Flap endonucleases pass 50-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 50-ends

Flap endonucleases (FENs), essential for DNA replication and repair, recognize and remove RNA or DNA 50-flaps. Related to FEN specificity for substrates with free 50-ends, but controversial, is the role of the helical arch observed in varying conformations in substrate-free FEN structures. Conflicting models suggest either 50-flaps thread through the arch, which when structured can only accommodate single-stranded (ss) DNA, or the arch acts as a clamp. Here we show that free 50-termini are selected using a disorder-thread-order mechanism. Adding short duplexes to 50-flaps or 30-streptavidin does not markedly impair the FEN reaction. In contrast, reactions of 50-streptavidin substrates are drastically slowed. However, when added to premixed FEN and 50-biotinylated substrate, streptavidin is not inhibitory and complexes persist after challenge with unlabelled competitor substrate, regardless of flap length or the presence of a short duplex. Cross-linked flap duplexes that cannot thread through the structured arch react at modestly reduced rate, ruling out mechanisms involving resolution of secondary structure. Combined results explain how FEN avoids cutting template DNA between Okazaki fragments and link local FEN folding to catalysis and specificity: the arch is disordered when flaps are threaded to confer specificity for free 50-ends, with subsequent ordering of the arch to catalyze hydrolysis. INTRODUCTION Structure sensing 50-nucleases are vital for DNA replication, repair, and recombination. Operating without regard to sequence, 50-nucleases recognize defined nucleic acid junctions and catalyze the hydrolysis of specific phosphate diester bonds (1–3). Exemplary junctions for 50-nuclease cleavage are formed during lagging strand DNA replication and long-patch base excision repair (lpBER), where 50-extensions (flaps) occur at adjacent duplexes (Okazaki fragments and lpBER intermediates) as a consequence of polymerase strand displacement synthesis. Divalent metal ion-dependent flap endonucleases (FENs), the prototypical 50-nuclease family members, are the enzymes that catalyze removal of 50-flaps. This hydrolytic processing yields 50-phosphorylated-nicked DNAs for subsequent ligation and during human replication must take place at least 50 million times per cell cycle. The importance of 50-flap elimination is demonstrated by the lethality of fen1 / ) knockouts in mammals (4). FENs endonucleolytically remove 50-flaps, thereby avoiding repetitive exonucleolytic processing. Even before structures of FEN proteins became available, it was suggested that FEN specificity for junctions with free 50-termini, and discrimination against other junctions lacking this feature that occur at replication forks, could be achieved by threading the 50-flap DNA through a hole in the protein (5). Yet this proposal has remained controversial, and the basis for end specificity has remained enigmatic. Subsequent structural studies did indeed reveal a hole in FEN proteins formed by helices linking the main DNA-binding domains straddling the active site (Figure 1A) (6,7). Known as the helical arch, this subdomain is partially disordered in some X-ray *To whom correspondence should be addressed. Tel: +44 11 42 229478; Fax: +44 11 42 229346; Email: [email protected] The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Present address: Peter Thompson, NIHR Trainees Coordinating Centre, Leeds Innovation Centre, 103 Clarendon Road, Leeds LS2 9DF, UK. Published online 8 February 2012 Nucleic Acids Research, 2012, Vol. 40, No. 10 4507–4519 doi:10.1093/nar/gks051 The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/nar/article-abstract/40/10/4507/2411615/Flap-endonucleases-pass-5-flaps-through-a-flexible by guest on 16 September 2017 structures (Figure 1B and Supplementary Figure S1A) (8–10). In