Tautomeric form, nucleotide bases

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-... 

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). 

Tautomerizations are also subject to the dictates of equation (16), with the more polar form increasingly favored in more polar media. This has been another successful target for FEP calculations, with applications ranging from 2-hydroxypyridine and histamine to nucleotide bases. In addition, the influence of solvation on simple carbenium ion rearrangements has been investigated by MC-FEP simulations. For the conversion of the classical to the non-classical 2-norbomyl cation in water and that of a tertiary cyclopentylcarbinyl cation to a secondary cyclohexyl cation in methylene chloride and THF, solvation differences of only ca, 1 kcal mol were obtained. Such ions are all relatively charge-delocalized and are consequently immune to significant differential solvation. 

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. 

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... 

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. 

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