Neighbouring bases in template influence base-pairing of isoguanine

Abstract

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

Replication of 2-hydroxyadenine-containing DNA and recognition by human MutSα

“…These findings are consistent with the known ability of DNA 2-OH-A to adopt multiple tautomeric forms that are influenced by temperature, solvent polarity and neighbouring bases [32][33][34]. In particular, a shift from the prevailing keto tautomer (N1-H) towards the enol form (O2H) is likely to affect the probability that the lesion is accommodated as 2-OH-A:T or with other partners through classical W-C bonding or wobble base pairs.…”

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs

“…In contrast, a 2-OH-dATP:rU base pair is well tolerated in the telomerase active site since 2-OH-dATP insertion opposite rU is only 2-fold less efficient than dATP. 2-OH-dATP can pair with thymine or uracil either in an enol tautomeric form with Watson–Crick hydrogen bonding, or in a keto form through wobble base pairing 51 , 52 . Similarly, HIV-1 and avian myeoblastosis virus RTs preferentially insert 2-OH-dATP opposite dT or rU more efficiently than replicative polymerases 53 , 54 .…”

Sequence determination of nucleic acids containing 5-methylisocytosine and isoguanine: identification and insight into polymerase replication of the non-natural nucleobases

“…A 2O–H tautomer of isoG (Figure 1B), complementary to T, has long been suspected of confounding replication of isoC–isoG (13,14,28–30). One problem that may result from isoG tautomerism is the difficulty of incorporating d Me isoC nucleotide opposite template disoG positions in the pyrosequencing reactions.…”

“…It is possible that isoG may not readily interconvert between tautomeric forms at the polymerase active site in the lower temperature pyrosequencing method, leading to problematic d Me isoC incorporation when isoG is locked in an alternate tautomeric form. The evident incorporation of d Me isoC opposite template disoG positions by the thermophilic polymerases may be the result of relatively more rapid interconversion of tautomers in the polymerase active site or a shifted tautomeric equilibrium [although the tautomeric equilibrium of disoG has been reported as unperturbed by variations in this temperature range (30)]. Curiously, if the 2O–H tautomer of isoG was present in the templates, it did not lead to significant dT incorporation opposite disoG in pyrosequencing (Supplementary Figure S1C–F).…”

“…Curiously, if the 2O–H tautomer of isoG was present in the templates, it did not lead to significant dT incorporation opposite disoG in pyrosequencing (Supplementary Figure S1C–F). Pyrosequencing and dye terminator sequencing suggest that the incorporation of dT nucleotide opposite template disoG by Family A polymerases, while apparently facile for a misincorporation event (14,30,37,38), is probably much slower than the incorporation of d Me isoC nucleotide opposite disoG.…”