Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I
Yuqian Shia, Homme W. Hellingaa, and Lorena S. Beesea,1
aDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710
Contributed by Lorena S. Beese, April 26, 2017 (sent for review March 23, 2017; reviewed by James M. Berger, Ben Luisi, and Stephen C. West)
Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure- active site, the mobile arch (16, 17). The scissile bonds in these 2+ specific 5′-nuclease superfamily. Its dominant, processive 5′–3′ exonu- complexes are located near two catalytic Mg ions in the active clease and secondary 5′-flap endonuclease activities participate in var- site, or appear to move toward them. A 5′-flap substrate, de- ious DNA repair, recombination, and replication processes. A single termined in a bacteriophage T5 FEN complex, is threaded through active site processes both recessed ends and 5′-flap substrates. By ini- an aperture in the interior of the mobile arch, also placing its scissile tiating enzyme reactions in crystals, we have trapped hExo1 reaction bond in close proximity to the two metals (21). Taken together, intermediates that reveal structures of these substrates before and these observations suggest that both substrate classes could adopt a after their exo- and endonucleolytic cleavage, as well as structures of common transition state within their shared active site (16, 17). uncleaved, unthreaded, and partially threaded 5′ flaps. Their distinctive This hypothesis has been difficult to test, because it is challenging 5′ endsareaccommodatedbyasmall,mobilearchintheactivesitethat to capture substrate or product complexes in conformations that binds recessed ends at its base and threads 5′ flaps through a narrow correspond to intermediates just before or after the cleavage step aperture within its interior. A sequence of successive, interlocking con- without using inhibitory metals that perturb coordination geome- formational changes guides the two substrate types into a shared re- tries (16, 17) or mutations that destroy a metal-binding site (21). action mechanism that catalyzes their cleavage by an elaborated Accordingly,thenatureofthetransition state(s) has not been variant of the two-metal, in-line hydrolysis mechanism. Coupling of established definitively (16, 17, 21). Furthermore, conformational substrate-dependent arch motions to transition-state stabilization sup- heterogeneity of the recessed or flap complexes observed in dif- presses inappropriate or premature cleavage, enhancing processing ferent experiments also prevented the direct structural comparisons fidelity. The striking reduction in flap conformational entropy is cata- required to test this hypothesis. Finally, no structures have been captured of threading intermediates, which might shed light on the lyzed, in part, by arch motions and transient binding interactions be- mechanisms by which disparate starting structures are guided into a tween the flap and unprocessed DNA strand. At the end of the common transition state. We report on time-resolved trapping of observed reaction sequence, hExo1 resets without relinquishing DNA 12 structures, representing intermediates in the hExo1 reaction binding, suggesting a structural basis for its processivity. cycle, which enabled us to address these questions. Rad2/XPG superfamily | DNA repair | flap endonuclease | crystallography | Results exonuclease hExo1 Structure. Previously, we have shown that the catalytically active N-terminal domain (residues 1–352) of hExo1 (16) forms a uman exonuclease 1 (hExo1) is a member of the RAD2/XPG ′ – H5 -structure specific nuclease superfamily (Exo1, FEN1, XPG, Significance and GEN1) that plays essential roles in three central aspects of genome maintenance (1–4): replication, repair, and recombination. Human exonuclease 1 (hExo1) is a 5′-structure–specific nuclease and The common catalytic core of this superfamily processes a variety of a member of the RAD2/XPG superfamily that plays important roles disparate DNA substrates (5–8), including nicked, gapped, single- in many aspects of genome maintenance. The means by which stranded 5′ flaps; Holliday junctions; and bubbles. The specificity of individual family members process multiple, structurally disparate substrate recognition and efficiency of subsequent cleavage is tai- substrates has been a long-standing question. The reaction in- lored to the biological function of each family member (1, 3–5). termediate structures reported here reveal that this remarkable feat hExo1 has dominant 5′–3′ exonuclease activity on nicked, gapped, is achieved by a series of orchestrated conformational changes that or recessed-end DNA, but also cleaves 5′-flap substrates endonu- guide disparate substrates into a common, catalytically competent cleolytically (7, 8). Its primary exonucleolytic activity is important in conformation, where they are cleaved by an enhanced variant of mismatch repair (1), double-stranded break repair (2), and the the two-metal, in-line hydrolysis mechanism. The observed motions DNA damage response (9). The secondary endonucleolytic activity not only enable exo- and endonucleolytic cleavage of gapped and of hExo1 is involved in processing immunoglobin class-switching re- 5′-flap substrates, respectively, but also encode unanticipated fea- combination intermediates (10), trinucleotide repeats (11), DNA- tures, including mechanisms that enhance processing fidelity and RNA hybrids such as Okazaki fragments (12), and ribonucleotide account for processivity. excision repair (13). hExo1 and the predominantly endonucleolytic
FEN1 family member exhibit partially overlapping functions, com- Author contributions: Y.S. and L.S.B. designed research; Y.S. and L.S.B. performed re- plicating assignment of their individual biological roles (4, 11–13). search; Y.S. and L.S.B. analyzed data; and Y.S., H.W.H., and L.S.B. wrote the paper. Exo1 knockout mice exhibit reduced survival, increased susceptibility Reviewers: J.M.B., Johns Hopkins Medical Institute; B.L., University of Cambridge; and S.C.W., to developing lymphomas (14), and sterility in both males and fe- The Francis Crick Institute. males (14). Defects in hExo1 are implicated in human cancers (15). The authors declare no conflict of interest. Structural studies of hExo1 (16) and FEN1 (17) have shown that Data deposition: The atomic coordinates and structure factors have been deposited in the asingleactivesitecleavesboth5′ recessed ends and 5′ flaps. The Protein Data Bank, www.pdb.org (PDB ID codes 5UZV, 5V04, 5V05, 5V06, 5V07, 5V08, different termini are accommodated by a process that “threads” 5′ 5V09, 5V0A, 5V0B, 5V0C, 5V0D, and 5V0E). flaps, but not recessed ends, through the protein (18–20). Structures 1To whom correspondence should be addressed. Email: [email protected]. of recessed substrates revealed that their 5′ phosphates are bound This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. at the base of a conformationally diverse structural element in the 1073/pnas.1704845114/-/DCSupplemental.
6010–6015 | PNAS | June 6, 2017 | vol. 114 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1704845114 Downloaded by guest on September 30, 2021 A Unprocessed strand Processed strand (Fig. 3 A and B).Inthesecondmotion,themobilearchundergoes the first of two rotations (A), thereby adopting “open” and “closed” 3′ conformations in stages 1 and 2, respectively (Fig. 3B and Fig. S1C). 5′ Following initiation by metal addition, we captured a third stage rIV–rVII HTH (structures ; Fig. 4 and Figs. S2 and S3) with four ap- 3′ H2TH proaches that slow down the reaction rate: in the wild-type enzyme + (K+/Na+ site) with Mn2 (rIV;Fig.4A–C and Fig. S2A), in a D173A mutant α4-α5 + + with cognate Mn2 ions (rV; Figs. S2B and S3)orMg2 (rVI; Fig. mobile arch C 2+ rVII D 5′ S2 ), and in a D225A mutant with Mn ( ; Fig. S2 ). At this stage, the DNA has twisted relative to stage 2. This motion “seats” C-terminal region the scissile phosphodiester bond in the active site next to two fully assembled metal-binding sites and positions a water molecule A B Active site nearby (Fig. 4 and Figs. S1 and and S3). We interpret this 2+ 2+ stage to represent a DNA substrate just before cleavage. (2 Mg /Mn ) In this catalytically competent DNA substrate complex, the 5’ Scissile bond 2+ 2+ B (Cleavage site) two catalytic Mg cations (or their Mn substitutions), M1 and f2 ∼ f1 Processed strand M2, are 3.6 Å apart. M1 is coordinated directly by D152, and d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 3’ through water-mediated interactions with D30 and D78. M2 is coordinated by D152, D171, and D173 (Fig. 4 and Fig. S2). The 3’ u3 u2 u1 d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 5’ Unprocessed strand scissile phosphodiester forms bridging interactions with both metals (Fig. 4 C–E). The water molecule is positioned appro- Fig. 1. hExo1 catalytic domain bound to DNA. (A) Protein–DNA complex: gray, priately for an in-line hydrolytic attack and is held in place by catalytic domain; cyan (circled), conserved active-site carboxylates; blue spheres, interactions with the N terminus, D225, and M2. catalytic metal ions; green, mobile arch; purple, H2TH; purple sphere, K+/Na+ site; Stage 3 formation is accompanied by a second rotation of the dark red, helix-turn-helix (HTH); pink, C-terminal region; gold, unprocessed mobile arch that “clamps” thescissilebondintoplace(B;Fig.3C strand; yellow, processed strand; red, scissile bond. This color coding scheme is and Fig. S1C). The clamped arch is stabilized by hydrogen bonds used throughout, unless indicated otherwise. (B) Nucleotide naming system. that “lock” a “latch” region at its base (Fig. 3C)andFig. S4A). The clamping motion engages two catalytically important residues, R92 and K85, to form ion pairs with a scissile bond oxygen, thereby A bean-shaped structure (Fig. 1 ). Its common core region is further stabilizing the developing transition state (Figs. 3C and 4D). structurally similar to FEN1 (16, 17). The duplex portion of the In stage 3, the 5′ phosphate of the processed strand is positioned DNA substrate is bound on the protein surface, tethered to a at the base of the mobile arch, where it is held in place by three + + – surface region that includes a K /Na -binding site [the helix interactions. First, it forms a hydrogen bond with the “junction – two-turn helix tether (H2TH)] (16). The active site is located guide” residue H36, which has flipped into a down conformation, near the point where the double-stranded DNA bifurcates into 5′ where it remains stacked with d1 (Fig. 4A and Fig. S2). Second, the and 3′ single strands (the dsDNA “junction”), the former of “arch guide” residue, Y32, flips into a down conformation, permit- which is processed (Fig. 1B). The two-helix (α4 and α5) mobile ting steric access of the 5′-terminal phosphate to the active site (Fig. arch and an immobile two-helix (α2 and α3) wedge domain that 4A and Fig. S2). Finally, a third metal binds the 5′ phosphate. The contains a hydrophobic region are situated at this junction (16). coordination sphere of this metal is completed by additional water- mediated interactions at the mobile arch base (Fig. 4A and Fig. S2). Time-Resolved Trapping of Reaction Intermediates. We have trapped We have also captured an intermediate along the path of stage DNA complexes at various stages of both exo- and endonucleolytic 3 formation (rIII;Fig.2A and C). In this structure, the mobile cleavage (Fig. 2A and Tables S1 and S2). Substrates (Table S3)can arch position is open, not clamped, and the latch is unlocked. The be captured in initial binding events by cocrystallization in the two metals are both present, and the arch guide residue, Y32, has absence of metals. Under favorable circumstances, various sub- rotated down (Fig. 2C and Fig. S4B). However, the scissile bond is strate intermediates and products can be obtained by initializing not positioned for cleavage: The metal that binds the 5′ phosphate 2+ 2+ the reaction in crystallo with addition of Mg (or Mn ,which is absent, and the two catalytic residues, R92 and K85, remain slows down the reaction rates and provides an anomalous signal disengaged. Although the hydrolytic water molecule is present, it that defines bound metal positions) and trapping intermediates by is not yet positioned for in-line attack. freezing the crystals in liquid nitrogen after different incubation Following stage 3, we captured a fourth stage that corresponds to time intervals. Mutations