Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family

Structures of Human Exonuclease 1 DNA Complexes Suggest a Uniﬁed Mechanism for Nuclease Family

Jillian Orans,1,3 Elizabeth A. McSweeney,1,3 Ravi R. Iyer,1,3 Michael A. Hast,1 Homme W. Hellinga,1 Paul Modrich,1,2 and Lorena S. Beese1,* 1Department of Biochemistry 2Howard Hughes Medical Institute, Box 3711 Duke University Medical Center, Durham, NC 27710, USA 3These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.cell.2011.03.005

SUMMARY sporadic tumors (Kolodner, 1995; Peltoma¨ ki, 2003). Failure to repair double-strand breaks can result in chromosomal rear- Human exonuclease 1 (hExo1) plays important roles rangements or deletions, leading to carcinogenesis and prema- in DNA repair and recombination processes that ture aging (Hartlerode and Scully, 2009). maintain genomic integrity. It is a member of the 50 The first characterized role of hExo1 was its exonuclease func- structure-specific nuclease family of exonucleases tion in human mismatch repair. hExo1 excises mismatches in this repair pathway and requires a nick 50 to the excision region and endonucleases that includes FEN-1, XPG, and 0 0 GEN1. We present structures of hExo1 in complex to perform 5 -3 hydrolysis on double-stranded DNA (Dzantiev et al., 2004; Genschel et al., 2002; Genschel and Modrich, with a DNA substrate, followed by mutagenesis 2003; Zhang et al., 2005). hExo1 interacts with a number of studies, and propose a common mechanism by MMR proteins, including MutLa and the DNA lesion recognition which this nuclease family recognizes and processes proteins MutSa and MutSb (Nielsen et al., 2004; Schmutte diverse DNA structures. hExo1 induces a sharp bend et al., 1998, 2001); these interactions directly modulate exonu- 0 in the DNA at nicks or gaps. Frayed 5 ends of nicked cleolytic activity (Genschel and Modrich, 2003, 2009). Binding duplexes resemble flap junctions, unifying the mech- of hExo1 to MutSa in a mismatch- and ATP-dependent manner anisms of endo- and exonucleolytic processing. is required for processive 50-30 hydrolysis (Genschel and Conformational control of a mobile region in the cata- Modrich, 2003). Additionally, studies in yeast suggest that lytic site suggests a mechanism for allosteric regula- Exo1 may also play a structural role in mismatch repair through tion by binding to protein partners. The relative stabilization of complexes containing multiple MMR proteins arrangement of substrate binding sites in these (Amin et al., 2001). In DBSR, hExo1 interacts with a different assembly of protein partners during homologous recombination enzymes provides an elegant solution to a complex (Mimitou and Symington, 2008; Zhu et al., 2008). Depletion of geometrical puzzle of substrate recognition and hExo1 results in an increase in the development of double-strand processing. breaks (Gravel et al., 2008; Nimonkar et al., 2008). hExo1 is a member of the 50 structure-specific nuclease family INTRODUCTION of metalloenzymes that are involved in multiple DNA repair pathways. This family includes flap endonuclease 1 (FEN-1), Human exonuclease 1 (hExo1) is essential for maintaining which participates in processing of Okazaki fragments; gap genomic stability by nucleolytic processing of DNA intermedi- endonuclease 1 (GEN1), involved in Holliday junction (HJ) resolu- ates involved in repair and recombination. hExo1 functions in tion; and xeroderma pigmentosum complementation group G several DNA repair pathways: it confers the primary exonuclease (XPG), which processes DNA bubble structures (Tomlinson activity employed in mammalian mismatch repair (MMR) et al., 2010). These proteins share a conserved N-terminal cata- (Genschel et al., 2002; Wei et al., 2003); it is involved in DNA lytic core nuclease region but exhibit individual preferences for resection during double-strand break repair (DSBR) (Zhu et al., structurally distinct DNA substrates. The C-terminal regions of 2008); and it is important for telomere maintenance through these proteins are divergent in sequence. Although structures promotion of recombination at transcription-induced telomeric of human FEN-1 and FEN-1 homologs have been determined structures (Vallur and Maizels, 2010b). Deficiency of mismatch (Ceska et al., 1996; Chapados et al., 2004; Devos et al., 2007; repair can have profound deleterious effects on human health, Dore´ et al., 2006; Feng et al., 2004; Hosfield et al., 1998; Hwang such as spontaneous mutability, hereditary nonpolyposis colo- et al., 1998; Matsui et al., 2002; Mueser et al., 1996; Sakurai et al., rectal cancer (HNPCC), and the development of 15%–25% of 2005), these lack either the assembled two-metal active site

