Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

Abstract

Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalysed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterised. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs containing two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover numbers for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilise the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concentrations below saturation with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.

The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

“…Step-Product release has been shown to be partially rate-limiting for T5FEN1-catalyzed reactions (23). To determine if this was also the case for hFEN1, single turnover (ST) experiments were performed using a quench flow apparatus under conditions whereby (23,48).…”

“…To determine if this was also the case for hFEN1, single turnover (ST) experiments were performed using a quench flow apparatus under conditions whereby (23,48). Reactions were initiated by combining equal volumes of enzyme and substrate in magnesium-containing buffer and quenched with sodium hydroxide.…”

“…Thus, all four steady-state hFEN1-catalyzed reactions studied herein were rate-limited by product release. The degree to which product release attenuates T5FEN1 multiple turnover rates has previously been assessed from the quotient of the ST rate and turnover number (k STmax /k cat ) (23). For the four hFEN1 substrates studied, the extent to which product release attenuated multiple turnover (MT) rates varied ϳ7-to 11-fold (Table 2).…”

“…Biochemical characterizations of FEN1 proteins from various organisms have shown that this family of nucleases can perform phosphodiesterase activity on a wide variety of substrates; however, the efficiency of catalysis on various substrates differs among the species. For instance, phage FEN1s prefer pseudo-Y substrates (22,23), whereas the archaeal and eukaryotic FEN1s prefer 5Ј-flap substrates (21,24,25), which have two dsDNA domains, one upstream and downstream of the site of cleavage, and a 5Ј-ssDNA protrusion (Fig. 1A).…”

“…Single Turnover Rapid Quench Experiments-Rapid quench experiments were conducted at 37°C using an RQF-63 device from HiTech Ltd. (Salisbury, UK) as previously described (23,48). Briefly, an 80-l aliquot of enzyme in reaction buffer (50 mM HEPES-K ϩ , pH 7.5, 0.1 mg/ml bovine serum albumin, 2.5 mM tris(3-hydroxypropyl)phosphine, 8 mM Mg(OAc) 2 , 100 mM KCl) was mixed with an equal volume of substrate in reaction buffer.…”