Spectroscopic Analysis of Polymerization and Exonuclease Proofreading By A High-Fidelity DNA Polymerase During Translesion DNA Synthesis

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

Epistatic Roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in Coping with Reactive Oxygen Species-Induced DNA Damage

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H2O2)-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H2O2-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed

Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity

DNA polymerases catalyze the addition of mononucleotides into a growing polymer using a DNA template as a guide for directing each incorporation event. The efficiency and fidelity of this biological process have been historically attributed to the ability of the DNA polymerase to coordinate proper hydrogen-bonding interactions between the incoming nucleotide with the templating nucleobase. However, the strength of this model has been weakened since several laboratories have demonstrated that non-natural nucleotides, i.e., those devoid of typical hydrogen-bonding capabilities, can be utilized by DNA polymerases with varying degrees of efficiencies. This review provides a comprehensive summary of current research efforts leading to the development and implementation of these analogs as probes for DNA polymerase function and activity. The ability of various non-natural purines and pyrimidines to be incorporated opposite templating nucleobases suggests that polymerization efficiency is not directly influenced by hydrogen-bonding interactions but rather by the overall shape and size of the formed base-pair. Conflicting evidence is obtained when the dynamics of nucleotide incorporation is assessed using nucleic acid containing permutations in hydrogen bonding capabilities or completely devoid of these interactions. With respect to replication opposite an abasic site, it appears that the π-electron surface area and desolvation properties of the incoming nucleotide play a significant role for facilitating incorporation. This information has lead to the development of new models for DNA polymerization as well as toward strategies for novel biotechnology platforms and unique chemotherapeutic agents

Non-Natural Nucleotide Analogs as Probes of DNA Polymerase Activity

DNA polymerases catalyze the addition of mononucleotides into a growing polymer using a DNA template as a guide for directing each incorporation event. The efficiency and fidelity of this biological process have been historically attributed to the ability of the DNA polymerase to coordinate proper hydrogen-bonding interactions between the incoming nucleotide with the templating nucleobase. However, the strength of this model has been weakened since several laboratories have demonstrated that non-natural nucleotides, i.e., those devoid of typical hydrogen-bonding capabilities, can be utilized by DNA polymerases with varying degrees of efficiencies. This review provides a comprehensive summary of current research efforts leading to the development and implementation of these analogs as probes for DNA polymerase function and activity. The ability of various non-natural purines and pyrimidines to be incorporated opposite templating nucleobases suggests that polymerization efficiency is not directly influenced by hydrogen-bonding interactions but rather by the overall shape and size of the formed base-pair. Conflicting evidence is obtained when the dynamics of nucleotide incorporation is assessed using nucleic acid containing permutations in hydrogen bonding capabilities or completely devoid of these interactions. With respect to replication opposite an abasic site, it appears that the π-electron surface area and desolvation properties of the incoming nucleotide play a significant role for facilitating incorporation. This information has lead to the development of new models for DNA polymerization as well as toward strategies for novel biotechnology platforms and unique chemotherapeutic agents

Enhancing The “A-Rule” of Translesion DNA Synthesis: Promutagenic DNA Synthesis Using Modified Nucleoside Triphosphates

Abasic sites are mutagenic DNA lesions formed as a consequence of inappropriate modifications to the functional groups present on purines and pyrimidines. In this paper we quantify the ability of the high-fidelity bacteriophage T4 DNA polymerase to incorporate various promutagenic alkylated nucleotides opposite and beyond this class of non-instructional DNA lesions. Kinetic analyses reveal that modified nucleotides such as N6-methyl-dATP and O6-methyl-dGTP are incorporated opposite an abasic site far more effectively than their unmodified counterparts. The enhanced incorporation is caused by a 10-fold increase in kpol values that correlates with an increase in hydrophobicity as well as changes in the tautomeric form of the nucleobase to resemble adenine. These biophysical features lead to enhanced base-stacking properties that also contribute toward their ability to be easily extended when paired opposite the non-instructional DNA lesion. Surprisingly, misincorporation opposite templating DNA is not enhanced by the increased base-stacking properties of most modified purines. The dichotomy in promutagenic DNA synthesis catalyzed by a high-fidelity polymerase indicates that the dynamics for misreplicating a miscoding versus a non-instructional DNA lesion are different. The collective data set is used to propose models accounting for synergistic enhancements in mutagenesis and the potential to develop treatment-related malignancies as a consequence of utilizing DNA-damaging agents as chemotherapeutic agents

Is A Thymine Dimer Replicated Via A Transient Abasic Site Intermediate? A Comparative Study Using Non-Natural Nucleotides

UV light causes the formation of thymine dimers that can be misreplicated to induce mutagenesis and carcinogenesis. This report describes the use of a series of non-natural indolyl nucleotides in probing the ability of the high-fidelity bacteriophage T4 DNA polymerase to replicate this class of DNA lesion. Kinetic data reveal that indolyl analogues containing large π-electron surface areas are incorporated opposite the thymine dimer almost as effectively as an abasic site, a noninstructional lesion. However, there are notable differences in the kinetic parameters for each DNA lesion that indicate distinct mechanisms for their replication. For example, the rate constants for incorporation opposite a thymine dimer are considerably slower than those measured opposite an abasic site. In addition, the magnitude of these rate constants depends equally upon contributions from π-electron density and the overall size of the analogue. In contrast, binding of a nucleotide opposite a thymine dimer is directly correlated with the overall π-electron surface area of the incoming dXTP. In addition to defining the kinetics of polymerization, we also provide the first reported characterization of the enzymatic removal of natural and non-natural nucleotides paired opposite a thymine dimer through exonuclease degradation or pyrophosphorolysis activity. Surprisingly, the exonuclease activity of the bacteriophage enzyme is activated by a thymine dimer but not by an abasic site. This dichotomy suggests that the polymerase can “sense” bulky lesions to partition the damaged DNA into the exonuclease domain. The data for both nucleotide incorporation and excision are used to propose models accounting for polymerase “switching” during translesion DNA synthesis

Summary of spontaneous and H₂O₂-induced base substitutions in <i>rpoB</i> that confer Rif^R.

<p>Results of <i>rpoB</i> DNA sequence analysis for spontaneous (<b>A</b>) and H₂O₂-induced (<b>B</b>) Rif^R<i>P. aeruginosa</i> mutants are summarized with respect to the types of nucleotide substitution observed. Frequency refers to the occurrence of each observed base substitution mutation as a function of the total number of spontaneous or H₂O₂-induced Rif^R mutants sequenced for each strain. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018824#pone-0018824-t002" target="_blank">Table 2</a> for details concerning the number of Rif^R clones analyzed for each strain, as well as the specific nucleotide position and substitution of each documented mutation.</p