Nucleic Acids Research

To explain biochemical and aenetic data on spontaneous nucleotide replacements in nucleic acid biosynthesis all the 8 mispairs in normal tautomeric forms have been considered. Possible B-conformations of DNA fragments containing each of such mispairs incorporated between Watson-Crick pairs have been found using computations of the energy of non-bonded interactions via classical potential functions. These conformations have no reduced interatomic contacts. The values of each dihedral angle of the sugar-phosphate backbone fal1 within the limits of those of double-helical fragments of B-DNA in crystals. These values differ from those of the corresponding angles for the low-energy polynucleotide conformations consisting of canonical pairs by no more than 30 (except for the fragment with the U:U pair for which the C^-Cj-O-P angle differs by about 50°). The difference in experimentally observed frequencies of various nucleotide replacements in DNA biosynthesis correlates with the difference in the energy of non-bonded interactions and with the extent of the sugar-phosphate backbone distortion for the fragments containing the mispairs which serve as intermediates for the replacements. INTRODUCTION Almost exclusively complementary (i.e. those forming A:U (T) or G:C pairs) nucleotides are incorporated into the newly synthesized chain in nucleic acid biosynthesis. The formation of any other pair is an error leading to a nucleotide replacement: transition, if a purine-pyrimidine mispair is formed, or transversion, if a purine-purine or pyrimidine-pyrimidine mispair is formed. Errors in DNA biosynthesis in vivo occur with a probability of 10 -10 per base pair per replication (1). Complex enzyme systems ensure extremely high accuracy in vi tro, too (2). To understand the processes of replication, repair and transcription and to be able to influence these processes (e.g. in chemeotherapy) it is important to elucidate vhat mechanisms ensure high accuracy of nucleic acid biosynthesis, to what extent accuracy is ensured by nucleic acid components, what is the role of synthesis enzymes and vhat are the pathways of infidelity. Two types of mispairs have been suggested as pathways of spontaneous mutations (3"9). Mispairs of the first type (3"5) have practically the same © IR L Press Limited, Oxford, England. 141 Nucleic Acids Research dimensions as the correct pairs but one base is in a rare tautomeric form. Such pairs could be incorporated into an undistorted DNA double h e l i x . Though soire experimental data do not contradict such a mechanism, there are many facts which cannot be explained w i th in i t s l i m i t s . Among these facts are: f i r s t , nucleotide replacements having pyri midine-pyri midine pairs as in te r mediate stages and the formation of such pairs in biosynthesis in v i t r o ; second, the error frequency in some systems is higher than the probab i l i t y of the rare tautomers (frequencies of d i f fe rent errors for a number of sys~terns are summarized in reviews (10,11)) and t h i r d , incorporation into DNA and RNA of base analogs wi th no plausible pairs of th is type (e .g. benzimidazol and some alkylated der iva t i ves) . Another way of nucleotide mispairing assumes that the bases are in the i r normal tautomeric forms (5"9) . Calculations of the in teract ion energy of nitrogen bases have shown that for each coplanar pair there are energy minima in which the mutual posi t ion of gl ycosyl bonds d i f fe rs from that in A:T and G:C pairs by no more than 3 A and 30°. These minima correspond to the forma t ion of two or one N-H...N and (or) N-H...0 hydrogen bonds. In some pairs the base is in syn-or ientat ion re la t i ve to sugar. Consideration of base pairs in normal tautomeric forms as intermediate stages of spontaneous mutations (6,7) suggests a qua l i t a t i ve explanation of a l l experimental data on spontaneous mutations involving replacenents of base pairs and on errors of nucleic acid synthesis in v i t r o . Such mispairs are characterized by a d i s placement of bases re la t i ve to the posi t ion occupied by complementary bases in Watson-Crick pairs and the displacement of bases requires d i s to r t i on of the sugar-phosphate backbone. I t is not evident a p r io r i that each mispair can be incorporated into the double hel ix without such a strong increase of energy that makes the incorporation of wrong nucleotides by th is mechanism prac t i ca l l y impossible. In previous papers (12-1't) we have demonstrated that d i f fe ren t types of mispairs containing bases in normal tautomeric forms can be incorporated in to the double he l ix without the appearance of reduced interatomic contacts and without a change of the sugar-phosphate backbone dihedral angles beyond the l im i ts character is t ic of double helices consist ing of A:T and G:C pa i rs . We have considered the incorporation into the B-conformation and A-conformation double helices of purine-pyrimidine (G:U, G:T), purine-purine ( I :A , G:A), purine-purine (syn) ( I :A syn, G:A syn) and pyrimidine-pyrimidine (C:U, C:T) pa i rs . Each of these pairs has two hydrogen bonds and the energy of base interact ion is close to that in the A:U pair or d i f f e r s by 1+2 kcal/mole.