Kinetic and biophysical characterisation of 5’-flap nucleases in DNA maintenance and repair

Genome fidelity is reliant on structure-specific nucleases to make accurate and reproducible incisions into
DNA intermediate structures during replication and repair processes. Organisms lacking structure-specific
nuclease activity are increasingly susceptible to genetic disease. Flap endonuclease 1 (FEN1) and Fanconi
anaemia nuclease 1 (FAN1) are structure-specific 5’-nucleases with preference for branched DNA structures,
hydrolysing the 5’-flap with metal ion dependency.

FEN1 is a well-characterised nuclease with preference for substrates bearing a 1-nucleotide 3’-flap and a
varying length 5’-flap, subsequently hydrolysing the phosphodiester bond 1-nucleotide into the 5’ duplex of
the substrate, referred to as the +1-1 position. Reproducible hydrolysis at the +1-1 position is crucial during
Okazaki fragment processing and long-patch base excision repair (LP-NER). In doing so, it creates a ligatable
product which is a substrate for DNA ligase. Protein and DNA conformational changes drive FEN1-mediated
catalysis and specificity. 3’-flap binding promotes ordering of the helical arch which encompasses the
threaded 5’-flap before catalysis. Additionally, DNA conformational changes at the +1-1 position are also
required to position the scissile phosphate within attacking distance of the catalytic metal ions, known as
active site transfer. A recent report has suggested that the mutant FEN1 D34A is rate-limited under excess
enzyme conditions by the DNA (and probably protein) conformational change. These experiments used a +1-
1 tandem 2-aminopurine (2-AP) reporter assay, describing a burst phase that precedes the +1-1 hydrolysis,
although this remains untested by other research groups. Furthermore, limited information is available
surrounding arginine and lysine residues (R239, K244 and R245 in human FEN1) in the HELIX-2-TURN-HELIX
(H2TH) DNA binding domain of FEN1 with regards to changes in kinetic rates and conformational change at
the +1-1/-1-2 positions. To answer these questions, a 2-AP reporter assay with the less severe D34N mutant
was performed but presented no evidence of a pre-cleavage burst phase, providing alternative findings to
Song et al. Furthermore, kinetic and exciton-coupled circular dichroism (ECCD) assays using 5’ fluorescein
(FAM) and 2-AP fluorescent labelled substrates respectively showed a modest reduction in multiple/single
turnover rates and change in ECCD signal in FEN1 mutants R239A, K244A and R245A, implying these residues
do not significantly alter FEN1 catalysis or active site transfer.
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FAN1 is a less well-characterised nuclease and is linked to interstrand crosslink (ICL) repair in Fanconi anaemia
(FA) by means of the Fanconi anaemia repair pathway (FA-pathway). Although human and bacterial homolog
structures are currently available, a considerable number of questions remain unanswered concerning FAN1
mechanism. Reports have demonstrated FAN1’s ability to catalyse the reactions of a plethora of DNA
substrates, showing preference for double-flapped substrates bearing a 5’-phosphorylated, 1-nucleotide 5’-
flap and an 8-nucleotide 3’-flap (DF(p1,8)). Hydrolysis of this substrate involves an N+3 incision model from
the 5’-flap terminal phosphate however the pattern alters depending on 5’-flap length. Despite this, in-depth
kinetic parameters for any FAN1 substrates have not yet been defined, raising questions as to the true FAN1
mechanism during 5’-flap processing. In this work, human FAN1 production was optimised and nuclease
assays were performed on various 5’-phopshorylated substrates. Our nuclease assays show concordance to
previous studies of an observed N+3 incision model for the DF(p1,8)3’FAM with multiple products produced.
However, only 1 product was produced for short double-flapped substrates and substrates lacking a
phosphorylated 5’-flap. Finally, the rate of FAN1 catalysis is hindered in the presence of a 5’FAM label
compared to 3’FAM labelled substrates, implying the need to explore other methods of product
measurement for FAN1-DNA reactions outside dHPLC for kinetic characterisation.