212 Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. required for catalysis or a DNA substrate. Consequently, many in reference to the nick at which the enzyme binds): the pre- questions concerning catalytic mechanism and substrate recog- nick DNA-binding region, which holds the double-stranded nition have remained unanswered. segment with the substrate strand that is cleaved; the post- Here, we present the structure of the hExo1 N-terminal cata- nick binding region, which in our complexes binds the single- lytic domain (residues 1–352) in complex with a 10 bp duplex stranded gap; the active site region containing a metal center with a three-base 30 single-strand extension. This structure at which DNA cleavage takes place; and a C-terminal segment mimics a gapped duplex and represents a model intermediate (Figure 1D). The DNA substrate is sharply bent at the dsDNA- structure in mismatch repair (Genschel and Modrich, 2003) and ssDNA junction (Figure 1C) by a protrusion of the protein is likely to correspond to a substrate in double-strand break (a2, a3)—the hydrophobic wedge, also observed in FEN-1 family repair (Nimonkar et al., 2008). hExo1 recognizes nicked, gapped, members (Chapados et al., 2004) —accounting for the speci- or blunt DNA in vitro (Genschel and Modrich, 2003; Lee and ficity of this enzyme family for nicked or gapped DNA. The Wilson, 1999). The active site accommodates both 50 ends and regions that are preserved and differ in the enzyme family are 50 flaps, which undergo exo- and endonucleolytic cleavage, shown in a structure-based sequence alignment (Figure 2A) of respectively (Lee and Wilson, 1999). Elucidating the structural FEN-1 family members with hExo1 and a structural alignment features by which the active site can accommodate such (Figure 2B) of Archaeoglobus fulgidus FEN-1 with hExo1. Partic- different substrates and activities is therefore essential for under- ularly noteworthy are the divergences in the post-nick binding standing this enzyme and other members in the FEN family. We region, which accommodates 30 nick termini or 30 flaps, and find that hExo1 binds at the junction of single- and double- the portion of the C-terminal domain of hExo1. stranded DNA by stabilizing a sharp bend that can be accommodated only in nicked or gapped DNA. A metal center cleaves the DNA Bending at Nicks or Gaps nicked strand immediately adjacent to this junction. The scissile Helices a2 and a3 are located at the boundary between the pre- bond is placed at the metal center if the last two bases at the 50 and post-nick regions of the DNA (Figure 1). In this region, the end of the double-stranded region fray. Such fraying converts DNA is sharply bent (90), and binding of intact duplex DNA a double-stranded region into a short single-stranded segment. is blocked by a number of hydrophobic residues that create Consequently, the 50 end of a nick could resemble the basal a ‘‘wedge’’ motif. We observe hydrophobic interactions between region of a flap, making endo- and exonucleolytic activities residues I40, A41 (a2), F58 and F62 (a3), and the C21 and A22 equivalent. Although we do not observe a 50 flap directly, the bases at the 30 terminus (Figure 3). A comparable interaction structure of the complex indicates a probable path whereby has been observed in A. fugidus FEN-1 DNA complex, which the flap strand exits out of the active site and binds along the has duplex instead of single-stranded DNA bound to the post- protein surface. The enzyme therefore solves the problem of nick binding region (Chapados et al., 2004). diverse substrate recognition and processing by setting up a series of surface sites that each bind and process the various The Pre-Nick DNA-Binding Region segments of structured DNA substrates. The pre-nick DNA-binding region is formed by a shallow surface The hExo1 catalytic domain shares 20% sequence identity groove that contains discrete patches of positive charge (Fig- with other FEN-1 family members. Much of the domain structure ure S1 available online). The DNA and protein make strikingly is structurally homologous with FENs, but we also identify impor- few contacts in this pre-nick region (Figure 3). The cleaved tant structural differences, which encode the functional special- DNA strand (‘‘substrate’’) makes interactions only in the vicinity izations that differentiate these enzymes from each other. of the active site and is completely solvated at the fourth base Analysis of these similarities and differences has enabled us to from the 50 terminus and beyond. The complementary, un- propose a unified model for substrate recognition and process- cleaved strand forms contacts with a helix-two turn-helix ing for the FEN enzyme family, based on the insight that (H2TH) motif (a10-a11), a modified version of the canonical a common relative geometrical arrangement of the active site, helix-hairpin-helix nucleotide-binding motif also observed in 50 binding site, and 30 binding site can accommodate and distin- FEN-1 structures (Ceska et al., 1996; Chapados et al., 2004; guish different structured DNA substrates. All family members Feng et al., 2004; Hosfield et al., 1998; Tomlinson et al., 2010). induce a sharp bend in the substrate for nick or gap recognition; This H2TH domain forms hydrogen bonds with the phosphate fraying at the 50 end of the duplex removes apparent differences oxygens of nucleotides C13 and T14 via the main-chain amides between nick and flap processing; and (non)accommodation of of residues 232–237, located in the turn region and at the begin- 50 or 30 flaps in surface binding sites further encodes substrate ning of a11. The T14 phosphate is also coordinated by a potas- specificity. The structure-based hypotheses generated by sium ion bound in the turn region of the H2TH motif. The K+- our observations and models are open to testing in future binding site (Figures 3A and 3C) is formed by interactions with experiments. two main-chain carbonyl groups (S222 and I233), the hydroxyl of S229, the DNA (T14 phosphate oxygen), and solvent (two RESULTS water molecules). This K+ coordination has not been observed in other 50 nuclease structures, determined in the absence of Overview of the hExo1 Structure DNA. It may therefore assemble only in the presence of DNA, The N-terminal domain of hExo1 forms a bean-shaped core with suggesting interactions that can be readily formed and reformed a helical protrusion and a number of surface grooves (Figures upon sliding in a processive processing mode of the enzyme. 1A–1D). The structure can be divided into four regions (defined This site is almost identical to one observed elsewhere in DNA

Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. 213 Figure 1. Structure of the hExo1 Catalytic Domain in Complex with a 50 Recessed End Substrate (A) Structure of the hExo1 catalytic domain bound to a gapped DNA substrate mimic. Conserved DNA-binding motifs are indicated: a4-a5 microdomain (‘‘helical arch’’), green; a2-a3 helix- loop-helix, red; K+ ion-binding site (blue) in helix- two-turn-helix (H2TH), purple. Two active site metal ions are shown (red). (B) Topology map of secondary structure elements in the hExo1 catalytic domain. (C) Simulated annealing omit electron density map calculated to 2.5 A˚ resolution contoured at 1s around DNA of the hExo1 D173A complex. Note the 90 bend of the 30 complementary DNA strand. DNA-binding motifs are colored as in (A). (D) Key functional features of the of hExo1-DNA complex. DNA-binding motifs are colored as in (B). C-terminal region is blue; hydrophobic wedge region of a2-a3 is shown in magenta; b6-b7 hairpin is teal. hExo1 DNA substrate is orange and yellow; modeled post-nick DNA from A. fulgidus FEN-1 structure (PDB 1RXW) is blue. A gap between the pre- and post-nick region is highlighted with a blue triangle. Active site residues are shown as sticks. Mn2+ (magenta) and K+ ions (blue) are highlighted. See also Figure S1.