that substantially lowered catalytic rates, the cleaved substrate with both products still in place (rVIII;Fig.4 + but retained metal binding, have aided trapping. A–C and Fig. S2E). This complex was captured using Mn2 in the + + D225A mutant. With wild-type enzyme and Mg2 or Mn2 ,we Recessed-End Cleavage. Using a substrate with a 5′ recessed end, we obtain mixtures of both substrate and product in our experimental have identified five major stages along the exonucleolytic reaction time series. In stage 4, neither the protein nor DNA has moved, but coordinate (Fig. 2 and Movie S1), each defined by one or more changes in the electron density around the scissile bond are con- experimental structures with distinct features. Stages 1 and 2 of + + sistent with its cleavage and incorporation of the attacking water the reaction were both obtained before initiation by Mg2 or Mn2 into the newly formed 5′ phosphate of the resulting product (Fig. addition, and are presumed to be in dynamic equilibrium with 4B). The M1 and M2 metals coordinate the termini of the two each other. In stage 1 (rI;Figs.2B and C and 3A and Fig. S1), the products: M2 binds the third oxygen (formerly the attacking water) dsDNA portion of the substrate at the d6 base pair is bound to the of the newly formed 5′ phosphate, and M1 binds the 3′ hydroxyl of H2TH tether, the u1 base contacts α2, but the junction does not the single-nucleotide product. BIOCHEMISTRY form contacts with the protein. Stage 2 (rII structure; Fig. 3) is characterized by two important motions relative to stage 1. First, a Product Release. In the final stage that we were able to capture combined translation and rotation (screw) of the DNA moves the (structure rIX; Fig. 2), we observed nucleotide monophosphate H2TH contact to the adjacent base (d7) in the dsDNA region (Fig. product release while retaining binding of the rest of the DNA 3A). This motion “engages” the junction at the d1 base pair with the substrate, accompanied by a partial reversal of the mobile arch junction guide residue H36, located on the α2-α3 wedge domain motions. This state is challenging to obtain, because in prolonged (Fig. 3A). It also organizes the unprocessed ssDNA strand to bend experimental timelines, crystals become disordered as synchrony is sharply around the wedge domain (16, 17), moving u1 into the lost and eventually dissolve. In state 5, the mobile arch has un- “hydrophobic wedge pocket” located between the α2- and α3-helices dergone the inverse B rotation and is now in its closed conformation.
Shi et al. PNAS | June 6, 2017 | vol. 114 | no. 23 | 6011 Downloaded by guest on September 30, 2021 Consequently, the two catalytic residues, R92 and K85, are dis- seated in the active site. Taken together, these results suggest that engaged from the DNA. The Y32 arch guide has flipped into its up f5I corresponds to an intermediate in which the flap base proximal position, reinstating the steric block to the active site. The dsDNA to the junction (f1) remains to be threaded. junction, newly formed at d2, is disengaged from the H36 guide. Flap Cleavage. We captured a threaded, endonucleolytically Flap Threading. Complexes with 5-nt (f5I)and2-nt(f2I) flap sub- cleaved, 2-nt flap complex, f2II, with its scissile bond placed in the + strates were captured in the absence of Mg2 . In both, the dsDNA active site (Fig. 5 C and D and Fig. S2F). In this structure, the is loosely bound to the H2TH tether. In f2I, neither processed nor mobile arch is clamped. We calculated electron density maps for unprocessed DNA single strands were observed, and the junction both uncleaved and cleaved models. In the former, a large (5.7- is not engaged. The protein conformation and dsDNA position sigma contour level) negative difference electron density peak was therefore closely resemble the stage 1 recessed substrate (rI). observed at the scissile bond, which was absent in the latter, con- In f5I, four of the five 5′-flap nucleotides (f2–f4) are threaded sistent with a cleaved product complex containing a 5′-phosphate. through an aperture within the mobile arch (Figs. 5 A, B, and D This threaded complex has therefore been trapped at stage 4, and Fig. S5) in which the helices have parted slightly to form a as defined by the recessed-end cleavage cycle analysis. As with small aperture. Both guide residues, Y32 and H36, are in their up recessed-ended substrates, cleavage occurred 1 bp inside the duplex conformation. The f2-f3 nucleotides are positioned inside the arch. region (Fig. 5B). Accordingly, the product is a three-base, single- Their bases are flipped relative to the duplex region, pointing to stranded 5′ flap (d1-f1-f2): d1 remains paired with the unprocessed the bottom of the arch. Their phosphates point toward a cluster of strand, d1 and f1 are inside the arch aperture, and the f2 base is arginines in α5. The f4 nucleotide is positioned beyond the arch disordered. The two nucleotides flanking scissile bonds in this and is not flipped. The f5 base is disordered. The f1 nucleotide complex and the exonucleolytic cleavage product of stage 4 (rIV)are forms a G-T mismatch with the u1 base of the unprocessed strand. near-perfectly superimposed (Fig. 4E and Fig. S2 E and F). The The dsDNA junction, formed here by the f1-u1 mismatch, is dis- environment of the scissile bond therefore is identical in 5′-flap engaged. The expected scissile bond (between d1 and d2)isnot cleavage and recessed-end substrates.