structured form, the arch is only large enough to accommodate singlebut not double-stranded (ds) DNA, appearing to account for FEN specificity. Support for a threading mechanism for end specificity came from biochemical experiments that suggested that forming a duplex within the 50-single-stranded (ss) flap or binding of proteins to this region of substrates prevented the FEN reaction (11,12). Structural studies of bacteriophage T4FEN bound to a pseudo-Y (pY) DNA substrate did show a 50-flap within the arch region (9). However, in this complex, the DNA did not occupy the divalent metal ion-free active site and one helix of the arch was disordered (Figure 1C and Supplementary Figure S1B). Nevertheless, models can be created using this structure by overlay with FENs crystallized with ordered arches showing the flap DNA passing through, although not yet positioned in the active site for reaction (Figure 1D), furthering controversy regarding a possible threading mechanism for specificity. In contrast, several studies have challenged the hypothesis that the helical arch enforces FEN specificity. Apparently in conflict with earlier literature, human FEN1 (hFEN1) has been demonstrated to endonucleolytically process so-called gapped flap substrates (13). Gapped flaps contain a short 50-region of duplex that cannot pass through a structured helical arch. Other biochemical studies on the question of FEN 50-flap accommodation have also produced results that are apparently at odds with a threading model (14,15). Thus, as an alternative to passage of substrate through the arch, this subdomain has been suggested instead to act as a clamp (3,7,14,16). One possible explanation of FEN specificity known as tracking in which FENs were proposed to initially interact with ss flaps either by threading or clamping and slide along these until junctions were encountered has been discredited (2,3,11–13,16–18). Deciphering the origins of FEN1 specificity for 50-flaps is made more complex by other 50-nucleases that are sequence related to FENs but have differing specificities (1–3,19). In humans, EXO1, the mismatch and resection 50-nuclease is most closely related to FEN1. EXO1 catalyses the processive hydrolyses of the 50-termini of gapped, nicked and blunt duplex DNAs. Like FEN1, EXO1 can also endonucleolytically remove 50-flaps (20). Another superfamily member XPG, the 50-nuclease of nucleotide excision repair (NER), acts upon bubble substrates (21). The major human Holliday junction resolvase is suggested to be GEN1, another superfamily member (22). However, neither NER bubbles nor four-way junctions possess free 50-termini in vivo. The 50-portion of these substrates could therefore not be passed through an arch. Recent structures of hFEN1 and hEXO1 bound to substrates and products in the presence of active site metal Figure 1. Structures of T5, T4 and human FENs with and without DNA. (A) Structure of T5FEN (1UT5) with transparent surface to highlight the helical arch and resulting hole above the active site bound divalent metals (black spheres). (B) Structure of hFEN1 (1UL1, X chain; pink) with transparent surface representations showing the disordered arch (missing arch residues, dotted lines; active site metal ions, black). (C) T4FEN structure in complex with a pseudo-Y (pY) substrate (2IHN) without metal ions. Based on alignment with a substrate-free T4FEN structure (1TFR), the location of active site divalent metal ions (black spheres) is shown along with template (brown) and 50-flap strands (yellow) of the pY and disordered residues (dotted lines). (D) Model of T5FEN (1UT5) in complex with a pY substrate with active site divalent metals, protein and DNA colored as in (A) and (C) based on alignment with the T4FEN-DNA structure (2IHN) shows that the 50-flap could go through the helical arch. Some steric clashes are observed suggesting conformational changes. (E) Structure of hFEN1 in complex with the product of reaction of a double-flap substrate (3Q8K). Template DNA (brown), the cleaved 50-flap DNA strand (yellow), and 30-flap DNA (purple) are shown with active site metal ions (gray) and a K ion (purple). (F) Active site of the hFEN1-product DNA complex (3Q8K) showing the 50-phosphate monoester product interacting with active site divalent metal ions (black spheres). Note, this nucleobase is not paired with the template. 