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.
| Identifer | oai:union.ndltd.org:kaust.edu.sa/oai:repository.kaust.edu.sa:10754/675121 |
| Date | 12 1900 |
| Creators | Althobaiti, Afnan |
| Contributors | Jaremko, Lukasz, Biological and Environmental Science and Engineering (BESE) Division, Khashab, Niveen M., Hamdan, Samir, Arold, Stefan T. |
| Source Sets | King Abdullah University of Science and Technology |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | 2024-01-24, At the time of archiving, the student author of this thesis opted to temporarily restrict access to it. The full text of this thesis will become available to the public after the expiration of the embargo on 2024-01-24. |
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