Principal Investigator: Prof. Lukasz Jaremko

Poster Presenter: Layan Alhindi

Lab: MD3 Lab 

Flap Endonuclease 1 (FEN1) dynamic structure and interactions with DNA and PCNA




DNA is prone to spontaneous alterations leading to mutations if left unrepaired, even under normal biological conditions. Since genetic stability is essential for an individual's survival, both mechanisms for repairing the numerous unintentional lesions that occur in DNA and the accurate process for DNA replication are necessary for maintaining genetic stability. Flap Endonuclease 1 is a central component of cellular DNA metabolism as it removes the 5' overhang flap in DNA repair. However, FEN1 is overexpressed in cancer cells; hence understanding the complex structural dynamics of FEN1 and its catalytic activity in solution and its interactions with DNA substrate will help to target FEN1 for therapeutic purposes. The dynamic and structural properties of proteins are essential to understand functions. The combination of Nuclear magnetic resonance (NMR) and Electron paramagnetic resonance (EPR) constitute a powerful tool for understanding structures, dynamics, and protein-nucleic acid interactions in solution. Both techniques can deliver detailed atomic level information on structure and dynamics at over 14 orders of magnitude of the time scales and amplitudes, from fast local vibrations, loop motions, and folding events, down to slow segmental movements. Here we utilize NMR to determine the structure and local fast and slow dynamics of FEN1. Moreover, with designed mutants for spin labels, we use EPR to determine the large-scale motions and their distribution of free FEN1 catalytic arms and mechanisms of DNA substrate recognition and PCNA binding. Our studies showed that FEN1 is a rigid well-folded enzyme with catalytic motifs exhibiting slow and fast segmental motions. We could assign 274 residues out of 380 amides and all isoleucine, alanine, and methionine methyl groups. We also showed that FEN1 interacts with PCNA to process the 5' flap of RNA primers during DNA replication, in which PCNA enhances the stability of FEN1, maintaining its activity. Moreover, our NMR and EPR experiments showed that DNA binding significantly attenuates the dynamics of FEN1 protein with no detectable effect in the EPR of PCNA binding to FEN1 or FEN1-DNA complex. Moreover, our FEN1 constructs and their DNA complexes are stable for up to 60 hours at room temperature, which eases the ligand screening studies for the FEN1 as a target for cancer therapies. Hence, this study emphasizes the importance of structural dynamics in decoding the protein-DNA interactions and mechanisms of DNA repair