structure could interact with the latter, including a hairpin loop between strands b6 and b7(Figure 1D). The 30 end of the missing strand is predicted to interact with the a2-a3 HLH region. In hExo1, the a2 helix makes an 90 bend at residue A41 not seen in FEN-1. In A. fulgidus FEN-1, the HLH2 region, which is absent in hExo1, interacts with phosphate groups of the 30 flap in a double-flap substrate (Chapados et al., 2004). It has not yet been determined whether such a double-flap substrate can be processed by hExo1. We postulate that these differences in interactions in the vicinity of the 30 nicked polymerase b (Pol b)(Figure S2)(Pelletier et al., 1994). In Pol b,it DNA strand play key roles in DNA substrate selection by prevent- has been suggested that this site facilitates movement of the ing, accommodating, or stabilizing binding of 30 flaps. protein along the DNA substrate backbone (Pelletier et al., 1996). The Active Site Our analysis of the active site is based on three structures of enzyme-DNA complexes: an inactive mutant (D173A) with Ca2+ The Post-Nick DNA-Binding Region (complex I) and two wild-type enzyme structures with Ba2+ In the post-nick DNA-binding region, the ssDNA segment of the (complex II) or Mn2+ (complex III) (see Figure S5 for metal depen- complementary (nonsubstrate) strand is contacted by a helix- dence and enzyme activity). The active site is located at the loop helix (HLH) motif (a2-a3) observed also in FEN-1. However, boundary between the pre- and post-nick DNA-binding regions a second HLH motif (HLH2) present in a number of FEN-1 struc- (Figure 1D). The FEN-1 enzyme family members are metalloen- tures is absent in hExo1 (Figure 2B). zymes, involving at least two Mg2+ cations in the active site In the gap-mimic substrate presented here, the uncleaved (Feng et al., 2004; Hwang et al., 1998; Mueser et al., 1996; DNA strand is present, but its partner DNA strand 30 to a nick Sakurai et al., 2005). Although one of the two metals in the active is absent. Nevertheless, it is clear that several elements in the site is absent in the mutant Ca2+ complex (Figure S3), the DNA is

214 Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. Figure 2. Structure and Sequence Align- ments of hExo1 with Other Human 50 Structure-Speciﬁc Nuclease Superfamily Members (A) Structure-based sequence alignment of human 50 structure-speciﬁc nuclease family members. Residues with assigned secondary structure are

colored as in Figure 1.310-helices are shown with diagonal lines. Note that the sequence of the large spacer region (insert) in hXPG is not shown. hExo1 residues mutated in this study are underlined and highlighted with purple arrows. Functionally important residues are indicated: conserved acidic residues in the active site, blue stars; K+-chelating residues, purple stars; interactions with complementary (nonsubstrate) DNA strand, black triangles; interactions with DNA substrate strand, red triangles; van der Waals contacts with DNA, gray circles. (B) Comparison of hExo1 with FEN-1. Alignment of structures of hExo1 (teal; motifs colored as in A) and A. fulgidus FEN-1 (gray; PDB ID 1RXW). Structures reveal signiﬁcant divergence in the C-terminal region (boxed) and the 30 ﬂap-binding motif of FEN-1 (magenta).

abolish activity, and D78A or D225A mutants alter substrate affinity (Lee et al., 2002)(Figures 4E and 4F). This loss of function has been observed also for all of the conserved active site acidic residues of human FEN-1 (Shen et al., 1996, 1997). The hydrolytic center is postulated to be similar to the classic two-metal mechanism that was originally described for the Klenow exonuclease activity (Beese and Steitz, 1991; Steitz and Steitz, 1993), although this has not yet been established definitively (Syson et al., 2008). If so, the scissile bond is expected to be located between the two metal centers. In the Ba2+ complex II, the scissile bond is close to, but not in direct contact with, the metal center (Figures 4A and 4C and Figure S3). We postulate that this complex corresponds to a ‘‘nascent substrate’’ in which an increase in ionic radius of Ba2+ relative to Mg2+ and slight distortions due to the phosphorothioate backbone prevent the phosphate oxygen bound similarly to the other structures, showing that the enzyme from being placed between the two metals. In Klenow fragment, binds at nicks in the absence of catalytic activity or a complete a similar situation has been interpreted as the consequence of metal center. differences between phosphodiester and phosphorothioate The metal positions in both the Ba2+ (complex II) and the Mn2+ coordination geometries (Brautigam and Steitz, 1998; Brautigam (complex III) complexes are well defined by anomalous scat- et al., 1999). In the Mn2+ complex III, the 50 phosphate of the tering (Figure S4). The metals are coordinated by five acidic resi- nicked strand interacts directly with the two metals (Figures 4B dues, which interact with site M1 (D30), site M2 (D171, D173, and and 4D and Figure S4). This complex is therefore likely to corre- D225), or both (D152). Alanine mutations in D78, D173, and D225 spond to a product conformation.

Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. 215 Figure 3. Interactions between the hExo1 Protein and DNA Substrate (A) Schematic of interactions between hExo1 and DNA in the complex I and II (nascent substrate) structures, colored as in Figure 1A. The hydrophobic wedge is at the ends of a2 and a3 (teal). The scissile phosphodiester bond is red. Hydrogen bonds between DNA substrate and protein side chains are indicated with arrows; main-chain hydrogen bonding residues are boxed. Residues coordinating K+ are underlined in teal; van der Waals contacts are indicated by dotted arcs. (B) Schematic of interactions between hExo1 and DNA in product structure (complex III). Two active site metals and K85 coordinate the terminal phosphate. The terminal base is ﬂipped. Disor- dered nucleotides A22 and T23 on the complementary strand are modeled in gray. (C) The K+-binding site in the H2TH domain. The

composite omit 2FoFc electron density for the protein (blue) and the DNA density (orange) are contoured at 1s. An anomalous difference Fourier map (magenta) of the Ba2+-substituted complex is superimposed (5s contour). See also Figure S2.

