Title: The role of protein and DNA dynamics in 5'-nuclease structure-selective substrate recognition and reactivity

Biography:

David completed his PhD at the age of 29 years from University of California, Los Angeles where he studied nucleic acid and protein structure using NMR spectroscopy. His first postdoc occurred at the City of Hope Beckman Research Institute where he conducted a detailed kinetic study of human flap endonuclease 1 (FEN1). Upon receiving a Marie Curie-Sklodowska fellowship, David moved to Sheffield to work in Jane Grasby’s laboratory where he studied the dynamics of FEN1 with and without substrate using NMR spectroscopy. He is the currently a research co-investigator in the Department of Chemistry at the University of Sheffield.

Abstract

During DNA replication and repair, structure-selective nucleases are required to process two-way junctions bearing overhangs or flaps formed in normal DNA metabolism. Failure to remove such structures efficiently results in genomic instability. One such process that exemplifies this is Okazaki fragment maturation, wherein the 5'-nuclease superfamily member, Flap endonuclease 1 (FEN1), hydrolyses 5'-flapped DNA, which are generated by polymerase d strand displacement synthesis, resulting in ligatable nicked-DNA intermediates (Image A). How FEN1 and related enzymes recognize their respective substrates and select the scissile phosphate diester has been an area of considerable debate for over a decade. More recent structural, biochemical and biophysical work has resulted in a plausible mechanistic model for FEN1 substrate and scissile phosphate diester selectivity. Moreover, the selection of substrate is achieved by three recognition processes; namely, (1) the ability of a two-way dsDNA junction to adopt a bent conformation on the enzyme surface and the discontinuity of the reacting strand such that (2) the 5'-flap may pass under the helical cap of the arch region and (3) a single nucleotide 3'-flap is recognized in a pocket on the enzyme surface (Image B). Once the substrate is recognized, a region in the protein that is disordered in the absence of substrate orders to form the helical arch (a4-a5), thereby delivering two catalytically crucial basic residues to the active site. Furthermore, the protein and DNA move concertedly to deliver the scissile phosphate to the requisite divalent metal ions in the active site by untwisting the helix.