title: Rate-determining Step of Flap Endonuclease 1 (FEN1) Reflects a Kinetic Bias against Long Flaps and Trinucleotide Repeat Sequences.
abstract: Flap endonuclease 1 (FEN1) is a structure-specific nuclease responsible for removing 5'-flaps formed during Okazaki fragment maturation and long patch base excision repair. In this work, we use rapid quench flow techniques to examine the rates of 5'-flap removal on DNA substrates of varying length and sequence. Of particular interest are flaps containing trinucleotide repeats (TNR), which have been proposed to affect FEN1 activity and cause genetic instability. We report that FEN1 processes substrates containing flaps of 30 nucleotides or fewer at comparable single-turnover rates. However, for flaps longer than 30 nucleotides, FEN1 kinetically discriminates substrates based on flap length and flap sequence. In particular, FEN1 removes flaps containing TNR sequences at a rate slower than mixed sequence flaps of the same length. Furthermore, multiple-turnover kinetic analysis reveals that the rate-determining step of FEN1 switches as a function of flap length from product release to chemistry (or a step prior to chemistry). These results provide a kinetic perspective on the role of FEN1 in DNA replication and repair and contribute to our understanding of FEN1 in mediating genetic instability of TNR sequences.
authors.affiliation: Department of Chemistry, Brown University, Providence, Rhode Island 02912.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Tarantino
authors.affiliation: Department of Chemistry, Brown University, Providence, Rhode Island 02912.
authors.forename: Katharina
authors.initials: K
authors.lastname: Bilotti
authors.affiliation: Department of Chemistry, Brown University, Providence, Rhode Island 02912.
authors.forename: Ji
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Chemistry, Brown University, Providence, Rhode Island 02912. Electronic address: sarah_delaney@brown.edu.
authors.forename: Sarah
authors.initials: S
authors.lastname: Delaney
mesh_terms: D001483:Base Sequence; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D018911:Trinucleotide Repeats
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D045585:Flap Endonucleases; C468317:FEN1 protein, human
keywords: DNA enzyme; DNA repair; DNA replication; base excision repair (BER); enzyme kinetics