Implementing fluorescence enhancement, quenching, and FRET for investigating flap endonuclease 1 enzymatic reaction at the single-molecule level.

ABSTRACT: Flap endonuclease 1 (FEN1) is an important component of the intricate molecular machinery for DNA replication and repair. FEN1 is a structure-specific 5' nuclease that cleaves nascent single-stranded 5' flaps during the maturation of Okazaki fragments. Here, we review our research primarily applying single-molecule fluorescence to resolve important mechanistic aspects of human FEN1 enzymatic reaction. The methodology presented in this review is aimed as a guide for tackling other biomolecular enzymatic reactions by fluorescence enhancement, quenching, and FRET and their combinations. Using these methods, we followed in real-time the structures of the substrate and product and 5' flap cleavage during catalysis. We illustrate that FEN1 actively bends the substrate to verify its features and

SUBMITTER: Sobhy MA 

PROVIDER: S-EPMC8385120 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature