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Interplay of Catalysis, Fidelity, Threading, and Processivity in the Exo- and Endonucleolytic Reactions of Human Exonuclease I

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Interplay of Catalysis, Fidelity, Threading, and Processivity in the Exo- and Endonucleolytic Reactions of Human Exonuclease I 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
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