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Master Thesis

La PolD, une ADN polymérase atypique d'arches et un nouvel outil prometteur pour les biotechnologies

Abstract : In all forms of life, DNA polymerases play a central role in genome replication, maintenance and repair. They are also valuable tools needed for many applications in molecular biology. All DNA polymerases were classified into seven distinct families based on their sequence homology: PolA, PolB, PolC, PolD, PolX, PolY and reverse transcriptases. The DNA polymerase family D, called PolD and whose structure has recently been resolved, remains the least well characterized. It has been discovered in thermophilic Archaea, and has interesting biochemical properties, exploitable by PCR (Polymerase Chain Reaction). It is an heterodimeric protein composed of a DP1 subunit responsible for the exonuclease activity, and a DP2 subunit responsible for the polymerization activity. The structures of DP1 and DP2 reveal that PolD is an atypical DNA polymerase. Indeed, the structure of the polymerase and exonuclease active sites differs from that of the other DNA polymerases characterized to date. PCNA (Proliferating Cell Nuclear Antigen) is a key player in the replication fork that dramatically improves the processivity of PolD both in vivo and in vitro. In this work, I was able to study the PolD and the PCNA of the Archaea Pyrococcus abyssi from several angles. At first, I worked on the PCNA alone to determine its structure by crystallography. Shortly after, I expressed and purified different PolD constructs in order to study the two proteins as part of their complexation using the bio-layer interferometry method. Crossing the data obtained allowed me to map their interactions and to propose a model of the ternary complex they form with DNA.
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Master Thesis
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Submitted on : Monday, September 30, 2019 - 5:33:41 PM
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Inès Hugonneau Beaufet. La PolD, une ADN polymérase atypique d'arches et un nouvel outil prometteur pour les biotechnologies. Sciences du Vivant [q-bio]. 2019. ⟨dumas-02301874⟩

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