Kulik, Anastasija (2019) Characterisation of Polymerase δ-interacting Protein 2 (POLDIP2), a novel human protein involved in modulating DNA replication/repair and other physiological functions. Masters thesis, University of Huddersfield.

Polymerase δ-interacting Protein 2 (POLDIP2) is composed of 368 amino acids, with a molecular weight of the 42 kDa and 37 kDa, respectively, before and after post-translational cleavage of the mitochondrial-targeting sequence (Hernandes, Lassègue and Griendling, 2017; Liu et al., 2003). POLDIP2 was shown to be involved in the replication of cell cycle, lung functioning, maintenance of cellular homeostasis (Hernandes et al., 2017). Also, POLDIP2 is involved in bone and DNA maintenance, neurodegenerative diseases, cancer and proliferation (Hernandes et al., 2017). Although, the POLDIP2 is a multifunctional protein it remains unknown how POLDIP2 can bind so many different proteins in different subcellular compartments. Moreover, it remains unknown if the two POLDIP2 forms (37 kDa and 42 kDa) have different functions. The POLDIP2 biochemistry remains completely untouched, therefore this project focused on structural and functional POLDIP2 characterisation.

All stages of recombinant POLDIP2 structure determination pipeline were attempted; starting from the POLDIP2 gene sub-cloning and ending with protein structure crystallization. The POLDIP2 structure was nearly solved (~99.8%) at 2.9 Å. The obtained POLDIP2 3D structure was used for the surface charge distribution and B factor analysis. The POLDIP2 B factor analysis and predicted/known secondary structure elements superimposition revealed that both rigid and flexible POLDIP2 secondary structure elements are involved in protein-protein interactions. The POLDIP2 B factor analysis was superimposed with known/predicted secondary POLDIP2 structure elements, revealing that both rigid and flexible POLDIP2 secondary structure elements are involved in protein-protein interactions.

Moreover, it was attempted to crystallise the POLDIP2 in complex with the proliferating cell nuclear antigen (PCNA), as POLDIP2 structure with a binding partner is required for the detailed understanding of POLDIP2 function. Furthermore, in vitro crosslinking and size exclusion chromatography were used to understand the PCNA-POLDIP2 binding stoichiometry. Finally, the initial negative grid staining of the PCNA-POLDIP2 complex for further cryogenic electron microscopy (cryo-EM) studies was attempted giving future work directions.

FINAL THESIS - KULIK.pdf - Accepted Version
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