Reaction coordinate: 1 2 3 4 5 A (Main stages) Tethered DNA Seated junction Assembled active site Cleaved product Reset enzyme Screw Twist Partial untwist dsDNA Anchored to K+ site Seated and anchored Wedged and anchored Seated and anchored Anchored u1 d1 d1 d1 d1 Unprocessed strand Prejunction stacked ( ), Junction stacked ( ), Junction stacked ( ), Junction stacked ( ), Prejunction stacked ( ), 3’-overhang disordered 3’-overhang bent 3’-overhang bent 3’-overhang bent 3’-overhang bent
{ + K site nucleotide d6 d7 d7 d7 d7 d7
DNA substrate DNA Scissile bond Disengaged Disengaged Disengaged Engaged Cleaved Disengaged Rotation A Rotation B Inverse rotation B Mobile arch Open Clamped Closed Partial clamped Clamped Closed Y32 flipped H36 flipped Y32 and H36 flipped { Y32, H36 guides Disengaged Engaged: up, up Engaged: down, down up, down down, down Disengaged: up, up
DNA binding DNA Latch Unlocked Unlocked Unlocked Locked Locked Unlocked R92, K85 Disengaged Disengaged Disengaged Engaged Engaged Disengaged Structural components { Two metals Empty Empty Occupied Occupied Occupied Less ordered Attacking water Absent Absent Present Inline Incorporated Absent Catalysis Experimental timeline: { )serutcurtS( Ir rII rIII rIV, rV, rVI, rVII rVIII rIX
f2I, f5I f2II B Junction seating Catalytic site assembly Exonucleolytic cleavage: Rotation B Rotation A 3′ Initial binding rII rIV ? Reset Bond cleavage Inverse rI rotation B
C rIX rVIII
d1 H36 H36 α2 H36 H36 H36 d2 Y32 9 Å Y32 Y32 Y32 Y32 D30 D171 3 Å 3 Å 2 Å G2 rI D173 rII rIII M1 M2 rIV rIX D78 D152 D225
Fig. 2. Exonucleolytic reaction cycle. (A) Assignment of structures observed in the experimental timeline (Named structures described in the main text are indicated at the bottom: r prefix, recessed-end substrates; f prefix, 5′-flap substrates) to stages along the proposed reaction coordinate (indicated at the top, numbered). At each stage, the states of major structural elements (left column) are described. Arrows indicate major motions. (B) Reaction cycle summary. The tethered DNA (Top Left; stage 1, rI) is seated at the dsDNA junction (Top Center; stage 2, rII). The scissile bond is then guided into the fully assembled active site (Top Right; stage 3, rIV) and cleaved (Bottom Right; stage 4, rVIII). The protein and DNA reset for the next cleavage cycle (Bottom Center; stage 5, rIX). (C) Structures (names indicated) illustrating the motion of the DNA along the reaction coordinate, relative to the position of the scissile bond (black square) in stage 3 (rIV). The thick dotted line indicates distance to scissile bond of the processed strand.
