Assuming that the efficiency of the incorporation of 5-methyl-2h-deoxyisocytosine-5h triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iGneighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3h-TXT-5h, 3h-GXG-5h, 3h-CXC-5h, 3h-CXT-5h, where X l iG) at 37, 50, 65 and 80 mC, we found that 3h-neighbours decrease the fraction of the keto tautomer in the order C G T, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5-20) primarily reflects the variability of K m values, since
2-Hydroxyadenine (A?) is formed from adenine during reaction of DNA with oxygen radicals. The enol form of A* pairs with T whereas the keto form with 5-methylisocytosine (iCM). We studied the influence of neighbouring bases in template, temperature and kind of polymerase on base-pairing of A" by comparison of relative efficiency of incorporation of d'ITP and diCMTP (calculated as the ratio of appropriate Vmax/Km) opposite A" site specifically located in oligodeoxynucleotide templates. We have revealed that the base pairing of A* is significantly influenced by template neighbours but not by temperature (3740°C) when studied in taq polymerase system.
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