Mutagenesis shows that, in addition to the metal centers, activity (Figure 4F), indicating that both residues participate, a number of other interactions are important for catalysis. R92 with H36 fulfilling a central role. on a4 interacts with the scissile bond in complexes I and II (Fig- ure 4C and Figure S3). The R92A mutant results in significant loss A Mobile a4-a5 Microdomain of activity (Figures 4E and 4F). A similar result was observed for As in FEN-1, a two-helix structure a4-a5 (also known as the FEN-1 from Pyrococcus furiosis and P.horikoshii (Allawi et al., helical arch, helical clamp, or I domain) forms one side of the 2003; Matsui et al., 2002). K85 on a4 interacts with the terminal active site (Ceska et al., 1996; Chapados et al., 2004; Devos phosphate in the product, but not the nascent substrate complex et al., 2007; Hosfield et al., 1998; Mueser et al., 1996; Sakurai (Figures 4C and 4D). In this study, we find that K85A also results et al., 2005). These helices contain a large number of positively in a dramatic loss of activity (Figure 4F). A similar observation has charged residues: in hExo1, a4 and a5 contain 11 positively been reported for the corresponding FEN-1 mutant (Sengerova´ charged residues (Figure S1). This segment binds the terminal et al., 2010). 50 phosphate of the substrate and provides residues that contribute to the fraying of the two end bases of the duplex. Duplex Fraying at the 50 Nick Terminus The mobility and ordering of the a4anda5 helices in the 50 nucle- In complex II, the terminal base no longer forms a canonical base ases vary greatly; they are disordered in a number of homolog pair with its partner on the template strand seen in complex I. The structures, and a disorder-to-order transition has been invoked terminal phosphate points toward R95 and R96 on the a4 helix upon flap substrate binding in FEN-1 (Devos et al., 2007; Dore´ (Figure S3). In the product complex (complex III), the terminal et al., 2006; Hosfield et al., 1998; Hwang et al., 1998; Matsui base has flipped out of the duplex. This conformation is stabi- et al., 2002; Sakurai et al., 2005). In the hExo1 structures presented lized by van der Waals interactions with R92 on a4 and a p-stack- here, a4, a5, and part of b3 form a mobile microdomain that adopts ing interaction with Y32 on a2(Figure 4D). The displaced base different conformations. Alignment of the three structures by their observed in complex III corresponds to the penultimate base in b sheet core reveals movements > 3 A˚ between equivalent Ca a prechemistry substrate that has the scissile bond positioned atoms of the mutant and product complexes (Figure S6). adjacent to the metal center (Figure 5). Accordingly, these observations suggest that the last two bases in the exonucleolytic The C-Terminal Region substrate are displaced out of their duplex interactions The C-terminal region (residues 285–345) (Figures 1B and 1D) (‘‘frayed’’). This fraying has two consequences: it positions the consists of strands b8andb9, helices a14 and a15, and loop scissile bond in the vicinity of the metal center, and it unifies regions that are stabilized by a network of H bonds and van der the mechanisms of exo- and endonucleolytic cleavage of nicks Waals interactions. This region adopts a structure that is distinct and flaps, respectively (see Movie S1). in sequence, fold, and location (on the opposite face of the mole- In all three structures, H36 interacts to a greater or lesser cule) from the corresponding region of FEN1 (Figure 2B). The extent with the terminal base and may therefore play a critical C-terminal region interacts with the active site through interactions part in the fraying process. The H36A mutant reduces activity at the base of a4 (L82, P83, and S84) and a9 (E150, Y149, Y157, 150-fold, whereas the Y32A mutant results in a 20-fold drop in and E154). In particular, E150 on a9 links C-terminal Q285 with

216 Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. Figure 4. Interactions in the hExo1 Active Site (A) Nascent substrate structure (complex II) with two Ba2+ ions (blue) coordinated by conserved carboxylates. Substrate-binding structural elements, metal ions, and N terminus are indicated. Structural elements are colored according to scheme in Figure 1. (B) Product structure (complex III) with two Mn2+ ions (magenta) bound at the active site. (C) Close-up of complex II active site (rotated 90 from A). Two Ba2+ ions (cyan) are coordinated by conserved carboxylate residues; the scissile phosphate is coordinated by R92. Given the limits of resolution of these structures, interactions (dashed lines) are approximate. (D) Close-up of complex III active site (rotated 90 from B). Two Mn2+ ions (magenta) are coordinated by conserved acidic residues and phosphate; the phosphate also interacts with K85. Approximate interactions within 2.8 A˚ are shown by dashed lines. (E) Activity of wild-type hExo1 catalytic domain determined by titration (concentrations indicated) against a ﬁxed concentration (25 nM) of a 50 recessed substrate (37C for 5 min), followed by separation using denaturing polyacrylamide gel electrophoresis and visualized by autoradiog- raphy (see Supplemental Information). (F) Activity of alanine mutants. Amount of DNA hydrolyzed was plotted as a function of protein concentration for Exo1 catalytic domain (WT) (black), K85A (green), D173A (red), H36A (light blue), R92A (magenta), and Y32A (orange). Error bars represent standard deviation from at least three replicates of the experiment. We cannot rule out contribution to residual activity by a contami- nating E. coli nuclease that could have copuriﬁed with the protein but consider this unlikely because all mutants were expressed at similar levels and displayed nearly identical chromatographic prop- erties. See also Figure S3, Figure S4, Figure S5, Fig- ure S6, and Movie S1.

