Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

As the essential enzymes in human bodies, DNA polymerases play a significant role in DNA replication, repair, genetic recombination, and reverse transcription. In 1956, the enzyme of DNA polymerase I, also named as Pol I, was discovered by Arthur Kornberg and colleagues. Subsequently, the Noble Committee had decided that the Noble Prize in Physiology or Medicine for 1959 was to be awarded to Kornberg for his excellent original work that describes the DNA replication process whereby the DNA polymerase copies the nucleotide sequence of a DNA template strand. Because of the complex enzyme structure in the DNA polymerase, it is a challenge to study the mechanism of the catalytic function and substrate selectivity of DNA. Recently, a new class of chemically modified deoxyribonucleoside triphosphate (dNTP) substrates has been developed to study the chemical mechanism of the replication fidelity and inhibition of human DNA polymerases. Here, we studied the models for nucleotidyl transfer reaction in aqueous solution to better understand this mechanism in DNA polymerase β. An unusual experimental result from Goodman’s group demonstrated two splitting lines for the linear free energy relationship (LFER) for the mispaired (W) and correctly base paired (R) analogues between the rate constant in the corresponding polymerase (kpol) and the highest pKa4 value of the bisphosphonic acid corresponding to the leaving group. In the following year, Kamerlin theoretically reported similar results for this mechanism in aqueous solution. When we designed our model system, we made it more biochemically

relevant. The calculated log k and log K values were found to depend linearly on the experimental pKa4 of the conjugate acid of the corresponding pyrophosphate or bisphosphonate leaving group. The scissile Pα–Olg bond length in studied methyl triphosphate analogues slightly increases with decreasing pKa4 of the leaving group; concomitantly, the CH3OPα(O2) moiety becomes more positive. These structural effects indicate that substituents with low pKa can facilitate both Pα–Olg bond breaking and the Pα–Onuc bond forming process, thus explaining the large negative βlg calculated for the transition state geometry that has significantly longer Pα–Onuc distance than the Pα–Olg distance. The extension of our model as well as broadening the range of studied mechanisms indicated the possibility of a break in the LFER curve for the mechanism that involves pre-equilibrium protonation of the γ-phosphate of dNTP substrate. It also confirmed that the mechanism that involves pre-equilibrium deprotonation of the nucleophile followed by the nucleophilic group attack that is the most possible α0γ0

mechanism in aqueous solution. Because this mechanism show similar slope of the LFER line like that observed in DNA polymerase β, this mechanism is also the most likely mechanism to occur in this polymerase.

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