50-Nuclease superfamily conserved active site carboxylates (red) and helical arch a4 Lys93 and Arg100 (blue) are shown. A tyrosine residue (Tyr40) from a2 stacks upon the unpaired nucleobase. 4508 Nucleic Acids Research, 2012, Vol. 40, No. 10 Downloaded from https://academic.oup.com/nar/article-abstract/40/10/4507/2411615/Flap-endonucleases-pass-5-flaps-through-a-flexible by guest on 16 September 2017 ions highlight the similarities between 50-nuclease superfamily members (Figure 1E) (2,3,23). Despite analogous structures for hFEN1 and hEXO1 complexes with product DNAs, which include conserved contacts between the cleaved 50-phosphate and helical arch residues (Figure 1F), differing interpretations for the requirement for threading versus clamping indicate that key questions regarding the basis for substrate specificities within the FEN-like 50-nucleases remain. Here, using functional studies with modified DNAs, we resolve how FENs accommodate the 50-region of substrates, demonstrate that processing of 50-gapped flaps is an hFEN1 activity that proceeds by the same mechanism and propose a universal model for departure of DNA from the active sites of 50-nuclease superfamily members. Moreover, our results explain how FENs can function to rapidly remove flaps during replication without risk of destroying template DNA between Okazaki fragments. MATERIALS AND METHODS Over-expression and purification of T5FEN and hFEN1 T5FEN and hFEN1 (wild-type and K93A) were over-expressed and purified as described (2,24). Synthesis and purification of oligonucleotide substrates Oligonucleotides (ODNs) were synthesized using an ABI model 394 DNA/RNA synthesizer or by DNA Technology A/S (Risskov, Denmark) using 50-fluorescein-CEphosphoramidite (6-FAM) or 30(6-FAM)-CPG to incorporate 50-FAM or 30-FAM, respectively, and biotin TEG phosphoramidite to add biotin (Link Technologies, Lanarkshire, UK). The long tether 30-biotin substrate [21 nt pY-30B] was constructed using 30-biotin TEG followed by three additions of spacer-CE-phosphoramidite-18. ODNs were purified by reverse-phase (RP) HPLC (Waters bridge 10 250mm C-18 column) using triethylammonium acetate buffers pH 6.5 with a gradient of acetonitrile. Purified ODNs were desalted using NAP-10 columns and subjected to MALDI-TOF mass spectrometry. Residual divalent metal ion contaminants were removed by treatment with Chelex resin. Experimental MWs were all within 3 Da of calculated. A complete list of ODNs is contained in Supplementary Figure S2. Determination of the rate of decay of enzyme substrate complexes Substrates were annealed as described (13,17). Enzyme and substrate were pre-incubated at 20 C (hFEN1) or on ice (T5FEN) for 2min in 25mM HEPES, pH 7.5, 50mM KCl, 2mM CaCl2, 1mM DTT and 0.1mg/ml BSA (hFEN; calcium buffer) or 25mM HEPES, pH 7.5, 50mM KCl, 1mM EDTA, 1mM DTT and 0.1mg/ml BSA (T5FEN; EDTA buffer) to form ‘premixed’ complexes. If required, five equivalents (with respect to [S]) of streptavidin (SA) were added before (‘blocked’ reactions) or after (‘trapped’ reactions) addition of enzyme and incubated accordingly for 5min. Increasing the concentration of streptavidin did not alter the outcome. Samples were warmed at 37 C, and the reaction was initiated by mixing with an equal amount of magnesium buffer as above but containing 16mM MgCl2 instead of CaCl2, (hFEN1) or 20mM MgCl2 instead of EDTA (T5FEN). The final concentrations of enzyme and substrate were 500 nM and 