Lys 85 on a4, which participates in catalysis (Figure S7). The a4 substrate, with the 50 end of the nicked strand placed within and a9 residues are conserved among the Exo1 subclass of 50 the active site, where exonucleolytic cleavage occurs. These nucleases but diverge from FEN-1 subclass nucleases (Figure 2A). observations enable us to propose a model for the interactions We postulate that these interactions couple catalysis to binding between hExo1 and MutSa in mismatch repair and to propose events that modulate the conformation of the entire C-terminal a uniﬁed mechanism for the endonucleolytic and exonucleolytic domain (which is absent in this structure) through binding interac- cleavage activities and substrate recognition for this protein tions with other proteins such as the MMR protein MutSa family. (Genschel and Modrich, 2003; Schmutte et al., 1998, 2001). In a re- a constituted reaction, MutS stimulates mismatch excision by full- Control of Catalytic Activity by Conformational Coupling length hExoI, but not the C-terminal deletion, consistent with the In the hExo1 structures presented here, a4, a5, and part of b3 proposed function of the C-terminal domain (Figures 6A and 6B). form a mobile microdomain that adopts different conformations (Figure S6) and contributes to substrate recognition and DISCUSSION cleavage (Figure 4). In other FEN family member structures determined in the absence of a DNA substrate, this region is Here, we report the structure of human Exonuclease 1 (hExo1) in disordered (Devos et al., 2007; Dore´ et al., 2006; Hwang et al., complex with a DNA substrate that represents a gapped 1998; Matsui et al., 2002; Sakurai et al., 2005). Binding

Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. 217 Figure 5. Assignment of Structures to Steps in the hExo1 Reaction Cycle (A) Comparison of active site in complex I (mutant), complex II (Ba2+ ‘‘nascent substrate’’), and complex III (Mn2+ ‘‘product’’) showing positions of DNA, metals, and H36. (B) Schematic representation of some mechanistically relevant interactions. Complex I contains one Ca+ ion at some distance away from the phosphate in the scissile bond. Complex II contains two Ba2+ ions in the active site and exhibits 50 base pair fraying. In both prechemistry structures, the scissile 50 phosphate interacts with R92. We propose that, in a catalytically active metal complex, the scissile phosphate is bound by K85. (C) Schematic representation of proposed hExo1 reaction cycle. After binding of the DNA and assembly of the metal sites (left), the scissile bond moves into the active site via phosphate coordination with two metal ions as well as K85 (center). Both the base poised for cleavage and the penultimate base are unpaired (frayed) from the duplex into the active site. Postchemistry, the 50 phosphate of the product strand is coordinated by two metal ions (right).

interactions that stabilize one conformation over another or In the absence of stimulatory factors, the full-length hExo1 is cause order/disorder transitions clearly have the potential to not very active but still binds to nicks or gaps (Genschel et al., exercise considerable control over nuclease activity. 2002; Genschel and Modrich, 2003; Lee and Wilson, 1999). We Parts of the a4/a5 helices are exposed and could form binding postulate that the C-terminal fragment that is partially absent in sites with protein partners, extended parts of DNA substrates. our structure functions as an autoinhibitory domain by modu- Other parts pack against the a2/a3 helices, which also form an lating the conformation of the a4/a5 microdomain. Upon binding extensive exposed surface that potentially can interact with part- to a mismatch, MutSa clamps to the dsDNA duplex and carries ners. Binding to either of these helical regions is likely to affect out a search via bidirectional one-dimensional diffusion along the the mobility and conformation of the a4 helix. The C-terminal DNA (Gradia et al., 1997, 1999). Upon encountering the pre- domain also could interact with a4/a5 microdomain, mediated bound hExo1 at a nick, MutSa captures the C-terminal domain, through contacts with the C-terminal region that are present in relinquishing its autoinhibitory interactions with the catalytic the structure determined here (Figure S7). MutSa and PCNA domain and activating exonucleolytic activity by enabling the interact with this domain (Liberti et al., 2011; Schmutte et al., a4/a5 microdomain to adopt a conformation that positions the 1998, 2001); similar interactions with other proteins could couple scissile bond over the metal center (see above). The activated hExo1 activity to other pathways such as double-stranded break hExo1-MutSa complex now returns to the mismatch site, driven repair (Doherty et al., 2005; Gravel et al., 2008; Nimonkar et al., by the unidirectional 50-30 hExo1 exonuclease activity. 2008; Sharma et al., 2003). A Model for 50 Flap Interactions A Model for the Interaction between hExo1 and MutSa Although hExo1 is predominantly exonucleolytic, it also can in Mismatch Repair process 50 flaps endonucleolytically. We do not observe a 50 The interaction between hExo1 and MutSa is critical for DNA flap directly, but our structure indicates that such a single- mismatch repair (Amin et al., 2001; Genschel et al., 2002; Nielsen stranded segment needs to be guided out of the crowded active et al., 2004; Schmutte et al., 2001; Wei et al., 2003). It has been site. Conflicting models have been proposed for the recognition well established that repair requires a nick at which hExo1 is re- and processing of 50 flap structures invoking either ‘‘tracking’’ of cruited by MutSa, initially bound at a mismatch as far as 1000 the nuclease along the single-stranded flap or ‘‘threading’’ of the base pairs away (Fang and Modrich, 1993; Genschel et al., flap through an aperture in the protein (Barnes et al., 1996; Ceska 2002; Genschel and Modrich, 2003). Furthermore, the C-terminal et al., 1996; Dervan et al., 2002; Gloor et al., 2010a, 2010b). Our (but not N-terminal) hExo1 domain interacts with MSH2 structures indicate that the protein must bind first at nicks, ruling (Schmutte et al., 1998, 2001). Based on the structural information out the tracking model, consistent with other observations (Gloor of the hExo1 DNA complexes and our observation that deletion et al., 2010b; Hohl et al., 2007). of the C-terminal hExo1 domain abolishes MutSa-provoked The threading model posits that the 50 flap passes through the excision, we propose a model for this recruitment process covalently closed arch formed by a4, a5, and their connecting (Figure 6C). loop. Although a model for such a path can be constructed by

