 66. J. D. Watson ‘The Double Helix,’ Weidenfeld and Nicholson London 1968. Abbreviations DNA deoxyribonucleic acid; RNA ribonucleic acid; t-RNA transfer (soluble) RNA; m-RNA messenger RNA; r-RNA ribosomal RNA; 5s-RNA RNA with sedimenta- tion coefficient of 5s; A adenosine; C cytidine; G guanosine; H hypoxanthine; I inosine; Y pseudouridine; T thymidine; rT ribothymidine; U uridine; U* uridine hydrate; dA deoxy- adenosine etc.; AMP CMP PA pC etc.5‘-phosphates; Ap Cp etc. 3‘-phosphates; poly-A polyadenylic acid ;d-pTpC etc. deoxyoligonucleotides. ’ Lord Todd,Chem. in Britain 1968 4 268; J. Hollander Nature 1968 218 791. A. Klug Nature 1968 219 808 880; L. D. Hamilton Nature 1968 218 633. J. Donohue Chem. in Britain 1968 4 468. J. E. Darnell Buct. Rev. 1968 32 262. A. Garen Science 1968 160 149. lo L. V. Crawford and M. P. G. Stoker (ed.) ‘The Molecular Biology of Viruses,’ C.U.P. London 1968; Symp. SOC.Gen. Microbiol 1968 18 125. H. Berger W. J. Brammar and C. Yanofsky J. Mol. Biol. 1968 34 219. l2 J. chim. Phvs.. 1968 65 No. 1. G.Michael Blackburn more readily than deoxyribose. Adenine which can be made from ammonium cyanide in liquid ammonia,14 is more reasonably formed in aqueous solutions of 4-aminoimidazole-5-carbonitrileand cyanide under conditions which could produce significant quantities of adenine and 4-aminoimidazole-5- carboxamide in a few years at -20 to 0".' Pyrimidines are less readily con- jured out of a 'primordial soup,' but a restricted reasonable route16 has been demonstrated from cyanoacetylene (1) via cyanovinylurea (2) to cytosine (3) and thence by hydrolysis to uracil (4).2HCNO HO HC =C-CN---OCN-C-NH.CH=CH.CNAH,N.C.NH.CH=CH-CN I1 II (1) 0 0 (2) (4) (3) There is still no convincing nucleoside synthesis under prebiotic conditions" but skirting this hurdle experiments on the phosphorylation of nucleosides have produced good yields of IMP from isopropylideneinosine and tri-n- butylammonium phosphate in non-aqueous solvent by the action of U.V.radiation. '* In dilute aqueous solutions the stable enol phosphate formed by addition of phosphate to cyanoacetylene slowly converts uridine into UMP.' Solid-state phosphorylation isanother prebiotic possibility :uridine and uridine- 2'(3')-phosphate at 160" yield a mixture of products including a significant amount of UpUpU 35 % of which has natural (3' + 5') linkages2' Greater preference is observed in UpUpU than in UpU formed for the (3'-,5') linkage ; this may be a clue to its evolution. Given poly-U as a template (another step in the dark) it is possible to achieve nucleotide condensations in aqueous solution by use of water-soluble carbodi- imides2 or phosphorimidazolides22 as condensing reagents.Three features l3 C. Ponnamperuma and N. W. Gabel Space Life Sciences 1968,1 65. l4 Y. Yamada I. Kumashiro andT. Takenishi J. Org. Chem. 1968,33,642. l5 R. A. Sanchez J. P. Ferris and L. E. Orgel J. Mol. Biol. 1968,38 121. l6 J. P. Ferris R.A. Sanchez and L. E. Orgel J. Mol. Biol. 1968,33 693. '' C. Reid L. E. Orgel and C. Ponnamperuma Nature 1968,216,936. '* Y. Sanno and A. Nohara Chem. and Pharm. Bull. (Japan) 1968,16,2056. l9 J. P. Ferris Science 1968 161 53. 2o J. MorBvek J. Kopeck$ and J. Skoda CON.Czech. Chem. Comm. 1968,33,960. 21 J. Sulston R.Lohrmann L. E. Orgel and H. T. Miles Proc. Nar. Acad. Sci. U.S.A. 1968 60 409. 22 B. J. Weimann R.Lohrmann L. E. Orgel H. Schneider-Bernloehr and J. E. Sulston Science 1968 161 387.Nucleic Acids 537 are noteworthy first the process is catalytic23 and enhances the rate of phosphodiester formation by an order of magnitude; second the system is species selective-poly-U facilitates only the homocondensation of adenosine units but not heterocondensation with C G or U; and third the phosphate linkage formed between A and pA is 96% (2‘ - 5‘) and that between d-pA and dA is mainly (5’ - 5’).24 Why did evolution select for (3‘ - 5’) phosphate esters?25 This and other speculative matters are expertly and lengthily dis- cussed by Crick,26 Orge1,27 and Woese.28*29 The clear conclusion is that selection among tenable but conflicting hypotheses cannot proceed without more facts. The whole problem of translation of information into control involves the interaction between nucleic acid bits and amino-acids three aspects of which are under scrutiny.Ralph3’ argues that interaction of amino-acid and its anticodon should involve the