6012 | www.pnas.org/cgi/doi/10.1073/pnas.1704845114 Shi et al. Downloaded by guest on September 30, 2021 Substrate Guidance. The processed strands of recessed ends and 5′ A d2 u1d1 d2 flaps are positioned in the active site by a sequence of successive, interlocking conformational changes of the mobile arch, the two u1 guide residues, and the DNA substrate (Fig. 2). In the fully as- d1 sembled active site, the mobile arch adopts two different forms: rIII 2+ rI d6 d6 recessed-end 5′ phosphates bind to a third Mg coordinated to its d7 d8 d7 d8 bottom, and 5′ flaps thread through an aperture that opens up in its interior. Together, this structural plasticity and the controlled motions enable the enzyme to guide topologically disparate sub- strate termini into a common reaction mechanism. BCα5 rI rIII rIV Conformational Entropy Reduction. Decrease in the substrate DNA α4 conformational diversity is a striking feature of the reaction as it α5 H36 proceeds toward its unique transition state. In stage 1 of the observed u1 ′ α4 timeline, recessed ends and unthreaded 5 flaps are both loosely held by the H2TH anchor. Their dsDNA junctions are disengaged from d1 R92 the protein, and their processed and unprocessed strands are disor- dered (e.g., rI, f2I). This disengaged state also accommodates partially u3 f I rIII K85 threaded flaps (e.g., 5 ), consistent with single-molecule studies of cleavage kinetics in FEN1 (18), which indicate that the threading M1 u1 reaction is catalyzed at this stage. The base pair mismatch observed in u2 M2 f5I between the unprocessed and partially threaded flap strands at the d1 S84 E150 entry of the mobile arch aperture suggests that threading is catalyzed, in part, by transient binding interactions between the two strands, d2 Y312 T281 thereby reducing flap conformational entropy. Subsequent engagement of the dsDNA junction with the protein Fig. 3. Mobile arch and DNA substrate motions before catalysis. (A)DNA and positioning of the processed strand in the active site dramatically motion (red arrows indicate screw motion) transitioning from stage 1 (Left, rI) reduce substrate conformational entropy. Unprocessed single strands to 2 (Right, rIII). The double-stranded section of the DNA translocates, shifting become ordered and are placed in the wedge domain hydrophobic the contact to H2TH from d6 to d7.InrIII, the dsDNA-ssDNA junction at the d1 pocket. At stage 3 (precleavage) and stage 4 (postcleavage), the base pair contacts the wedge domain. The unprocessed single strand (u1–u3) is processed ends of both recessed-end and 5′-flap substrates adopt a disordered in rI (dashed line) and moves in rIII to place u1 between the α2- and single conformation within the active site. α3-helices, organizing u2-u3 to form an ordered, sharp bend around the Flap threading is an example of a “fly-casting” mechanism that α2-α3 wedge. (B) Mobile arch transitions (red arrows, rotation A) from the open enhances the kinetics of searching for states with low conforma- (Top;stage1,rI)toclosed(Bottom;stage2,rIII) conformation. (C)Mobilearch tional entropy. An initial binding event establishes a search volume transitions(redarrows,rotationB)fromtheclosed(stage2,rIII, light green) to without restricting internal motions. Subsequent formation of in- clamped (stage 3, rIV, dark green) conformation, placing the scissile bond termediates progressively reduces conformational degrees of free- next to metals M1 and M2 in a catalytically competent geometry. R92 and dom (25). The loosely tethered, conformationally diverse complexes K85 contribute to transition-state stabilization. E150 always orients K85 (only (rI and f2I) represent the initial, high-entropy encounter complex. shown in clamped form for simplicity). The clamped conformation is “latched” Mismatch formation in f5I may represent a postulated intermediate by S84, T281, and the Y312 main chain carbonyl. that narrows the “binding funnel” of available conformations as threading proceeds. These structures therefore present a remark- Conclusions able experimental manifestation of intermediates proposed in the fly-casting model for entropy reduction. Shared Catalytic Mechanism. We trapped structures of DNA complexes representing the exonucleolytic reaction just before and directly after Mobile Arch Motions. We identified three mobile arch conforma- the chemical cleavage step, and also captured an endonucleolytic tional states: open, closed, and clamped (the open and closed states cleavage product. In both recessed-end and 5′-flap substrates the are ensembles around an average structure, and the clamped state is scissile phosphodiester bond is located 1 bp in from the double- near unique). Each is associated with different substrate confor- stranded junction (Fig. 4 and Fig. S2), consistent with the dominant mational degrees of freedom. In the open state, the DNA is held cleavage site identified in solution (7, 8, 17). This bond is positioned loosely. In the closed state, the dsDNA junction is engaged and the between two metals in the active site that are ∼3.6 Å apart, which is two single strands are ordered. In the clamped state, the active site the correct distance for catalysis by a two-metal mechanism, as de- is fully assembled, positioning the scissile bond, attacking water, two scribed originally for the 3′-5′ exonucleolytic reaction of Klenow metals, and mobile catalytic residues in a catalytically competent fragment (KF) DNA polymerase (22, 23). In substrate complexes, a geometry. Mobile arch motions therefore progressively restrict water molecule is positioned for in-line attack of the phosphodiester substrate conformational degrees of freedom as the processed bond, whereas it is absent in products, having been incorporated into strand is guided into the active site. the newly formed 5′ phosphate (Fig. 4 A–C). The structural similar- In other family members, the mobile arch also is important for ities to KF enabled us to construct a model of the transition state substrate recognition and processing, and undergoes disorder/ (Fig. 4D), which revealed the clamped mobile arch transiently order transitions in FEN1 and in GEN1 (17, 21, 26). Arch mo- bility enables hExo1 activity to be manipulated allosterically. For
positions two key catalytic residues (16, 24), K85 and R92, to stabilize BIOCHEMISTRY instance, poly(ADP ribose)-binding of R93 within the arch ap- the negatively charged trigonal plane formed by three oxygens in the erture is an important known control point for hExo1 (27, 28). transition state. The two bases flanking the scissile bond in the ′ recessed-end and 5 -flap products are placed identically in the active Guide Residues. The side chains of H36 and Y32 switch between “up” E E F site (Fig. 4 and Fig. S2 and ). These observations therefore and “down” conformations. These residues are located on the clearly establish that exo- and endonucleolytic cleavage share a α2-helix of the immobile wedge domain, adjacent to the mobile arch. common reaction mechanism in which nucleolysisiscatalyzedbythe H36 is a principal attachment point for seated dsDNA junctions, well-known two-metal, in-line hydrolysis mechanism (22, 23), but is interacting with the d1 base of the processed strand, and H36A is elaborated by additional features that couple mobile arch motions 150-fold reduced in catalytic activity (16). The Y32 conformation with transition-state stabilization. controls 5′-end binding, and Y32A is 20-fold reduced in activity (16).