218 Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. Figure 6. Model of 50 Exonuclease Activity in Human DNA Mismatch Repair (A) MutSa-stimulated mismatch provoked excision. Activation of hExoI by MutSa was scored as a function of MutSa concentration in the presence of 280 fmol MutLa, 900 fmol replication protein A (RPA), and 27 fmol of either full-length hExoI (top) or hExoI catalytic domain (bottom) with heteroduplex (left) or homoduplex (right) DNA substrates. After incubation for 5 min at 37C, reaction products were digested with NheI and ClaI and sepa- rated by electrophoresis on a 1% agarose gel (see Supplemental Information). Excised molecules are resistant to cleavage by NheI and are indicated by arrows. (B) Extents of excision on G-T (closed circles) and A-T (open circles) substrates are plotted for full- length hExoI (black) and hExoI catalytic domain (red). Quantitation is based on a single represen- tative experiment, depicted in (A). (C) A model for the interaction between MutSa and hExoI. MutSa is bound to a mismatch (pink dia- mond); hExo1 (red) is prebound at a nick and bends the DNA. The hExo1 C-terminal domain is depicted as an unstructured ensemble connected by a ﬂexible linker. MutSa is shown in green (MSH2) and blue (MSH6) with its putative hExo1- binding region (Schmutte et al., 2001) in purple. Following mismatch recognition, ﬁrst MutSa searches for hExo1 via bidirectional, one-dimensional diffusion along the DNA (search). Next, MutSa encounters hExo1 prebound at a nick and interacts with the C-terminal domain (capture), thereby alleviating the autoinhibition of the Exo1 (activate). Excised nucleotides are shown as black circles. Finally, the entire complex travels back toward the site of the mismatch, driven by the unidirectional 50-to-30 exonucleolytic cleavage of DNA (return). RPA (orange) binds to single- stranded regions of DNA uncovered by hExo1 activity. See also Figure S7.

linear extension of the DNA in the currently observed complexes a4(Figure 7A, path 1). We propose that slight rearrangements (Figure 7A, path 3), such a model raises a number of questions in the highly mobile a4 helix could position the scissile bond of concerning the energy source for this process (how the single- a flap over the metal center. In path 2, the mobility of the a4/a5 stranded segment is pulled through the aperture in the absence microdomain opens a cleft between it and the a2 region, thereby of a driving force such as ATP hydrolysis), accommodation of the providing flap exit path (Figure 7A, path 2). Neither proposed flap prior to threading (as the enzyme binds to the nick prior to path for 50 flaps invokes threading through the protein; instead, threading a flap), and potential divergence in the evolution of flap interactions take on the characteristic of a conventional substrate recognition and processing mechanisms in the super- binding event, consistent with recent biochemical experiments family (XPG and GEN1 cut bubbles and HJs, respectively, which (Gloor et al., 2010a) and enabling development of a unified do not have free ends and cannot thread). A nonthreading path mechanism for the superfamily (see below). involving binding to a surface groove or cleft (which may subse- quently close through a conformational change) could provide A Unified Model for Substrate Discrimination a mechanistically simpler alternative. A path can be constructed and Nuclease Activities in the FEN Family by extending a single-stranded 50 end of the substrate strand to FEN family members recognize or distinguish between five DNA follow approximately the natural curvature of the DNA backbone structures: simple nicks, gaps and 30 overhangs (hExo1 and set up in the double-stranded segment in the 50 binding region, FEN), 50 flaps (FEN, hExo1, and GEN1), 50 and 30 double flaps allowing the DNA to exit the active site by passing in front of (FEN), bubbles (XPG), and HJs (GEN). Bubbles and HJs can be

Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. 219 Figure 7. A Unified Model for Substrate Recognition in the 50 Structure-Specific Nuclease Family (A) Models of interactions between hExo1 and a 50 flap DNA substrate. The observed 50 recessed (pre-nick) DNA complex is shown in yellow (complementary strand) and orange (substrate strand). Two alternate paths (orange arrows) for accommodation of a 50 flap in hExo1 are shown; path 1 (orange arrow) shows a path that extends the 50 DNA flap in the nascent substrate complex in front of mobile helix a4; path 2 (orange arrow) passes through a cleft between the a4/ a5 microdomain and the a2 helix, which could open up if the microdomain moves. A third path (blue arrow) threads the DNA through the protein, below the arch formed by the loop connecting a4 and a5, which cannot accommodate bubbles or HJs in a unified model. (B) Schematic representation of the unified substrate-binding model. Selected structural elements that define alternate possible paths 1 or 2 are colored. thought of as logical extensions of the combined 50 and 30 For those members of the enzyme family that are regulated by double-flap substrates: a bubble is covalently connected to interactions with protein partners, we propose that the mobile a double-flap substrate, and a HJ is a fused double flap (Fig- a4/a5 microdomain plays an important role in such control ure 7B). The similarities in the hydrolytic mechanism and diversi- mechanisms either through direct interactions or indirectly ties in substrate recognition are reflected in the patterns of though the C-terminal domain. The concerted motions of the structure (Figure 1) and sequence alignments (Figure 2). a4 and a5 helices together with part of b3 appear to terminate All FEN family members recognize nicks or gaps. Consequently, at the highly conserved G79, which may act as a part of a hinge. the hydrophobic wedge, which induces a sharp bend in the DNA at The DNA substrate complexes of hExo1 presented here begin such sites, is structurally conserved, although its sequence to clarify a number of longstanding questions in the mechanism diverges. The nucleolytic mechanism is similarly conserved of the FEN nucleases. All family members induce a sharp bend in among the family members, as indicated by the presence of the the substrate for nick or gap recognition; fraying at the 50 end of acidic patch for binding metals in the catalytic site. This conserva- the duplex removes apparent differences between nick and flap tion extends to residues involved in fraying at the 50 ends of nicks, processing and position the scissile phosphate; and surface which unifies the exo- and endonucleolytic activities. binding sites encode recognition of 50 and 30 DNA substrate All of the DNA substrates possess a pre-nick duplex DNA region; structures through conventional binding interactions. Within the binding region for this element is also highly conserved. The this general framework, recognition of simple nicked (or gapped) K+-binding site is postulated to facilitate sliding of DNA in a proces- ends, flaps, and covalently connected flaps (bubbles) or HJs can sive mode (but not in XPG). With the exception of hGEN1, these be accommodated. Accommodation of additional structures in interactions are conserved across all FEN family members. flap regions, such as double-stranded segments, or telomeric GEN1 processes HJs and may not depend on processivity. G4 DNA (Vallur and Maizels, 2010a, 2010b) will depend both The diversity of 50 and 30 substrate structures presents on the distance from the nick or gap junction and potential inter- a geometrical puzzle for a common mechanism of DNA process- actions with other parts of the particular enzyme such as the ing. It is impossible to thread all of these varied substrates highly divergent C-terminal domain. The elegant simplicity by through a conserved, covalently closed aperture located within which nature has solved complex topological puzzles in the a4-a5 helical arch. In particular, bubbles or HJs cannot be substrate recognition and processing is remarkable. threaded without introducing a covalent break in either the 0 DNA or the protein. However, it may be possible to pass a 5 EXPERIMENTAL PROCEDURES extension out of the active site without needing to invoke threading through the protein (see above), providing a unified model of Cloning, Expression, Purification, and Activity Determination structured DNA substrate processing: 50 and 30 ends are all of hExo1 Catalytic Domain Constructs Wild-type, mutant, and selenomethionine-labeled Exo1 N-terminal domains recognized by grooved surface features (Figure 7B). a a (residues 1–352) were cloned, expressed, and purified as described in the The 4/ 5 microdomain is highly divergent among the family Supplemental Information. Exonuclease assays were carried in 20 mM Tris- 0 members and encodes recognition of the 5 flap or its equivalent HCl (pH 7.6), 0.75 mM HEPES-KOH, 120 mM KCl, 5mM MgCl2, 250 mg/ml 0 in bubbles and HJs. Similarly, 3 DNA substrate features are bovine serine albumin, 1.5 mM ATP, 1 mM glutathione, 60 mM dithiothreitol, encoded by the interactions with highly divergent a2/a3 region and 1% glycerol. Activities of wild-type and mutant Exo constructs were 0 and the post-nick segment. If the 30 end of the nicked strand is compared by incubating 25 nM radiolabeled 5 recessed DNA substrate with m bound in this region, then 30 flaps could be limited in length (or 0.005–5 nM protein in 5 l for 5 min at 37 C. Reaction products were analyzed by denaturing polyacrylamide gel electrophoresis (Figure 4E). not be able to bind). Conversely, surface grooves or clefts could guide flaps, bubbles, or HJs. hExo1 does not appear to have such Crystallization, Data Collection, and Structure Determination a a groove (consistent with its function), but an insertion in the 2 Wild-type and mutant proteins were cocrystallized (see Supplemental and a3 helices could form these. Such insertions are indicated Information) with a 50 recessed end substrate (top strand, 50-(p)-TCGACTA for XPG and GEN in the structure-based sequence alignment. GCG; bottom strand, 50-CGCTAGTCGACAC) in the presence of Ca2+;Ba2+

220 Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. and Mn2+ were introduced by exchange in crystal soaks. Diffraction data were Energy Sciences, under contract number W-31-109-Eng-38. Use of the collected at The Advanced Photon Source (APS); at Argonne National Labora- SIBYLS beamline at the Advanced Light Source, Lawrence Berkeley National tory, beamlines 22-ID and 22-BM (SER-CAT); and at The Advanced Light Laboratory, was supported, in part, by the DOE program Integrated Diffraction Source (ALS) at Lawrence Berkeley National Laboratory, beamline 12.3.1 Analysis Technologies (IDAT) and the DOE program Molecular Assemblies (SIBYLS). Experiments were conducted at 100 K. Native diffraction data Genes and Genomics Integrated Efficiently (MAGGIE) under contract number were collected to 2.5 A˚ resolution, and selenomethionine data were collected DE-AC02-05CH11231 with the U.S. Department of Energy. to 3.3 and 3.4 A˚ resolution at l = 0.9794 A˚ at the selenium K edge. Data were scaled in space group P21212 using HKL2000 (Otwinowski, 1998). The struc- Received: October 31, 2010 ture of the hExo1 catalytic domain (D173A) DNA complex was determined by Revised: January 25, 2011 selenium-SIRAS experimental phasing (Hendrickson et al., 1990). Two Se Accepted: March 1, 2011 peak data sets and a native data set were used to determine experimental Published: April 14, 2011 phases with SHARP (Bricogne et al., 2003). The model contained two molecules in the asymmetric unit, and five selenium sites per monomer were REFERENCES observed. The model was built manually in COOT (Emsley and Cowtan, 2004). Initial solvent-flattened maps lacked side-chain density and connec- Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., tivity and were improved using partial model phase combination and b factor Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C. sharpening in CNS (Bru¨ nger et al., 1998). PHENIX Autobuild (Adams et al., (2002). PHENIX: building new software for automated crystallographic struc- 2002) was used to improve initial model and electron density. The structure ture determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954. was refined using CCP4 (Winn et al., 2003) and CNS with a maximum likelihood target and phase probability distribution. Allawi, H.T., Kaiser, M.W., Onufriev, A.V., Ma, W.P., Brogaard, A.E., Case, Wild-type crystals diffracted to 3.1 A˚ resolution and were scaled in the D.A., Neri, B.P., and Lyamichev, V.I. (2003). Modeling of flap endonuclease interactions with DNA substrate. J. Mol. Biol. 328, 537–554. P43212 space group using HKL2000. Wild-type barium derivative data were measured at l = 1.2 A˚ , and manganese derivative data were collected at l = Amin, N.S., Nguyen, M.N., Oh, S., and Kolodner, R.D. (2001). exo1-Dependent 1.0 A˚ or 1.25 A˚ . Molecular replacement phases were calculated using PHASER mutator mutations: model system for studying functional interactions in (Storoni et al., 2004) using the D173A mutant structure as a model. Initial low- mismatch repair. Mol. Cell. Biol. 21, 5142–5155. resolution refinement was carried out in CNS using deformable elastic network Barnes, C.J., Wahl, A.F., Shen, B., Park, M.S., and Bambara, R.A. (1996). (DEN) restraints (Schro¨ der et al., 2007). The hExo1 D173A structure was input Mechanism of tracking and cleavage of adduct-damaged DNA substrates as a reference model. DNA was built manually in COOT using electron density by the mammalian 50-to30-exonuclease/endonuclease RAD2 homologue 1 maps from this refinement. Metal ion positions were identified using phased or flap endonuclease 1. J. Biol. Chem. 271, 29624–29631. anomalous difference Fourier maps. Structures were refined using PHENIX Beese, L.S., and Steitz, T.A. (1991). Structural basis for the 30-50 exonuclease and CNS, with a maximum likelihood target and phase probability distribution. activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. Final model coordinates were checked with MOLPROBITY (Davis et al., 2004). EMBO J. 10, 25–33.