recognition of the aminoacyladenylate by the appropriate t-RNA-hence an explanation for the invariant location of U next to the anticodon (pp. 554-559). Next the binding of nucleotides to poly- peptides shows3’ that poly-L-arginine binds pG more effectively than PA pC or pU. Lastly Gabba~~~ finds that double helices of poly-I poly-C are thermally stabilised by interaction with lysyl dipeptides or aminoacyldiamines. Dilysine is ten times more effective than lysine and L-amino-acids are superior to their D-enantiomers. Alongside this chiral specificity should be noted the partial resolution of DL-adenosine by template-controlled condensation with D-adenosine-5’-phosphorimidazole.33 These two developments appear to simplify the problem of chiral evolution in the biosphere.Chemistry of Bases.-Transfer RNA continues to be a good source of new minor bases. 2-Thiocytosine (5) and 5-methylaminomethyl-2-thiouracil(6) have been isolated34 as their 2’(3’)-nucleotides from t-RNAE. coli 5-carboxymethyl-(7) and 5(6)-carbomethoxymethyl-2-thiouraci136(8) nucleotides have been identified as products from t-RWAYeast and 2’-0N(4)-dimethylcytidine 23 J. Sulston R. Lohrmann L. E. Orgel and H. T. Miles Proc. Nut. Acad. Sci. U.S.A. 1968,59 726. 24 H. Schneider-Bernloehr R. Lohrmann J. Sulston B.J. Weimann L.E. Orgel and H. Todd Miles J. Mol. Biol. 1968 37 151. 25 Anon. Nature 1968 218 523. 26 F. H. C. Crick J. Mol. Biol. 1968,38 367. ” L. E. Orgel J. Mol. Biol. 1968 38 381. 28 C. R. Woese Proc. Nut. Acad. Sci. U.S.A. 1968 59 110. ” See also M. Nei Nature 1969 221,40; J. E. Edstrom ibid. 1968 220 1196; D. C. Reanney and R. K. Ralph J. Theoretical Biol. 1968 21,217. 30 R. K. Ralph Biochem. Biophys. Res. Comm. 1968,33,213. 31 K. G. Wagner and R. Arav Biochemistry 1968 7 1771. 32 E. Gabbay and R. Kleinman J. Amer. Chem. SOC.,1968,90,7123; E. J. Gabbay R. Kleinman and R. R. Shimshak ibid. p. 1927. 33 H. Schneider-Bernioehr R. Lohrmann L. E. Orgel J. Sulston and B. J. Weimann Science 1968,162,809. 34 J. Carbon H. David and M. H.Studier Science 1968,161 1146. 35 M. W. Gray and B. G. Lane Biochemistry 1968 7 3441. 36 L. Baczynskyj K. Biemann and R. H. Hall Science. 1968. 159. 1481. G. Michael Blackburn is found in 16s-RNA from E. ~oli.~’ N(6)-isopentenyl-2-The cyt~kinin,~~ methylthioadenosine (9) isolated from crude t-RNA and ~ynthesised,~~ originates in t-RNAz:o,i.40 Cultures of L. aciduphilus but not of yeast incor- porate [2-14C]mevaIonic acid into N(6)-isopentenyladenosine (lo) which is a minor base in several t-RNA species (pp. 554-559).41 Sfj H (9) R = MeS (7) (8 (10) R = H r = P-D-ribofuranosyl 4-Thiouracil residues in t-RNA are selectively modified by eth~leneimine~~ and by N-eth~lmaleimide,~~ and the reduction of 4-thiouridine dihydrouridine and N(4)-acetylcytidine with sodium r3H1borohydride gives characteristic products from each; this permits estimation of these bases in t-RNA.44 Such reduction of t-RNA,,,,, without further degradation produces changes in biological activity.45 In contrast to the random methylation of bases by dimethyl s~lphate,~~ a purified enzyme catalyses methylation of certain base sequences in methyl- 37 J.L. Nichols and B. G. Lane Biochim. Biophys. Acta 1968 166 605. 38 J. P. Hegelson Science 1968 161 974. 39 W. J. Burrows D. J. Armstrong F. Skoog S. M. Hecht J. T. A. Boyle N. J. Leonard and J. Occolowitz Science 1968 161 691. 40 F. Harada H. J. Gross F. Kimura S. H. Chang S. Nishimura and U. L. RajBhandary Biochem. Biophys. Res. Comm. 1968,33 299. 41 A.Peterkofsky Biochemistry 1968 7 472; F. Fittler L. K. Kline and R. H. Hall ibid. p. 940. 42 B. R. Reid Biochem. Biophys. Res. Comm. 1968,33,627. 43 J. Carbon and H. David Biochemistry 1968 7 3851. 44 P.Cerutti J. W. Holt and N. Miller J. Mol. Biol. 1968 34 505. 45 P. Cerutti Biochem. Biophys. Res. Comm. 1968 30,434; M. Molinaro L. B. Sheiner F. A. Neelon and G. L. Cantoni J. Biol.Chem. 1968 243 1277. 46 R.A. Zakharyan T. V. Venkstern and A. A. Bayev Biokhimiya 1967 32 1068. Nucleic Acids 539 deficient t-RNA and nucleotides Ap2MeGPCp Gp1MeApApUp and Apl MeApApUp have been ~haracterised.~~ Much effort in cancer research is currently focussed on the alkylation of DNA and RNA both in uitro and in uiuo. Carcinogens which effect such alkyla- tion include N-nitro~odimethylamine,~~’*~ N-methyl-N-rnethyl~rethane,~~ N-nitroso-toluene-p-sulphonamide,so N-methyl-N-nitroso-N’-nitroguani- dine,49-ethyleneiminium nitrogen mustards,53 sulphur mustards methyl methanesulphonate and dimethyl s~lphate,~~‘? and N-hydroxyfluoren- 2-ylacetamide.’’Although the carcinogenic polycyclic aromatic hydrocarbons are not recognised alkylating agents they bind to DNA after incubation and treatment with hydrogen peroxide.56 Similar binding has been observed to result under the influence of U.V. irradiati~n.~~ Since it is known that the K-region epoxides have little carcinogenic power,58 the possibility that aromatic peroxides are the intermediates in DNA alkylation by these hydrocarbons must be considered.With the exception of N-hydroxyfluoren-2-y1acetamide,’ all alkylations of hydrogen-bonded base pairs give predominantly 7-alkylguanosinet Bi-functional sulphur mustards produce bis-2-(7-guanyl)ethyl sulphide (1 1). 54a Lesser amounts of 1- and 3-alkyladenosines and 1-methylcytidine are found.48b Organ specificity is exhibited by certain reagents ;dimethyl sulphate acts mainly on brain cells and dimethylnitrosamine can alkylate up to 1% of G residues in rat liver RNA but is ten times less active on kidney nucleic Some of these results are consistent with the hypothesis that there is a casual relationship between alkylation of DNA and carcin~genesis,~~~ but firm con- clusions are as yet premature. However in one case at least there is good evi- dence for a direct relati~nship.~’ After treatment of the skin of a mouse with P-propiolactone the initiation of tumourigenesis correlates well with covalent binding of the p-lactone to skin DNA.Isolated DNA yields 7-(2-carboxyethyl)- guanine (12) on hydrolysis. Two pyrimidine ring substitution reactions have been investigated. Uracil 47 B. C. Baguley and M. Staehlin Biochemistry 1968 7. 45. 48 (a)P. D. Lawley P. Brooks P. N. Magee V. M. Craddock and P. F. Swann Biochim. Biophys. Actu 1968 157 648; (b)W. Lijinsky J. Loo,and A. E. Ross Nature 1968 218 1174. 49 P. D. Lawley Nature 1968 218 580. R. R. McCalla Biochim. Biophys. Actu 1968 155 114. P. Chandra A. Wacker R. Sussmuth and F. Lingens 2.Nuturforsch 1967 22b 512; V. M. Craddock Biochem. J. 1968 106,921 ;F.Lingens J. Rau and R. Sussmuth Z. Nuturforsch. 1968 23b 1565; B. Singer H. Frankel-Conrat J. Greenberg and A. M. Michelson Science 1968 160 1235. 52 C. C. Price G. M. Gaucher P. Koneru R. Shibakawa J. R. Sowa and M. Yamaguchi Biochim. Biophys. Acta 1968 166 327. s3 K. W. Kohn and C. L. Spears Biochim. Biophys. Actu 1967 145 734. 54 (a)P. D. Lawley and P. Brookes Biochem. J. 1968,109,433;(b)P. F. Swann and P. N. Magee ibid. 110 39. 55 C. M. King and B. Phillips Science 1968 159 1351. 56 C. E. Morreal T. L. Dao K. Eskins C. L. King and J. Dienstag Biochim. Biophys. Actu 1968,169,224. ” P. 0.P. Ts’o and P. Lu Proc. Nut. Acad. Sci. U.S.A. 1964 51,272. J. A. Miller and E. C. Miller Lab. Invest. 1966 15 226. 59 N. H. Colburn and R. K. Boutwell Cancer Res.1968 28 642. G.Michael Blackburn II?-.X JL ““Y2 Q (13) (14) and uridine rapidly exchange hydrogen at C-5 in acid6’ and in alkaline6‘ solu-tion. An examination of the mechanism for 2-hydroxyprimidine and its deri- vatives suggests that hydrogen exchange is consequent upon hydration of the 5,6-double bond.62 This is supported by hydrogen exchange in the photo- hydrate of UMP6 and of cytidine. 64 0(6),5’-Cyclonucleosidesare produced from 5-halogenopyrimidine nucleosides with strong base.65 Thus 5-iodouri- dine (13) readily obtained by iodination of uridine,66 is converted into 1-[0(6),5‘-cyclo]-p-D-ribofuranosylbarbituric acid (14) with potassium t-but- oxide.67 Both addition4imination and heteryne mechanisms have been con- sidered but the observed68 base-catalysed exchange of C-6 hydrogen in 5-fluoropyrimidine nucleosides seems best interpreted in favour of the latter mechanism.Nucleosides and Nucleotides-Much synthetic work continues to be directed at the modification of nucleosides in either the base or the sugar fragment. The 2’-C-methyL6’ and 3’-C-methyl-ribo~ides~~ and 3’-deoxy-3’- C-hydroxymethyl-erythro-furanoside”of adenine have been prepared and 5’-homothymidine has been made by extension of the sugar.72 An elegant syn- thesis of the carbon isostere of UMP 6’-deoxyhomouridine-6’-phosphonic “ K. Kusama J. Biochem. (Japan) 1968 63 561. S. R. Heller Biochem. Biophys. Res. Comm. 1968 32 998. A. R. Katritzky M. Kingsland and0. S. Tee J. Chem. SOC.(B),1968 1484.63 R. W. Chambers J. Amer. Chem. SOC.,1968,90,2192. 