Shi et al. PNAS | June 6, 2017 | vol. 114 | no. 23 | 6013 Downloaded by guest on September 30, 2021 A D Base d2 Base d2 d1 d2 d1 O H36 d1 H36 H36 d1 O d2 O O O R92 N O N D171 rIV rVIII f II G2 2 N O P D30 K85 (N-terminus) D30 D30 N OH Y32 Y32 α4 O O O N Y32 S172 O W S172 S172 O O M2 D171 D171 D30 O W M1 O D225 D171 O G2 G2 G2 O W O O O u1 O D173 D173 D78 D173 D78 D78 D173 D78 D152 D152 D225 D152 A225 D152 D225 D30 BC5′ 5′ D171 E D171 G2 M1 Y32 G2 D171 D173 D171 D30 M1 M2 D152 M2 D30 D30 3′ 5′ D173 D78 D78 D/A225 D78 D152 K85 D78 D173 D173
+ Fig. 4. Uncleaved and cleaved substrate complexes. (A) Comparison of uncleaved recessed-end DNA substrate (Left; rIV, wild-type enzyme with Mn2 ) with its exonu- 2+ 2+ cleolytically cleaved product (Center; rVIII, D225A mutant with Mn ) and the endonucleolytically cleaved 5′-flap product (Right; f2II, wild-type with Mg ). Purple spheres, Mn2+; green spheres, Mg2+; gold sphere, attacking water (also Fig. S3); red spheres, waters coordinated to metals; green, cleaved nucleotide or flap products. With exception of the cleaved bond, the active-site structures remain similar before and after cleavage, consistent with states that bracket a cleavage step: Note that the d1 base remains in contact with H36 and all bases remain paired. (B) Simulated annealing omit map (blue, 1σ contour) and anomalous difference maps (red, 3σ contour) unambiguously identify uncleaved (Left, rIV) and cleaved (Right, rVIII) scissile bonds. The red arrow indicates the cleaved bond. (C) Superposition of the phosphate in the uncleaved (rIV) and cleaved (rVIII) scissile bonds shows incorporation of the attacking water (gold, red arrow) into newly formed 5′ phosphate after catalysis. (D) Proposed transition state for an enhanced, in-line, two-metal mechanism. The oxygen atoms of the phosphate in the scissile bond form a trigonal plane, stabilized by the two metals (M1 and M2), K85, and R92. Two bipyramidal apices are formed by the attacking water and 3′ oxygen of the leaving group. M2 and the protein N terminus (G2) orient the attacking water. The invariant carboxylates organize the transition geometry by direct and water-mediated (W) interactions to the metals, K85, N terminus, and attackingwater.(E)Two
nucleotides flanking the scissile bond of the exonucleolytic (rVIII, purple) and endonucleolytic (f2II, green) product complexes are almost perfectly superimposed.
As the mobile arch transitions from the closed state to the clamped and d2. Formation of the clamped mobile arch conformation also state and the seated substrate rotates into the active site, H36 flips requires that Y32 flips to its down position either to maintain flap from the up conformation to the down conformation. This motion threading or to enable steric accessibility of the recessed-end 5′ maintains the contact with d1, and therefore is a key contribution to phosphate-binding site. Both H36 and Y32 therefore function as the proper seating of the scissile bond, which is located between d1 steric guides that couple mobile arch motions to seating of substrates.
A BCu1 f I α5 f I α4 f II 5 5 u2 2 α4 u2R95 u1 f4 R96 R93 R95 d1 d2 α2 f1 R93 H36 α4 d1 d2 f4 Y32 f2 f3 f1 f2f3 α6 D30 D78 3′ Initial binding 3′ palF gnidaerht dnoB egavaelc D Junction seating, 3′ catalytic site assembly
5′
f2I f5I f2II
Fig. 5. Threading of uncleaved and cleaved 5′ flaps through an aperture formed by the mobile arch (green) and α2(red).(A and B) Two views of the uncleaved, partially threaded, five-residue 5′ flap (blue). (A) View through the aperture into the active site. Four positively charged residues (R92, R93, R95, and R96; only R96 is shown) are located on the face of α4 pointing to the ssDNA and may stabilize threaded flap nucleotides. (B) Side view tracing the path of the flap out of the double-stranded region through the aperture. The red arrow indicates the expected scissile bond (d1-d2). The unprocessed strand is partially disordered (dashed line). Both Y32 and H36 are in their up conformations and disengaged. The f2 and f3 bases straddle either side of the aperture. At the entrance of the arch, u1 and f1 form a mispair. The f2 and f3 bases point in the opposite direction (“flipped”)relativetof1. The f4 base does not form extensive contacts with the protein, and
f5 is disordered. R93 can interact with poly(ADP ribose), and is located adjacent to the threaded 5′-flap path. (C) Side view of the cleaved 5′-flap f2II.Thef1 base has threaded through the aperture and is flipped relative to d1 (part of the cleaved flap), and f2 is disordered. The aperture straddles f1 and d1.(D) Threading and
endonucleolytic cleavage. (Left) Initially, the dsDNA region is loosely tethered by H2TH (f2I). (Center) Next, the processed strand threads through the mobile arch (f5). (Right) Following dsDNA junction seating, the scissile bond is guided into the assembled active site and cleaved (f2II).
