Abstract
Human flap endonuclease 1 (FEN1), one of the structure-specific 5' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5' nuclease mechanisms. This is achieved by coordinating threading of the 5' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage.
Highlights
Structure-specific 50 nucleases are highly conserved phosphodiesterases—endo- and exonucleases—that recognize a diverse range of DNA and RNA structures and play a central role in all aspects of DNA metabolism (Finger et al, 2012; Grasby et al, 2012; Tsutakawa and Tainer, 2012)
We found that bending of the DF substrate requires threading of the 50 flap into the capped-helical gateway of flap endonuclease 1 (FEN1), structuring it to select for a 50 flap with ssDNA, verifying the full base-pairing at the flap junction and binding to the one-nucleotide 30 flap
Observing DNA Bending by FEN1 We characterized the dynamics of DNA bending by FEN1 using single-molecule Forster resonance energy transfer (smFRET) with a variety of fluorescently labeled substrates
Summary
Structure-specific 50 nucleases are highly conserved phosphodiesterases—endo- and exonucleases—that recognize a diverse range of DNA and RNA structures and play a central role in all aspects of DNA metabolism (Finger et al, 2012; Grasby et al, 2012; Tsutakawa and Tainer, 2012). Eukaryotic members of this superfamily include: flap endonuclease 1 (FEN1), a DNA replication and long patch base excision repair protein; Exo, a mismatch repair protein; Xeroderma pigmentosum complementation group G protein (XPG), a nucleotide excision repair protein; and gab endonuclease 1 (GEN1), a homologous recombination protein. It is of significant interest that we understand how these highly conserved proteins recognize and attack a diverse range of DNA structures
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