Janska, A; (2017) Structural and biochemical characterisation of DNA polymerase epsilon and its role in DNA replication. Doctoral thesis , UCL (University College London).

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Abstract

DNA is copied by the replisome, a multi-protein assembly that couples polymerase and helicase activities. In eukaryotes, there are three main replicative polymerases (α, ε, δ) and the CMG (Cdc45-MCM-GINS) helicase is a three-member complex, where MCM (minichromosome maintenance) constitutes the motor while Cdc45 (cell division cycle 45) and GINS (go-ichi-ni-san) are essential cofactors. Pol ε is unique in that the protein is essential while cells where the catalytic domain is deleted become severely sick yet are still viable. Therefore, it was believed to have an additional role apart from DNA synthesis and proposed to recruit GINS to MCM together with Sld2 (synthetic lethal with Dpb11) and Dpb11 (DNA polymerase B-binding protein subunit 11) as part of the pre-LC (pre-loading complex). The role of Pol ε in helicase assembly was investigated by biochemical approaches and its structure was determined by electron microscopy both in isolation and in complex with CMG in order to investigate the helicase-polymerase coupling. The structural part of this work was performed in collaboration with Alessandro Costa’s Macromolecular Machines Laboratory and all microscopy work as well as data analysis was performed by the collaborators. The non-catalytic part of Pol ε was found to bind Sld2, Dpb11 and GINS while the catalytic domain was found to interact with Sld3. These results support the notion that the essential role of Pol ε is to aid CMG assembly. Although the pre-LC was reconstituted in vitro and independently of origins, it is still not clear whether its formation is strictly required. Pol ε was found to be made up of two lobes connected by a thin linker, which spatially separates the catalytic and non-catalytic portions of the assembly. The inactive lobe appears anchored at the front of the helicase while the catalytic lobe extends towards the side of CMG and adapts two conformations. Such positioning of the polymerase deviates from the classical model of the replisome, where polymerases trail behind the helicase and raises questions about the path of DNA through this assembly. The role of the conformational switch is not known, but it may be important for substrate engagement by Pol ε.

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