6014 | www.pnas.org/cgi/doi/10.1073/pnas.1704845114 Shi et al. Downloaded by guest on September 30, 2021 Processing Fidelity. The clamped mobile arch conformation posi- be expected if catalytic and processing fidelity mechanisms are tions the two key catalytic residues K85 and R92, which are im- common to all members, whereas the arch encodes substrate portant for catalysis (16, 24). This state cannot form with incorrectly guidance specializations. or incompletely bound substrates such as partially threaded 5′ flaps, in which the dsDNA junction is not seated and the guide residues Materials and Methods are not triggered. Coupling transition stabilization to arch motions Crystallization and in Crystallo Enzyme Reaction. The wild-type and mutant therefore enhances processing fidelity by suppressing inappropriate proteins were prepared as described previously (16). Oligonucleotides (In- or premature substrate cleavage. tegrated DNA Technologies, Inc.; Table S3) were prepared as previously de- scribed (16). The 5′ ends of processed strands were terminated with a Processivity. Nucleotide monophosphate product release is accom- phosphate; all other 5′ and 3′ termini were protected with a phosphorothioate panied by partial reversal of the conformational sequence without linkage. Complexes were prepared by combining 150 μMwith200μMDNA relinquishing bound DNA. The mobile arch unclamps, and the substrate and incubating at 4 °C for 30 min. Crystals were obtained by sitting- dsDNA junction disengages, resulting in a loosely held substrate drop vapor diffusion at 17 °C, mixing 100 μLofcomplexwith100μLofpre- tethered at H2TH. This resetting motion suggests a structural basis cipitant solution [100 mM NaOAc (pH 7.0), 10 mM KCl, 2–4% PEG 4000]. Most for the observed processivity of hExo1 exonuclease activity (29, 30). crystals with recessed-end DNA appeared within 1 h after setting up trays, and ′ – Reseating would translocate the protein along the DNA by 1 bp, crystals with 5 -flap substrates appeared after 2 4 d. Sixteen hours after their analogous to the motion of the substrate engagement step that appearance, crystals were transferred into cryoprotectant solution (mother followed initiation of the experimental timeline (Movie S2). liquor with 35% ethylene glycol) for 10 min. For time-resolved experiments, the reaction was initiated by transfer into cryoprotectant supplemented with 10–20 mM MgCl or MnCl . Crystals were flash-frozen in liquid nitrogen to Evolution of Substrate Guidance Mechanisms. 2 2 The residues encod- stop the reaction at varying time intervals (∼1–600 s). ing the enhanced two-metal mechanism are highly conserved in the RAD2/XPG family. These residues include seven carboxylates Data Collection, Structure Determination, and Refinement. Diffraction data that coordinate the two metals, either directly (D152, D171, were collected at 100 K at the Advanced Photon Source (Argonne National and D173) or through a bound water (D30 and D78); D225, Laboratory; beamlines 22-ID, SER-CAT) or the Advanced Light Source (Lawrence which orients the attacking water; and E150, which undergoes a Berkeley National Laboratory; beamline 12.3.1, SIBYLS); all crystals diffracted conformational change in the clamped conformation, orienting to 2.2–2.9 Å resolution. Structures were determined as described previously K85. Where tested, their mutagenesis results in loss of activity (SI Materials and Methods and Tables S1 and S2). (16, 31). The K85 and R92 that encode transient stabilization of the transition state are highly conserved, whereas the rest of the ACKNOWLEDGMENTS. This work was supported, in part, by NIH Grants R01 mobile arch sequence is not. This pattern of conservation might GM091487 and P01 CA092584.
