Quantifying the energetic contributions of desolvation and π-electron density during translesion DNA synthesis

Abstract

This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2′-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of π-electron density and solvation energies. In general, the Kd, app values for hydrophobic non-natural nucleotides are ∼10-fold lower than those measured for isosteric hydrophilic analogs. In addition, kpol values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and π-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.

Insights into the Roles of Desolvation and π‐Electron Interactions during DNA Polymerization

“…This mechanism is further supported as the K m value for the other hydrophobic analogue, 5-MeCITP, is~25-fold lower than its hydrophilic counterpart, 5-CITP (compare 5.4 mm versus 125 mm, respectively). Similar observations were reported with the high-fidelity bacteriophage T4 DNA polymerase, [8] and this commonality suggests that nucleobase hydrophobicity plays a vital role in the binding affinity of the incoming nucleotide with members of the A and B families of DNA polymerases.…”

“…[8] The first class of analogues (5-NITP, 5-CITP, and 5-MeCITP) were used to quantify the role of nucleobase hydrophobicity and desolvation. The second class (5-PhITP, 5-F5-PhITP, and 5-F4OMe-PhITP) were used to evaluate the importance of p-electron density and size variation.…”

“…Collectively, this study demonstrated that the catalytic efficiency, k pol /K d,app , for incorporating these non-natural nucleotides is governed by their intrinsic hydrophobicity and degree of p-electron density. [8] This information was used to propose a model in which the binding affinity of the nucleotide is controlled by nucleobase desolvation while p-electron density facilitates the conformational change step preceding phosphoryl transfer.…”

Inhibition of Translesion DNA Synthesis as a Novel Therapeutic Strategy to Treat Brain Cancer

“…5-nitroindolyl-2 0 -deoxyriboside (5-NIdR), 5-nitroindolyl-2 0 -deoxyriboside triphosphate (5-NITP), 3-Eth-5-NIdR, and 3-Eth-5-NITP were synthesized and purified as previously described (15,16). DNAs including that containing an abasic site were obtained from Operon and purified as described (46).…”

Template independent synthesis of nucleic acid libraries

“…Terminal deoxynucleotidyltransferase (TdT), due to its property of template-independent nucleotide addition, looked like the appropriate tool to build random libraries of nucleic acid molecules [9,10]. It has been reported in the literature that protruding, recessed or blunt-ended double or single-stranded DNA molecules serve as its substrate [11,12]. TdT, catalyzes the addition of dNTPs to the 3' endof single-stranded and double stranded DNA molecules [13][14][15][16].…”