64 L. Grossman Photochem. and Photobiol. 1968 7,727. 65 B. A. Otter E. A. Falco and J. J. Fox Tetrahedron Letters 1968,2961. 66 H. Yoshida J. Duval and J.-P. Ebel Biochim. Biophys. Acta 1968,161 13. 67 D. Lipkin C. Cori and M. Sano Tetrahedron Letters 1968 5993. R. J. Cushley S. R. Lipsky and J. J. Fox Tetrahedron Letters 1968 5393. 69 S. R. Jenkins B. Arkon and E. Walton J. Org. Chem. 1968,33,2490. 70 R. F. Nutt M. J. Dickinson F. W. Holly and E. Walton J. Org. Chem. 1968,33 1789. 71 E. J. Reist D. F. Calkins and L. Goodman J. Amer. Chem. SOC.,1968,90,3852. ’I2 G. Etzold G. Kowollik and P. Langen Chem. Comm. 1968,422. Nucleic Acids 541 acid (16) has been accomplished in four stages from 5’-aldehydo-2,3’-0-iso- propylideneuridine (15).The AMP analogue can also be made by this route.73 0 Reagents:i Ph,P=CH*PO(OPh) ;ii Pd-BaS0,-H ;iii PhCH,.ONs; iv Pd-H,-H+ The three nucleotide antibiotics toyokamycin (17) sangivamycin (18) and tubercidin (19) have been ~ynthesised~~ and new pyrimidine nucleoside anti- biotics have been identified. Polyoxin A and B,75active components of agri-cultural antifungal agents have the unique structures (20a and 20b). Gougero- tin has the revised structure (21).76 \ (17) R = CN (18) R = CONH (19) R = H r = P-D-ribofuranosyl 0 CH,OH L &H OH (20b) R = €€ 73 G. H. Jones and J. G. Moffatt J. Amer. Chem. Soc. 1968 90 5337. 74 R. L. Tolman R. K. Robins and L.B. Townsend J. Amer. Chem. SOC.,1968,90 524. 75 K. Isono and S. Suzuki Tetrahedron Letters 1968 1133. 76 J. J. Fox,Y. Kuwada and K. A. Watanabe Tetrahedron Letters 1968 6029. 542 G. Michael Blackburn A synthesis of pseudouridine (22) and of 5-p-D-ribofuranosyluridine(23) has been described,77 though the latter product does not appear to be identical with the naturally occurring material.78 A related synthesis of 6-azapseudouridine failed when the ribityl fragment in (24) cyclised in an unexpected fashion given (25).79 A novel preparation of purine nucleosides provides a-anomers virtually free of the p-isomers :N(6)-octanoyladenine is heated with the boron trichloride complex of methyl ribofuranoside in chloroform. a-Adenosine is obtained after deacylation with sodium methoxide.80 (22) R = H (25) (23) R = P-D-ribofuranosyl The readily available nucleoside thiophosphates promise to be good probes for many enzyme reactions.Thus adenosine-5‘-thionophosphateis a poor substrate for enzymic deamination or phosphorolysis but as an activator for phosphorylase b it is more effective than AMP.81 The introduction of one thio- group into the valine codon GpUpU markedly lowers its capacity to bind t-RNAVa’ to ribosimes.82 Uridine-2’,3’-cyclothiophosphate(26) is hydrolysed in alkali to uridine-2’(3’)-thiophosphate,whereas acid hydrolysis results in significant loss of sulphur.83 The diastereoisomers of (26) can be separated by crystallisation one binds to ribonuclease as strongly as does uridine cyclic phosphate but both are hydrolysed by the enzyme more slowly than the natural substrate.The complete retention of sulphur in the product has strong implica- tions for the stereochemistry at phosphorus in the mechanism of action of ribon~clease.~~ Oligonucleotides.-Well established methods of synthesis leave some room for improvement. A third oligonucleotide synthesis using phosphotriesters has been reported phenyl phosphate triesters can be hydrolysed by alkali but are stable under milder conditions appropriate for the removal of 0-acetyl groups.85 One of the abiding problems in ribonucleotide synthesis is the pro- pensity of (3’ + 5‘) phosphate diesters to isomerise into (2’ + 5’) linked nucleo- 77 D. M. Brown M. G. Burdon and R.P. Slatcher J. Chem. SOC.(C) 1968 1051. A. W. Lis and E. W. Lis Fed. Proc. 1964 23 532. ’’ M. Bobek J. FarkaS and F. Sorm,Tetrahedron Letters 1968 1543. Y. Furukawa K. Imai and M. Honjo Tetrahedron Letters 1968 4655. A. W. Murray and M. R. Atkinson Biochemistry 1968 7 4023. V. LisL F. Eckstein and J. Skoda CON. Czech. Chem. Comm. 1968,33,2734. 83 F. Eckstein and H. Gindl Chem. Ber. 1968 101 1670. 84 F. Eckstein F.E.B.S. Letters 1968 2 85. 85 C. B. Reese and R. Saffhill Chem. Comm. 1968,767. Nucleic Acids 543 tides. This can be avoided successfully by careful selection of the conditions for removal of protecting groups. 