1. Genschel J, Bazemore LR, Modrich P (2002) Human exonuclease I is required for 5′ and 17. Tsutakawa SE, et al. (2011) Human flap endonuclease structures, DNA double-base 3′ mismatch repair. J Biol Chem 277:13302–13311. flipping, and a unified understanding of the FEN1 superfamily. Cell 145:198–211. 2. Nimonkar AV, Ozsoy AZ, Genschel J, Modrich P, Kowalczykowski SC (2008) Human 18. Sobhy MA, Joudeh LI, Huang X, Takahashi M, Hamdan SM (2013) Sequential and exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair. Proc multistep substrate interrogation provides the scaffold for specificity in human flap Natl Acad Sci USA 105:16906–16911. endonuclease 1. Cell Reports 3:1785–1794. 3. Potenski CJ, Niu H, Sung P, Klein HL (2014) Avoidance of ribonucleotide-induced 19. Patel N, et al. (2012) Flap endonucleases pass 5′-flaps through a flexible arch using a mutations by RNase H2 and Srs2-Exo1 mechanisms. Nature 511:251–254. disorder-thread-order mechanism to confer specificity for free 5′-ends. Nucleic Acids 4. Qiu J, Qian Y, Chen V, Guan MX, Shen B (1999) Human exonuclease 1 functionally Res 40:4507–4519. complements its yeast homologues in DNA recombination, RNA primer removal, and 20. Dervan JJ, et al. (2002) Interactions of mutant and wild-type flap endonucleases with mutation avoidance. J Biol Chem 274:17893–17900. oligonucleotide substrates suggest an alternative model of DNA binding. Proc Natl – 5. Ip SC, et al. (2008) Identification of Holliday junction resolvases from humans and Acad Sci USA 99:8542 8547. yeast. Nature 456:357–361. 21. AlMalki FA, et al. (2016) Direct observation of DNA threading in flap endonuclease – 6. Evans E, Fellows J, Coffer A, Wood RD (1997) Open complex formation around a le- complexes. Nat Struct Mol Biol 23:640 646. ′ ′ sion during nucleotide excision repair provides a structure for cleavage by human XPG 22. Beese LS, Steitz TA (1991) Structural basis for the 3 -5 exonuclease activity of Es- cherichia coli DNA polymerase I: A two metal ion mechanism. EMBO J 10:25–33. protein. EMBO J 16:625–638. 23. Steitz TA, Steitz JA (1993) A general two-metal-ion mechanism for catalytic RNA. Proc 7. Lee BI, Wilson DM, 3rd (1999) The RAD2 domain of human exonuclease 1 exhibits 5′ Natl Acad Sci USA 90:6498–6502. to 3′ exonuclease and flap structure-specific endonuclease activities. J Biol Chem 274: 24. Pickering TJ, Garforth SJ, Thorpe SJ, Sayers JR, Grasby JA (1999) A single cleavage 37763–37769. assay for T5 5′–>3′ exonuclease: Determination of the catalytic parameters for wild- 8. Keijzers G, Bohr VA, Rasmussen LJ (2015) Human exonuclease 1 (EXO1) activity type and mutant proteins. Nucleic Acids Res 27:730–735. characterization and its function on flap structures. Biosci Rep 35:e00206. 25. Shoemaker BA, Portman JJ, Wolynes PG (2000) Speeding molecular recognition by 9. Tomimatsu N, et al. (2012) Exo1 plays a major role in DNA end resection in humans using the folding funnel: The fly-casting mechanism. Proc Natl Acad Sci USA 97: and influences double-strand break repair and damage signaling decisions. DNA 8868–8873. Repair (Amst) 11:441–448. 26. Liu Y, et al. (2015) Crystal structure of a eukaryotic GEN1 resolving enzyme bound to 10. Vallur AC, Maizels N (2008) Activities of human exonuclease 1 that promote cleavage DNA. Cell Reports 13:2565–2575. of transcribed immunoglobulin switch regions. Proc Natl Acad Sci USA 105: 27. Zhang F, Shi J, Chen SH, Bian C, Yu X (2015) The PIN domain of EXO1 recognizes poly – 16508 16512. (ADP-ribose) in DNA damage response. Nucleic Acids Res 43:10782–10794. 11. Vallur AC, Maizels N (2010) Complementary roles for exonuclease 1 and Flap endo- 28. Cheruiyot A, et al. (2015) Poly(ADP-ribose)-binding promotes Exo1 damage recruit- – nuclease 1 in maintenance of triplet repeats. J Biol Chem 285:28514 28519. ment and suppresses its nuclease activities. DNA Repair (Amst) 35:106–115. 12. Levikova M, Cejka P (2015) The Saccharomyces cerevisiae Dna2 can function as a sole 29. Myler LR, et al. (2016) Single-molecule imaging reveals the mechanism of Exo1 nuclease in the processing of Okazaki fragments in DNA replication. Nucleic Acids Res regulation by single-stranded DNA binding proteins. Proc Natl Acad Sci USA 113: 43:7888–7897. E1170–E1179. 13. Sparks JL, et al. (2012) RNase H2-initiated ribonucleotide excision repair. Mol Cell 47: 30. Genschel J, Modrich P (2003) Mechanism of 5′-directed excision in human mismatch 980–986. repair. Mol Cell 12:1077–1086. 14. Wei K, et al. (2003) Inactivation of Exonuclease 1 in mice results in DNA mismatch 31. Lee Bi BI, Nguyen LH, Barsky D, Fernandes M, Wilson DM, 3rd (2002) Molecular in- repair defects, increased cancer susceptibility, and male and female sterility. Genes teractions of human Exo1 with DNA. Nucleic Acids Res 30:942–949. BIOCHEMISTRY Dev 17:603–614. 32. Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66:125–132. 15. Bayram S (2014) The exonuclease 1 Glu589Lys gene polymorphism and cancer sus- 33. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in os- ceptibility: Evidence based on a meta-analysis. Asian Pac J Cancer Prev 15:2571–2576. cillation mode. Methods Enzymol 276:307–326. 16. Orans J, et al. (2011) Structures of human exonuclease 1 DNA complexes suggest a 34. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- unified mechanism for nuclease family. Cell 145:212–223. molecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221.
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