Rational Attempts to Optimize Non-Natural Nucleotides for Selective Incorporation Opposite an Abasic Site

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

2013

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High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2'-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis.

Section: Incorporation Of 5 Napitp Opposite Normal and Damaged Dnacontrasting

confidence: 71%

Section: Incorporation Of 5 Napitp Opposite Normal and Damaged Dnasupporting

confidence: 66%

Section: Incorporation Of 5 Napitp Opposite Normal and Damaged Dnamentioning

confidence: 99%

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Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis

Devadoss

Lee

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

2013

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“…Thus, the reported catalytic efficiencies reflect upper estimates based upon the rate constant ( k obs ) measured divided by the highest concentration of nucleotide tested. Values taken from [d] Berdis,9 [e] Zhang et al,5b [f] Reineks and Berdis,5a [g] Zhang et al,5c [h] Zhang et al,5d [i] Zhang et al,5e…”

Section: Resultsmentioning

confidence: 99%

The Use of Non‐natural Nucleotides to Probe Template‐Independent DNA Synthesis

McCutcheon

2007

ChemBioChem

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The vast majority of DNA polymerases use the complementary templating strand of DNA to guide each nucleotide incorporation. There are instances, however, in which polymerases can efficiently incorporate nucleotides in the absence of templating information. This process, known as translesion DNA synthesis, can alter the proper genetic code of an organism. To further elucidate the mechanism of template-independent DNA synthesis, we monitored the incorporation of various nucleotides at the "blunt-end" of duplex DNA by the high-fidelity bacteriophage T4 DNA polymerase. Although natural nucleotides are not incorporated at the blunt-end, a limited subset of non-natural indolyl analogues containing extensive pi-electron surface areas are efficiently utilized by the T4 DNA polymerase. These analogues possess high binding affinities that are remarkably similar to those measured during incorporation opposite an abasic site. In contrast, the k(pol) values are significantly lower during blunt-end extension when compared to incorporation opposite an abasic site. These kinetic differences suggest that the single-stranded region of the DNA template plays an important role during polymerization through stacking interactions with downstream bases, interactions with key amino acid residues, or both. In addition, we demonstrate that terminal deoxynucleotide transferase, a template-independent enzyme, can efficiently incorporate many of these non-natural nucleotides. However, that this unique polymerase cannot extend large, bulky non-natural nucleotides suggests that elongation is limited by steric constraints imposed by structural features present within the polymerase. Regardless, the kinetic data obtained from using either DNA polymerase indicate that template-independent synthesis can occur without the contributions of hydrogen-bonding interactions and suggest that pi-electron interactions play an important role in polymerization efficiency when templating information is not present.

“…The triphosphates were prepared as previously described (24,25,28,33) starting from the corresponding nucleosides. Triphosphorylation was initiated by forming the 5′-monophosphorodichloridated intermediate by the dropwise addition of POCl 3 in the reaction mixture containing the 2′deoxyribonucleoside (0.07 mmol) and 1,8- bis (dimethylamino)naphthalene (0.11 mmol) dissolved in 0.37 ml of trimethylphosphate pre-chilled at 0°C.…”

Section: Methodsmentioning

confidence: 99%

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

Motea

Lee

2010

Nucleic Acids Research

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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.