86 Terminal phosphomonester functions also require special consideration 3’-phosphates are best generated from their benzyl esters87 and 5’-phosphates can be produced by selective cleavage of trichloroethyl esters88 or through removal of a terminal nucleoside protecting group by means of periodate oxidation and p-elimination.89 Nonetheless it is obvious that oligoribosides will continue to be made best by enzymic transcription of synthetic DNA. Khorana’s progress towards gene synthesis already appears irresistible. The methods of synthesis described in last year’s report” have led to the synthesis9 of the eicosadeoxynucleotide (27) by the block condensation of an appropriately protected oligonucleotide with a free 3’-hydroxy-group and 5’-phosphates of tri- and tetra-nucleotides as shown in the Scheme. Khorana considers this to be the maximum chain length that current methods of chemical synthesis and purification allow for deoxynu~leotides.~~ Similar methods are MMTrGpApA + pCpC -MMTrGpApApCpC PGPGPA I MMTrGpApApCpCpGpGpApGpApCpT-pGpApCpT MMTrGpApApCpCpGpGpA PCPTPAPC I MMTrGpApApCpCpGpGpApGpApCpTpCpTpApC PCPAPTPG I MMTrGpApApCpCpGpGpApGpApCpTpCpTpApCpCpApTpG (27) 86 B.E. Griffin M. Jarman and C. B. Reese Tetrahedron 1968,24,639; B. E. Griffin and C. B. Reese ibid. p. 2537; H. P. M. Fromageot C. B. Reese and J. E. Sulston ibid. p. 3533. 87 F. Cramer and G. Schneider Annalen 1968 717 193. A. Franke F. Eckstein K.-H. Scheit and F. Cramer Chem. Ber. 1968 101,944. 89 F. Kathawala and F. Cramer Annalen 1968 712 195. 90 G. M. Blackburn and M. J. Waring Annual Reports 1967 B 64,479. 91 N.K. Gupta E. Ohtsuka V. Sgaramella H. Biichi A. Kumar H. Weber and H. G. Khorana Proc. Nat. Acad. Sci. U.S.A. 1968 60,1338. 92 H. G. Khorana Biochem. J. 1968 109 709. 544 G. Michael Blackburn used to make the second eicosanucleotide which has ten bases complementary to the first and is of opposite polarity so that together (28) the two oligomers span the length of residues 21-50 of the gene for t-RNA$is (p. ). This molecular duplex has two 'sticky' ends which can complex with an added complementary oligonucleotide. Thus the nonanucleotide (29) com-bines with (28) to form a stable trimolecular complex which 'melts' at 42". This is effectively a segment of a DNA helix with one single-strand break and application of the ligase ('sealing' enzyme) from bacteriophage T4 joins together the eicosa- and nona-nucleotides to give a molecule of 29 nucleotide units.A similar operation after the addition of the heptanucleotide (30) gives the DNA duplex (31). The success of these operations is monitored first by the incorpora- tion of the 32P-label from (29) into the product second by nearest neighbour analysis and third by addition of the nucleotide residues complementary to the single-strand termini of (31) by use of DNA polymerase from E. coli. Thus the dC residue at the right-hand end of (31) and the dA dG and dC at the other are specifically added by the polymerase enzyme to complete the thirty-base double-stranded DNA molecule. Detailed experiments show that a 'cohesive' end of 5 or 6 deoxyribonucleo-tide residues provides sufficient overlap for the ligase extension procedure.93 Thus blocks of decanucleotides can be added step by step to elongate either end of the molecule and leave a new cohesive end the addition of a pentanuc- leotide d-CpTpApApG to (28)can be followed by the further extension of the left-end by use of decanucleotides corresponding to t-RNAA'" sequence 46-55 then 51-60 and so on to completion of the gene.Using very similar techniques a second group is intent on the synthesis of the gene corresponding to chain A of bovine insulin.94 Thus far the protected duodecanucleotide d-pTpTpApApTpTpApCpApApTpA has been prepared. Syntheses on polymer supportsg have been comparatively unsuccessful measured against the achievements in peptide synthesis.The best support for nucleotide studies appears to be a highly cross-linked rigid polystyrene- divinylbenzene copolymer. Even with this the efficiency of addition of monomers falls alarmingly by the third or fourth deoxynucleotide residue.96 Nevertheless cellulose-bound polynucleotides have opened some interesting opport~nities.~' Their use as solid-state primers for enzymic polymerisations includes the synthesis of a polymer-bound single-strand DNA complementary to a given soluble single-strand DNA.98 Details of sequence determination are described elsewhere [Part (ii)] but the potential application of mass spectrometry for this purpose is heralded by 93 N. K. Gupta E. Ohtsuka H. Weber S. H. Chang and H. G. Khorana Proc. Nut. Acad.Sci. U.S.A. 1968,60,285. 