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

Abstract

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 pi-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 pi-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 pi-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.

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

“…In order to obtain biochemical support for this conclusion, we asked if recombinant DinB protein catalyzed accurate bypass of a model cis-syn thymine cyclobutane dimer in vitro . As a control for these experiments, we utilized an exonuclease proofreading-deficient form of the bacteriophage T4 Pol (T4 exo – Pol), which incorporates dATP exclusively opposite the 3′-dT of the dimer, yielding almost exclusively a 14-mer product ([48]; see Fig. 5, panels B & C).…”

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

“…This feature provides constraints on the derived kinetic parameters such that K d, app values represent close approximations to true binding affinities for the non-natural nucleotides. In addition, we have demonstrated through elemental effects on nucleotide incorporation (10,46), pulse chase (47,48) and fluorescence analyses (49) that k pol values reflect the conformational change step that precedes phosphoryl transfer. As a result, measurable differences in these kinetic parameters as a function of non-natural nucleotide can be used to quantify the importance of various biophysical features.…”

Mechanisms of DNA Polymerases

“…Extension beyond both DNA lesions is inefficient and reduced 1,000-fold compared to extension beyond a correct base pair. 123,137 In addition, the kinetics for excising dAMP from either DNA lesion are identical and significantly faster than dAMP excision from an unmodified thymine. 123,137 This enhancement likely reflects the enzyme's ability to partition the misaligned primer from the polymerase active site into its exonuclease domain and contributes toward the lack of extension beyond either mispair.…”