Nucleotides tautomeric forms

Overall NMR and NQR spectroscopy data thus indicate that the diketo structure predominates for uracil, thymine, and their nucleosides or nucleotides. These studies have failed to detect other tautomeric forms of these compounds. 

Starting from the comparative study of the ionization constants of uracil itself as well as of its several methylated or ethylated derivatives (representing models of tautomeric forms), it may be seen (Table XVII) that uracil and uridine exist in aqueous solution in the diketo form 32. The pX values are not known for the model tautomers 27, 29, and 30, but these forms have been ruled out on the basis of UV studies. Recently the ionization constants of uracil, thymine, their derivatives and nucleotides were determined over the range 10-50°, and thermodynamic enthalpy, entropy, and free energy changes for protonation and depro-tonation of these compounds have been evaluated.93-95,332... 

All commonly occurring bases in nucleotides are capable of existing in two tautomeric forms, which differ by the placement of a proton and some electrons. For example, guanosine can undergo a change from a keto form to an enol form as shown in figure 23.3. The keto form is so strongly favored that it is difficult to detect even trace amounts of the enol form at equilibrium. Similarly, the keto forms of thy-... 

Nucleoside analogues do not always behave as expected. 5-Bromouracil (6.a) effectively forms base pairs with guanine nucleotides. This surprising observation has been explained by invoking a different tautomer of 6.a. Draw a different tautomer of 6.a and show how it can effectively base pair to a guanine nucleotide. (Tautomerization theory Topal, M. D., Fresco, J. R. Base Pairing and... 

Mistakes in base incorporation can be made this is largely a result of the transient existence of tautomeric forms of the bases (Chap. 7). If at the instant of insertion of a new nucleotide by DNA polymerase the base in the template shifts to its rare tautomeric form, which has altered base-pairing specificity, an incorrect nucleotide may be added to the chain e.g., one containing guanine instead of adenine opposite the enol form of thymine. 

This enzyme has a proofreading role. At a low random frequency, incorrect bases (in the form of nucleotides) are inserted into the growing DNA chain. This results from the existence of rare tautomeric forms of the four bases, which, if occurring transiently in the template at the moment of insertion of an incoming nucleotide, will cause a mistake in base pairing. When such a template nucleotide shifts back to its preponderant form, a base pair mismatch results. The 3 —>5 exonuclease recognizes the mismatch and catalyzes the hydrolytic removal of the nucleotide from the end of the chain before elongation resumes. 

Tautomeric enol and imino forms of bases occur only rarely, and can lead to mutations. It should be emphasized that in none of the above described mismatch base pairs is there any evidence for the existence of rare tautomeric forms. For the A-C pair, protonation at (adenine)N(l) appears more probable than the imino form (Fig. 20.6). However, conclusive evidence is still lacking because hydrogen atoms cannot be located at the attainable resolution of about 2 A. Moreover, in none of the crystal structures of the nucleosides and nucleotides or of the bases themselves is there any evidence of the enol-imino tautomers (Part II, Chaps. 15, 16, 17). 

Infrared spectra of heavy-water solutions have been used to determine the tautomeric forms of nucleosides, nucleotides, and polynucleotides and to study the... 

Uracil reacts with hydrazine to give pyrazol-3(2if)-one (944) and urea N-methyl- and dimethyl-hydrazine behave similarly to give the 2-methyl- and 1,2-dimethyl derivatives. The reactions of hydrazines with uridine and related nucleosides and nucleotides is well studied (67JCS(C)1528). The tautomerism and predominant form of uracil are discussed in Section 2.13.1.8.4. 

The nucleoside formed from hypoxanthine and ribose is known as inosine (Ino or I) and the corresponding nucleotide as inosinic acid. Further substitution at C-2 of -H by -OH and tautomerization yields xanthine (Xan). Its nucleoside is xanthosine (Xao, X). A similar hydroxylation at C-7 converts xanthine to uric acid, an important human urinary excretion product derived from nucleic acid bases. 

From the observed rate of appearance of point mutations (one mutation per 106 gene duplications), we can estimate that one mutation occurs per 109 replications at a single nucleotide site. Point mutants tend to "back mutate," often at almost the same rate as is observed for the forward mutation. That is, one in 109 times a mutation of the same nucleotide will take place to return the code to its original form. The phenomenon is easy to understand. For example, if T should be replaced by C because the latter formed a minor tautomer and paired with A, the mutation would appear in progeny duplexes as a GC pair. When this pair was replicated, there would be a finite probability that the C of the parental DNA strand would again assume the minor tautomeric structure and pair with A instead of G, leading to a back mutation. 

As shown in the biosynthesis of granaticin, a hydride shift occurs intramolecularly. This process is mediated by an enzyme-bond pyridine nucleotide. A concerted abstraction of H-4 as a hydride in la and a C-5 deprotonation in 2a leads to the 4,5-enol ether 3a. The reduced form of the pyridine nucleotide transfers the hydride to C-6, simultaneously releasing a hydroxide to give 4a. Final tautomerization yields the dTDP-4-keto-6-deoxy-sugar in v-xylo configuration 4a. In other enzymes of the oxidoreductase type, the active site may show a different configuration. Thus, the intermediate 3a can be protonated from above at C-5 to yield the l-arabino isomer of 4a [2]. The stereochemistry of this mechanism was demonstrated by double labelling (cf. l-4b series), and as a net result proved a suprafacial 4—>6 hydride shift. 

The 3 —>5 exonuclease activity of DNA polymerase I, at least, functions to proofread for such mistakes. After the incorrect base is incorporated, it will not remain hydrogen-bonded to the tautomeric base in the template once the latter returns, almost immediately, to its more stable form. The 3 — 5 exonuclease activity shows a strong preference for a frayed or non-hydrogen-bonded end and removes the misincorporated nucleotide before chain growth proceeds further. DNA polymerase III holoenzyme also has the potential to proofread by the same mechanism. 

---