94 S.A.Narang S. K. Dheer and J. J. Michniewicz,J. Amer. Chem. SOC. 1968 90 2702. 95 T. Shimidzu and R. L. Letsinger J. Org. Chern. 1968,33 708. 96 F. Cramer and H. Koster Angew. Chem. Internut. Edn. 1968 7 473. 97 P. T. Gilham Biochemistry 1968 7,2809. 98 T. M. Jovin and A. Kornberg J. Biol. Chem. 1968 243 250. SCHEME G-T-A-C -C-C-T-C-T-C-A-G-A-G-G-C-C-A-A-G-OH 5’ IIIIIIIIII S’HO-G-C-T-C-C-C-T-T-A-G-C -A-T-G-G-G-A-G-A-G 3’HOG-G-G-A-A-T-C pT-C -T-C-C-G-G-T-T-OH 3‘ ‘i‘ (30) ligase (29) .................... t i C G A i G-G-G-A-A-T-C-G-T-A-C-C-C-T-C-T-C-A-G-A-G-G -C-C-A-A-G z-b .................... I I I I I I I I I I I I I I I I I I I I I I I I I I...... G~-T-C-C-~-T-T-A-G-C-A-T-G-G-G-A-G-A-G-T~-T-€-C-G-G-T-T i......C i (31) MMTr = 5’-monomethoxytrityl. Amino-groups are protected by acylation with anisoyl for C benzoyl for A and isobutryl for G respectively. The 3‘-OAc at the end of each new block is removed by hydrolysis before condensation of the next block. (The phos- phates are omitted from deoxynucleotides (28H31) for clarity.) G. Michael Blackburn studies on purines and pyrimidine^,^' nucleosides and nucleotides,'" and dinucleoside phosphates. lo' Photochemistry.-Sensitised photochemistry has clarified the mechanism of pyrimidine dimer formation. Cytosine uracil thymine and orotic acid all exhibit triplet state dimerisation in solution. '02 Dimethylthymine gives all four cyclobutane products on photosensitised dimeri~ation."~ A detailed study of the solvent effects on the relative yields of the four and the influence of triplet quenching additives suggests a complex pattern of behaviour in which all dimers are formed independently from both singlet and triplet states with different adducts showing a preference for the one or the other.'04 The singlet pathway becomes important at high concentration of monomer.'03 Thymine dimers frozen in a glass at 80°K can be monomerised by irradiation at 248 nm. Subsequent irradiation at 280 nm. reforms dimers with high quantum yield and with quenching of monomer fluorescence. This ingenious experi- ment105 suggests a mechanism for dimerisation in which a singlet excimer is formed involving two suitable oriented thymines some 2.8 A apart which collapses to a cyclobutane dimer before fluorescent emission.The properties of all four thymine dimers have been surveyed.'06 X-Ray diffraction structures are available for cis-syn dimers of dimethylthymine'07 and uracil.lo8 Whereas the major thymine photoproduct from native DNA is the cis-syn dimer," irradiation of denatured DNA yields lesser amounts of a second dimer identified as the trans-syn isomer (32) which is not cleaved by the photoreactivating enzyme. '09 Although the cyclobutane dimers have received 0 0 Y-NH 02% KNH 0 0 (3 2) (33) 99 M. H. Studier R. Hayatsu and K. Fuse Analyt. Biochem. 1968 26 320. loo J. A. McCloskey A. M. Lowson K. Tsuboyama P. Krueger and R. N. Stillwell J. Amer. Chem. Soc. 1968,90,4182. lo' D.F. Hunt C. E. Hignite and K. Biemann Biochem. Biophys. Res. Comm. 1968 33 378. lo' C. L. Greenstock and H. E. Johns Biochem. Biophys. Res. Comm. 1968,30 21. H. Morrison A. Feeley and R. Kleopfer Chem. Comm. 1968,358. lo4 H. Morrison and R. Kleopfer J. Amer. Chem. SOC.,1968,90,5037. lo5 A. A. Lamola and J. Eisinger Proc. Nat. Acad. Sci. U.S.A. 1968 59 46. lo6 M. A. Herbert J. C. LeBlanc D. Weinblum and H. E. Johns Photochem. and Photobiol. 1968 9 33. lo' N. Camerman and A. Camerman Science 1968 160 1451. Io8 E. Adman M. P. Gordon and L. H. Jensen Chem. Comm. 1968 1019. log E. Ben-Hur and B. Ben-Ishai Biochim. Biophys. Acta 1968 166 9. Nucleic Acids 547 most attention a minor photoproduct of thymine' lo is assigned structure (33) and on heating is dehydrated to give (34).The pyrimidine photohydrates whch are formed from singlet excited states,'" have had their structures confirmed both by n.m.r. analysis' l2 and by chemical degradation of both cytidine and uridine hydrates. '' The transient formation of unstable cytidine hydrates can be assayed by reaction with hydroxy- lamine to give N(4)-hydroxycytidine ;'l4 photoreduction of thymine and uracil dimers with sodium [3H]borohydride provides a radioisotope marker for photodimerisation. ' ' The mechanisms of photoreduction and hydrogenolysis of pyrimidine nucleosides and their photoproducts have been reviewed. 