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  • jzl In this report we evaluate if this molecular mechanism

    2020-01-15

    In this report, we evaluate if this molecular mechanism is used universally by high-fidelity DNA polymerases during the replication of DNA lesions that are structurally distinct from abasic sites. This was approached by quantifying the kinetic parameters for the incorporation of modified and non-natural analogs opposite 8-oxo-G catalyzed by gp43exo−. We chose 8-oxo-G since the oxidized DNA lesion possesses dual coding properties as it can jzl pair with dCTP when in the anti conformation or with dATP when in the syn conformation (Fig. 1b). However, like an abasic site, several replicative DNA polymerases such as gp43 and human DNA polymerases including pol δ and pol γ efficiently misincorporate dATP opposite 8-oxo-G [31], [32], [33], [34], [35]. In the case of pol γ, for example, adenosine-2′-deoxyriboside monophosphate (dAMP) is stably inserted and frequently elongated despite the presence of rigorous exonuclease proofreading activity with the mitochondrial DNA polymerase [35]. At face value, the preferential misinsertion of dATP opposite both types of lesions suggests that a common mechanism is used to replicate damaged DNA. Indeed, the results generated here with 8-oxo-G demonstrate that the binding affinity of the incoming deoxynucleoside triphosphate (dNTP) for gp43exo− is controlled by the overall hydrophobicity of the nucleobase. However, the rate constant for polymerization is regulated by different biophysical features that are dependent upon whether the DNA lesion is miscoding or non-instructional. Specifically, during the replication of non-instructional lesions, the rate constant for the polymerization step is controlled by π-electron density present on the incoming nucleotide, whereas the data presented here show that hydrogen-bonding interactions play a much larger role with miscoding lesions such as 8-oxo-G. Collectively, these studies provide additional insight into how different molecular forces are used by high-fidelity DNA polymerases during the misreplication of structurally distinct DNA lesions.
    Results
    Discussion TLS is an essential biological process that allows both prokaryotic and eukaryotic cells to cope with unrepaired DNA lesions. While it is clear that TLS is important for maintaining cell viability, the molecular mechanisms accounting for how certain DNA polymerases efficiently replicate different forms of damaged DNA still remain enigmatic. This study addresses this question by quantitatively comparing the ability of a high-fidelity DNA polymerase to replicate miscoding versus non-instructional DNA lesions. The results from these studies provide a new model highlighting the role of nucleobase hydrophobicity during the replication of structurally distinct DNA lesions. In addition, these analyses provide insight into how nucleobase desolvation is used differentially by high-fidelity and specialized DNA polymerases and how these differences provide a viable explanation as to why specialized DNA polymerases are more efficient at TLS compared to high-fidelity polymerases. Finally, the results of this study also demonstrate that inappropriate modifications to nucleotide pools can enhance pro-mutagenic DNA synthesis catalyzed by high-fidelity DNA polymerases. By inference, this activity may facilitate the initiation of genetic diseases, the most notable of which is cancer. While the studies described here have focused on defining how a high-fidelity DNA polymerase replicates damaged DNA, an important question remains as to the activity of specialized DNA polymerases during the replication of 8-oxo-G. Indeed, we previously demonstrated that pol eta relies heavily on hydrogen-bonding interactions when replicating 8-oxo-G [37]. As discussed below, this current study shows that the high-fidelity DNA polymerase, gp43, relies extensively on nucleobase desolvation to efficiently replicate the oxidized DNA lesion. This dichotomy suggests that high-fidelity and specialized DNA polymerases use different molecular forces to replicate damaged DNA. We are currently examining the ability of other specialized DNA polymerase such as human pol iota and pol kappa to utilize these modified nucleotide substrates to provide additional insight into the molecular mechanism(s) of TLS. However, our discussions below first address if a unified mechanism exists that accounts for the ability of high-fidelity DNA polymerases to replicate structurally distinct DNA lesions.