5 nM, respectively. For ‘trapped’ and ‘premixed’ reactions, sampling was carried out using quench flow apparatus (RQF-63 quench flow device, Hi-Tech Sci Ltd., Salisbury, UK). After time delays of 6.4ms to 51.2 s, quench (8M Urea containing 80mM EDTA) was added. ‘Blocked’ reactions were sampled manually. Reactions were analyzed by dHPLC equipped with a fluorescence detector (Wave system, Transgenomic, UK) as described (13,17,25,26). After quenching the presence of SA did not alter the dHPLC retention time with tetrabutyl ammonium bromide as the ion-pairing reagent (Supplementary Figure S3). The formation of product formed over time (Pt) was fitted to Equation (1), to determine the first-order rate constant (k) where P1 is the amount of product at end point: Pt 1⁄4 P1 1 e kt ð1Þ Competition experiments Competitor ODNs were pY or double-flap substrates without FAM label or biotin (Supplementary Figure S2). Enzyme and FAM–biotin–substrate were incubated at 20 C for 2min (hFEN1) or on ice for 2min (T5FEN) in either calcium buffer (hFEN1) or EDTA buffer (T5FEN) as above. For ‘trapped’ reactions, five equivalents of SA were added followed by incubation for a further 1min. Competitor substrate was then added, and the mixtures were incubated for 10min at 37 C. Increasing this time to 20min had no impact on the outcome. An equal volume of magnesium buffer (as above) was added to initiate reaction producing final concentrations of enzyme (500 nM), FAM-labeled substrate (5 nM) and competitor (2.5 mM, T5FEN; 5 mM, hFEN1). Reactions were sampled, quenched and the amount of product determined as above. In experiments where streptavidin and/or competitor were not added an equal volume of appropriate buffer was, and all samples underwent identical incubations. Preparation of azide-alkyne ODN 9 -(50-O-Dimethoxytrityl-20-deoxyribofuranosyl)-N2[(dimethylamino) methylidene]-2-amino-6-methylsulfonyl purine-30-(2-cyanoethyl-N,N-diisopropyl)-phospho ramidite (27) was used to construct an ODN with 30-FAM and a 50-alkyne (6-Hexyn-1-yl-(2-cyanoethyl)-(N,Ndiisopropyl)-phosphoramidite (Glen Research) using mild/fast deprotection phopshoramidites for dC, dA and dG. Following 1 mmol scale synthesis, the CPG-bound ODN was treated with 200 ml of 11-Azido-3,6,9trioxaundecan-1-amine:acetonitrile:DBU at a ratio of 9:9:2 at 37 C for 48 h with gentle mixing. Concentrated NH3(aq) (1ml) was then added and the mixture left for Nucleic Acids Research, 2012, Vol. 40, No. 10 4509 Downloaded from https://academic.oup.com/nar/article-abstract/40/10/4507/2411615/Flap-endonucleases-pass-5-flaps-through-a-flexible by guest on 16 September 2017 a further 72 h at room temperature. After evaporation to dryness, the residue was suspended in water (150ml) and extracted with diethyl ether (3 500ml). The aqueous layer was removed and then purified by HPLC as described above. MW (AA-HP) 11 173.56 calculated; 11 176 found. Preparation of triazole ODN Reactions contained AA-HP (10 nmol) mixed with CuSO4.5H2O (750 nmol) sodium ascorbate (30 mmol) and tris-3-hydroxypropyltriazolylamine (28) (21mmol) in a total volume of 1ml with NaCl (final concentration 0.2M) and were incubated at room temperature overnight with gentle mixing. The reaction mixture was desalted (NAP-10) and purified by RPHPLC under denaturing conditions (as for other ODNs but at 55 C). The triazole ODN (Z-HP) eluted 3.3min earlier than AA-HP (Figure 5C). Determination of the rates of the reaction of triazole cross-linked gapped substrates Kinetic analysis was carried out using GAP DF-AA and GAP DF-Z substrates at a concentration of 50 nM, with 5 pM WT hFEN1 or 2.5 nM K93A at 37 C in 50mM HEPES, pH 7.5, 100mM KCl, 8mM MgCl2, 1mM DTT and 0.1mg/ml BSA. Samples of reaction mixture were quenched in an equal volume of 250mM EDTA, pH 8.0. Reactions were analyzed as above. Initial rates of reaction were obtained from plots of amount of product versus time.