'l6 Much recent work on the molecular biology of U.V. lesions in DNA has been surveyed.'' While photohydrates are primarily mutagenic (p.548) pyrimidine dimers appear to be less lethal than was thought. In a population of E. coli cells which lack the enzyme for repairing U.V. damage a dose of U.V. radiation sufficient to create 50 pyrimidine dimers in each DNA molecule only kills 63% of the bacteria. Experiments indicate that the DNA of the survivors' progeny has gaps in one strand but that these defects slowly disappear during incubation. ''*Other studies demonstrate that thymine dimers in bacterio- phage DNA can be handed on efficiently from parent to progeny under non- repair conditions and suggest that fewer than one in five thymine dimers is lethal.' '' Pulse radiolysis of uracil'20 and thymine12' in aqueous solution causes the addition of hydroxyl radicals to the pyrimidine.Thymine forms the tertiary radical which in the presence of oxygen is converted into 5-hydroperoxy-6- hydroxydihydrothymine (35). The use of hydroxyl scavengers shows that while HO' and H' react destructively with thymine the solvated electron does not.'22 y-Radiolysis of solutions of AMP produces the unusual cyclonucleoside phos- phate (36).'23 Base Pairing and Stacking.-The specificity of base pairing through hydrogen bonds is most simply observed in non-aqueous solvents. Dimethyl sulphoxide appears to be an excellent choice and n.m.r. investigations of cytidine and guanosine give value of -6 kcal. M-' for the enthalpy of base pairing while showing no evidence of base stacking.'24 A similar value has been measured 'Io A. J. Varghese and S.Y. Wang Science 1968 160 186. '" I. H. Brown and H. E. Johns Photochem. and Photobiol. 1968,8 273. '" W. J. Wechter and K. C. Smith Biochemistry 1968 7 4064. 'I3 N. Miller and P. Cerutti Proc. Nat. Acad. Sci. U.S.A. 1968 59 34. G. D. Small and M. P. Gordon J. Mol. Biol. 1968,34 281. T. Kunieda L. Grossman and B. Witkop Biochem. Biophys. Res. Comm. 1968,33,453. '16 B. Witkop Photochem. and Photobiol. 1968 7 813. I" Photochem. and Photobiol. 1968 7 578 ff. ''13 W. D. Rupp and P. Howard-Flanders J. Mol. Biol. 1968 31 291. 'I9 W. Sauerbier and M. Hirsch-Kauffmann Biochem. Biophys. Res. Comm. 1968 33 32. R. M. Danziger E. Hayon and M. E. Langmuir J. Phys. Chem. 1968,72 3842. P. T. Emmerson and R. L. Willson J. Phys. Chem. 1968 72 3669. H. Looman and J.Blok Radiation Res. 1968 36 1. '" K. Keck 2.Naturforsch. 1968 23b 1034. R. A. Newmark and C. A. Cantro J. Amer. Chem. SOC. 1968 90 5010. G. Michael Blackburn ..XoH OL OH (35) for the interaction of 9-ethyladenine and 1-cyclohexyluracil in pure chloro- form.12' This pairing is weakened by the presence of ethanol in the solvent and base pairing between monomers appears to be excluded in aqueous solution.126 There is little evidence for complexing between di- tri- or tetra-nucleotides and their complements in water. The formation of a 2:l complex between GpGpC and GpCpC is possibly non-specific since GpGpC self-aggregates strongly.127 This is interpreted to indicate that codon :anticodon interactions must be augmented by ribosome and t-RNA interactions.The biological effects of barbiturates may relate to the observation that 9-ethyladenine forms specific hydrogen-bonded complexes with phenobar- bital barbital and thiopental which are much more stable than its complex with l-cyclohexyluracil.128 The X-ray structure of a 2 1 complex of 8-bromo- g-ethyladenine and phenobarbital shows Watson-Crick hydrogen-bonding between bases. 12' The tautomeric constant for N(4)-hydroxycytosine derivatives (37) favours the imino-form (38) for N(4)-aminocytosine the amino-form (39) is more stable than the imino-form (40). Thus the mutagenic action of hydroxylamine on DNA can be identified with the transformation of a cytosine residue (41) which base pairs with guanine into an N(4)-hydroxycytosine unit (38) which tautomerically resembles uracil and so will base-pair with adenine.On repli- cation this change will result in progeny DNA containing an A residue where the parent had a G.130 Since modification of (41) to (39) by hydrazine does not involve a tautomeric change hydrazine would be predicted to be an indifferent mutagen-as observed. Changes in base pairing consequent on the photohydration of pyrimidines are environment dependent. in vitro Polypeptide synthesis shows that uracil hydrate U* codes as cytosine when it is in the first position in a codon triplet but that in the centre of the triplet it codes as C with only 20 % efficiency. A striking example of this change in base pairing is provided by the reversal 12' J. S. Binford and D.M. Holloway J. Mol. Bol. 1968 'l 91. 