Genomic DNA is under constant assault by environmental factors that introduce a variety of DNA lesions. Cells evolved several DNA repair and recombination mechanisms to remove these damages and ensure the integrity of the DNA material. A variety of specific proteins, called nucleases, processes toxic DNA structures that deviate from the heritable duplex DNA as common pathway intermediates. DNA-induced protein ordering is a common feature in all DNA repair nucleases. Still, the conformational requirement of the DNA and the protein and how they control the catalytic selectivity of the nuclease remain largely unknown. This study focus on the bases of catalytic activity of a protein belongs to the 5’ nuclease super-family called the human Flap endonuclease 1 (FEN1); it removes excess 5’ flaps that are generated during DNA replication. hFEN1 mutations and over-expression had been linked to a variety of cancers. This thesis aims to study the structural and dynamic properties of free hFEN1 and the catalytic activity of DNA-bound hFEN1 in solution utilizing the modern high-resolution multidimensional Nuclear Magnetic Resonance (NMR) spectroscopy. It was possible to depict the secondary structure and backbone conformation in solution of wild type (WT) hFEN1 by the usage of the improved list of assigned resonances, derived from the NMR 2D and 3D ¹⁵N-detected experiments and compared to the assignment with the previously published resonance assignment (BMRB id: 27160). I was successfully assigned the new spectrum and enhanced it by assigning seven more residues. Moreover, we tested the interaction of 1:10 ratio of hFEN1-Ca2+ with DNA by the ¹³C-detected 2D CACO experiment. The results indicate hFEN1:DNA interaction. Furthermore, parts of hFEN1 get more ordered/structured once DNA appears, thus we recorded the protein flexibly by 2D ¹H-¹⁵N TROSY-HSQC using the relaxation rate parameters: longitudinal R1, transverse R2 complemented with ¹⁵N-{¹H} NOEs (heteronuclear Overhauser enhancement). It was found that the overall molecular architecture is rigid, and the highest flexibility lies in the α2-α3 loop and arch (α4-α5) regions. Further analysis is needed to understand more profoundly the activity of hFEN1 in an atomic level by inducing mutations and testing the protein in various environmental conditions.

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DNA needs to be accurately copied during DNA replication for a normal cell function. Errors during DNA replication can cause genomic instability that can lead to cancer. To avoid mistakes during the process of DNA replication, nuclease enzymes can act as molecular scissors in removing lethal DNA structures. Therefore, Flap endonuclease 1 (FEN1) is an enzyme that can cleave the 5’flap primer during Okazaki fragment maturation. However, studies have shown that overexpression of FEN1 is associated with different types of cancer. Thus, targeting FEN1 represents a potential for enhancing cancer therapy. However, structural investigation of FEN1 and factors that influence DNA binding need to be comprehensively studied at the molecular level before designing an inhibitor. Thus, this thesis aimed to investigate and compare the catalytic behavior of FEN1wt, FEN1K93A, and FEN1D181A in different experimental conditions. We have found that the activity of FEN1 is affected by the presence of divalent metal ions such as Ca2+ and Mg2+ by performing enzymatic assays. Using the microscale thermophoresis technique, we determined the dissociation constants for FEN1 proteins. Additionally, we performed a thermal shift assay in different conditions which gave us additional insights into the stability of the protein-DNA complex in FEN1. We have found that protein-DNA complex in FEN1D181 is more stable than FEN1wt and FEN1K93A by having a higher melting temperature. Lastly, I used the NMR technique to map the conformational changes within FEN1 proteins upon interacting with divalent metal ions such as Mg2+ ions. To do this, I performed a series of Mg2+ ions titration for FEN1 (WT, K93A, and D181A) using a 2D 1 H 15N TROSY-HSQC experiment to monitor the chemical shifts changes to the chemical environment around the N-H backbone amides of the protein. We have found that both WT and K93A FEN1 proteins interact in a similar way with Mg2+ ions, i.e., explicitly targeting first the higher affinity catalytic site, then spreading around several unspecific low-affinity sites across the protein; however, we observed only the unspecific and weak milli molar binding affinity in FEN1D181A across the entire protein surface upon interacting with Mg2+ ions.