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. 5 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.