126 T. N. Solie and J. A. Schellman J. Mol. Biol. 1968 33 61. S. R. Jaskunas C. R. Cantor and I. Tinoco Biochemistry 1968 7 3164. 12* Y. Kyogoku R. C. Lord and A. Rich Nature 1968,218 69. S.-H. Kim and A. Rich Proc. Nut. Acad. Sci.,U.S.A.,1968 60,402. 130 D. M. Brown M. J. E. Hewlins and P. Schell J. Chern. SOC.(0,1968 1925. F. P. Ottensmeyer and G. F. Whitmore J. Mol. Biol. 1968,38 1. Nucleic A cids 549 (37) R = OH (38) R = OH (39) R = NH (40)R = NH2 (41)R = H of a lethal mutation with U.V. irradiation. Bacteriophage h containing an amber mutation infect a host bacterium and produce m-RNA which contains a nonesense codon UAG. This has the effect of terminating protein synthesis at the amino-acid of the codon preceding UAG-with lethal consequences for the phage.A dose of U.V. radiation a short time after the phage has begun to make the defective m-RNA converts some of the nonesense triplet UAG into the hydrated form U*AG. This behaves in protein synthesis like CAG and codes for glutamine. Thus normal protein synthesis is restored and the bacteriophage multiply.' 32 An investigation of the kinetics of base pairing can determine whether the rate of certain biological processes is limited by this factor. T-Jump methods'33 show that the rates of formation of dimers U :A U :U and A :A are essentially diffusion controlled. The different stabilities of these three complexes are governed by different rates for their dissociation. The kinetics of base pairing and co-operative helix formation have been measured for oligomers from tri- to deca-nucleotides giving time constants from to sec.134 The rate of combination of poly-A and poly-U to give a double helix decreases with increasing temperature and reaches zero 0.2" below the 'melting' temperature of the complex.However the rate of dissociation of the duplex is complicated and exhibits a minimum value at T,.13' This behaviour requires a revision of the mathematical models for stacking and suggests that the requirement for helix growth is a stable nucleus of more than one base pair. Single-strand stacking in ribotrinucleotides shows that stability depends not only on the nature of the bases involved but also on their sequence.'36 Thus UpApGp stacks better than ApUpGp.Purines tend to stack better than pyrimidines; this fact has led to the prediction that hairpin turns in single- stranded RNA should beexpected in pyrimidine rather than in purine regions. 137 The interactions of polymers with monomers are not as simple as first appeared. Adenosine interacts with poly-U below 26" to give a rigid A:2U complex probably involving co-operative hydrogen-bonding and A-A F. P. Ottensmeyer and G. F. Whitmore J. Mol. Biol. 1968,38 17. 133 G. G. Hammes and A. C. Park J. Amer. Chem. Soc. 1968 90,4151. 134 M. Eigen J. chim. Phys. 1968 65 53. 13' R. D. Blake L. C. Klotz and J. R. Fresco J. Amer. Chem. Soc. 1968,90 3556. S. Aoyagi and Y. Inoue J. Biol.Chem. 1968 243 514. 137 R. C. Davis and I. Tinoco Biopolymers 1968 6 223.G.Michael Blackburn base stacking stabilisation. Above 26" there is non-co-operative base stacking with some intercalation of adenosine into the poly-U.'38 Intercalation is best seen in n.m.r. studies of poly-U and purine interactions-where no hydrogen- bond base pairing is p~ssible.'~' Other work shows that 3,4-benzpyrene intercalates between adjacent bases in the DNA helix. Finally the complexes formed at low temperature betwen poly-U and adenine nucleotides are in- soluble in water. 141 While this phase-transition contributes to the stability of the complex and provides a useful mechanism for prebiotic sequestration of AMP it impedes further study of these interesting complexes. 13* B. W. Bangerter and S. I. Chan Proc.Nat. Acad. Sci.U.S.A.,1968,650 1144. 139 B. W. Bangerter and S. I. Chan Biopolymers 1968 6,983. S. A. Lesko,A. Smith P. 0.P. Ts'o and R. S. Umans Biochemistry 1968 7 434. 141 P. 0.P. Ts'o and M. P. Schweizer,Biochemistry 1968,7,2963;P. 0.P. Ts'o and W. M. Huang ibid. p. 2954.