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    Structural and Dynamic Profiles of the WT hFEN1 in solution

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    Name:
    MS_Thesis_Fatimah_Almulhim_Summer 2020.pdf
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    2.653Mb
    Format:
    PDF
    Description:
    MS Thesis
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    Type
    Thesis
    Authors
    Almulhim, Fatimah F. cc
    Advisors
    Jaremko, Mariusz cc
    Committee members
    Falqui, Andrea cc
    Saikaly, Pascal cc
    Program
    Bioscience
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Date
    2020-06
    Embargo End Date
    2020-07-05
    Permanent link to this record
    http://hdl.handle.net/10754/664008
    
    Metadata
    Show full item record
    Access Restrictions
    At the time of archiving, the student author of this thesis opted to temporarily restrict access to it. The full text of this thesis became available to the public after the expiration of the embargo on 2020-07-05.
    Abstract
    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.
    Citation
    Almulhim, F. F. (2020). Structural and Dynamic Profiles of the WT hFEN1 in solution. KAUST Research Repository. https://doi.org/10.25781/KAUST-Y3K6P
    DOI
    10.25781/KAUST-Y3K6P
    ae974a485f413a2113503eed53cd6c53
    10.25781/KAUST-Y3K6P
    Scopus Count
    Collections
    Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; Theses

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