Abstract
Most models accounting for the efficiency and fidelity of DNA polymerization invoke the use of either hydrogen bonding contacts or complementarity of shape and size between the formed base pair. This report evaluates these mechanisms by quantifying the ability of a high-fidelity DNA polymerase to replicate 5-nitroindole, a purine mimetic devoid of classic hydrogen bonding capabilities. 5-NITP acts as a universal nucleotide since it is incorporated opposite any of the four natural nucleobases with nearly equal efficiencies. Surprising, the polymerization reaction is not reciprocal as natural nucleotides are poorly incorporated opposite 5-nitroindole in the template strand. Incorporation opposite 5-nitroindole is more efficient using natural nucleotides containing various modifications that increase their base stacking potential. However, 5-substituted indolyl nucleotides that contain pi-electron and/or hydrophobic groups are incorporated opposite the non-natural nucleobase with the highest catalytic efficiencies. The collective data set indicate that replication of a non-natural nucleobase is driven by a combination of the hydrophobic nature and pi-electron surface area of the incoming nucleotide. In this mechanism, the overall hydrophobicity of the incoming nucleobase overcomes the lack of hydrogen bonding groups that are generally required for optimal DNA polymerization. However, the lack of hydrogen bonds between base pairs prevents primer extension. This final aspect is manifest by the appearance of unusually high pyrophosphorolysis activity by the T4 DNA polymerase that is only observed with the non-natural nucleobase in the template. These results highlight the importance of hydrogen bonding interactions during primer extension and pyrophosphorolysis.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.