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.
Highlights
Flap endonuclease 1 (FEN1) removes 5Ј-flaps during DNA replication and repair
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 Trinucleotide repeat (TNR) sequences
DNA Substrates—DNA substrates containing either mixed sequence or (CAG)n flaps capable of flap equilibration were used to determine the kinetic parameters of FEN1 (Table 1)
Summary
FEN1 removes 5Ј-flaps during DNA replication and repair. Results: With increasing flap length, rate of removal decreases, and the rate-determining step changes. 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. Defining the kinetic parameters of FEN1 activity on biological substrates is increasingly important because we and others have proposed that FEN1 contributes to genetic instability when DNA replication or long patch base excision repair is initiated in regions of trinucleotide repeat (TNR) DNA, such as CAG sequences [15,16,17,18,19,20]. We elucidate inherent limitations of FEN1 and how those limitations can mediate or promote disease-initiating TNR expansion
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call