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    Using single molecule fluorescence to study substrate recognition by a structure-specific 5’ nuclease

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    Dissertation_Fahad Rashid_Final.pdf
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    Description:
    Dissertation
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    Type
    Dissertation
    Authors
    Rashid, Fahad cc
    Advisors
    Hamdan, Samir cc
    Committee members
    Habuchi, Satoshi cc
    Di Fabrizio, Enzo M. cc
    Laporo, Joseph
    Program
    Bioscience
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Date
    2018-12
    Embargo End Date
    2019-05-12
    Permanent link to this record
    http://hdl.handle.net/10754/630218
    
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    Access Restrictions
    At the time of archiving, the student author of this dissertation opted to temporarily restrict access to it. The full text of this dissertation became available to the public after the expiration of the embargo on 2019-05-12.
    Abstract
    Nucleases are integral to all DNA processing pathways. The exact nature of substrate recognition and enzymatic specificity in structure-specific nucleases that are involved in DNA replication, repair and recombination has been under intensive debate. The nucleases that rely on the contours of their substrates, such as 5’ nucleases, hold a distinctive place in this debate. How this seemingly blind recognition takes place with immense discrimination is a thought-provoking question. Pertinent to this question is the observation that even minor variations in the substrate provoke extreme catalytic variance. Increasing structural evidence from 5’ nucleases and other structure-specific nuclease families suggest a common theme of substrate recognition involving distortion of the substrate to orient it for catalysis and protein ordering to assemble active sites. Using three single-molecule (sm)FRET approaches of temporal resolution from milliseconds to sub-milliseconds, along with various supporting techniques, I decoded a highly sophisticated mechanism that show how DNA bending and protein ordering control the catalytic selectivity in the prototypic system human Flap Endonuclease 1 (FEN1). Our results are consistent with a mutual induced-fit mechanism, with the protein bending the DNA and the DNA inducing a protein-conformational change, as opposed to functional or conformational selection mechanism. Furthermore, we show that FEN1 incision on the cognate substrate occurs with high efficiency and without missed opportunity. However, when FEN1 encounters substrates that vary in their physical attributes to the cognate substrate, cleavage happens after multiple trials During the course of my work on FEN1, I found a novel photophysical phenomena of protein-induced fluorescence quenching (PIFQ) of cyanine dyes, which is the opposite phenomenon of the well-known protein-induced fluorescence enhancement (PIFE). Our observation and characterization of PIFQ led us to further investigate the general mechanism of fluorescence modulation and how the initial fluorescence state of the DNA-dye complex plays a fundamental role in setting up the stage for the subsequent modulation by protein binding. Within this paradigm, we propose that enhancement and quenching of fluorescence upon protein binding are simply two different faces of the same process. Our observations and correlations eliminate the current inconvenient arbitrary nature of fluorescence modulation experimental design.
    Citation
    Rashid, F. (2018). Using single molecule fluorescence to study substrate recognition by a structure-specific 5’ nuclease. KAUST Research Repository. https://doi.org/10.25781/KAUST-SB502
    DOI
    10.25781/KAUST-SB502
    ae974a485f413a2113503eed53cd6c53
    10.25781/KAUST-SB502
    Scopus Count
    Collections
    Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; Dissertations

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