CATALYSIS AND INHIBITION OF DNA POLYMERASE ETA, A TRANSLESION POLYMERASE IMPORTANT FOR CHEMOTHERAPEUTIC EFFICACY
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Date
2014-10-07
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Johns Hopkins University
Abstract
The first objective of my thesis was to characterize the mechanism of Pol η catalysis at the molecular level, specifically concerning alignment and deprotonation of the primer. A new technique, time resolved crystallography, has enabled me to visualize atomic details of the active site in a time-lapse series during Pol η catalysis. A number of moieties have been proposed to deprotonate the primer 3´-OH during catalysis, including the S113 residue and the transient water. My work identifies a key structural transition from a ground state to a reactant state conformation that’s critical for efficient deprotonation and catalysis, and identifies the S113 residue as the mediator of this transition. Additionally, I collected data that demonstrated that a previously proposed “transient water” was not solely responsible for primer deprotonation, and suggests that the general base can be varied in the polymerase catalysis mechanism.
The second objective of this thesis was characterization of Pol η as a target for inhibition. Platinum-based chemotherapies are among the most successful anti-cancer treatments available currently, but current platinum chemotherapeutics are only effective against some cancer types and are limited by acquired resistance in cancers. Due to Pol η’s proven role as a resistance gene against chemotherapy treatment in cancers, Pol η inhibition represents a promising therapeutic goal. In this work I identified a novel druggable pocket in the Pol η thumb domain and verified the inhibitory potential of this binding site biochemically. Additionally, I present an investigation of the effect of phenanthriplatin adducts, a monofunctional platinum based chemotherapeutic candidate, on replication and the ability of DNA polymerases to replicate past a site-specific phenanthriplatin lesion. Among a panel of DNA polymerases, only Pol η was able to bypass the phenanthriplatin lesion, although with a very low efficiency. Kinetic and structual studies of the different steps in TLS past phenanthriplatin lesions reveal the nature of the interaction of Pol η with phenanthriplatin platinated DNA and elucidate the mechanism by which the structure of phenanthriplatin inhibits TLS. This work demonstrates the therapeutic potential of monofunctional platinum chemotherapeutics such as phenanthriplatin in overcoming known cancer resistance mechanisms.
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Polymerase, chemotherapy, trans lesion synthesis