Mismatch-Provoked Excision Assays Brautigam, C.A., and Steitz, T.A. (1998). Structural principles for the inhibition of the 30-50 exonuclease activity of Escherichia coli DNA polymerase I by phos- Mismatch-provoked excision reactions were carried out in the buffer phorothioates. J. Mol. Biol. 277, 363–377. described in the Experimental Procedures and contained 24 fmol of a 6440 bp circular G-T heteroduplex (or control A-T homoduplex) with a strand Brautigam, C.A., Sun, S., Piccirilli, J.A., and Steitz, T.A. (1999). Structures of 0 break located 128 bp 50 to the mismatch. Reactions (20 ml) were assembled on normal single-stranded DNA and deoxyribo-3 -S-phosphorothiolates bound 0 0 ice by addition of 1 ml each of MutSa, MutLa, and RPA, diluted as described to the 3 -5 exonucleolytic active site of DNA polymerase I from Escherichia (Genschel and Modrich, 2003), to 16 ml of a solution containing all other coli. Biochemistry 38, 696–704. components except ExoI. Reactions were initiated by addition of 1 ml of ExoI Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M., and Paciorek, W. (2003). directly to the above solution on ice and were immediately transferred to Generation, representation and flow of phase information in structure determi- a37C water bath and incubated for 5 min. Samples were deproteinized by nation: recent developments in and around SHARP 2.0. Acta Crystallogr. D Proteinase K treatment followed by phenol extraction. Extent of excision Biol. Crystallogr. 59, 2023–2030. was scored by NheI resistance assay as described (Genschel et al., 2002). Bru¨ nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse- Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. ACCESSION NUMBERS (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921. Atomic coordinates and structure factors for hExo1 complexes I, II, and III have Ceska, T.A., Sayers, J.R., Stier, G., and Suck, D. (1996). A helical arch allowing been deposited in the Protein Data Bank under ID codes 3QE9, 3QEA, and 0 single-stranded DNA to thread through T5 5 -exonuclease. Nature 382, 90–93. 3QEB, respectively. Chapados, B.R., Hosfield, D.J., Han, S., Qiu, J., Yelent, B., Shen, B., and Tainer, J.A. (2004). Structural basis for FEN-1 substrate specificity and SUPPLEMENTAL INFORMATION PCNA-mediated activation in DNA replication and repair. Cell 116, 39–50. Davis, I.W., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2004). Supplemental Information includes Extended Experimental Procedures, seven MOLPROBITY: structure validation and all-atom contact analysis for nucleic figures, two tables, and one movie and can be found with this article online at acids and their complexes. Nucleic Acids Res. 32(Web Server issue), W615– doi:10.1016/j.cell.2011.03.005. W619.

ACKNOWLEDGMENTS Dervan, J.J., Feng, M., Patel, D., Grasby, J.A., Artymiuk, P.J., Ceska, T.A., and Sayers, J.R. (2002). Interactions of mutant and wild-type flap endonucleases We thank Anita Changela, Shannon Kelly, and Miaw-Sheue Tsai for assistance with oligonucleotide substrates suggest an alternative model of DNA binding. with cloning and purification. This work was supported by National Institutes of Proc. Natl. Acad. Sci. USA 99, 8542–8547. Health grants R01 GM091487 to L.S.B., R01 GM45190 to P.M., and P01 Devos, J.M., Tomanicek, S.J., Jones, C.E., Nossal, N.G., and Mueser, T.C. CA092584 to L.S.B. and P.M. P.M. is an Investigator of the Howard Hughes (2007). Crystal structure of bacteriophage T4 50 nuclease in complex with Medical Institute. Use of the Advanced Photon Source was supported, in a branched DNA reveals how flap endonuclease-1 family nucleases bind their part, by the U.S. Department of Energy, Office of Science, Office of Basic substrates. J. Biol. Chem. 282, 31713–31724.

Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. 221 Doherty, K.M., Sharma, S., Uzdilla, L.A., Wilson, T.M., Cui, S., Vindigni, A., and Lee, B.I., Nguyen, L.H., Barsky, D., Fernandes, M., and Wilson, D.M., III. Brosh, R.M., Jr. (2005). RECQ1 helicase interacts with human mismatch repair (2002). Molecular interactions of human Exo1 with DNA. Nucleic Acids Res. factors that regulate genetic recombination. J. Biol. Chem. 280, 28085–28094. 30, 942–949. Dore´ , A.S., Kilkenny, M.L., Jones, S.A., Oliver, A.W., Roe, S.M., Bell, S.D., and Liberti, S.E., Andersen, S.D., Wang, J., May, A., Miron, S., Perderiset, M., Pearl, L.H. (2006). Structure of an archaeal PCNA1-PCNA2-FEN1 complex: Keijzers, G., Nielsen, F.C., Charbonnier, J.B., Bohr, V.A., et al. (2011). Bi-direc- elucidating PCNA subunit and client enzyme specificity. Nucleic Acids Res. tional routing of DNA mismatch repair protein human exonuclease 1 to replica- 34, 4515–4526. tion foci and DNA double strand breaks. DNA Repair (Amst) 10, 73–86. Pub- Dzantiev, L., Constantin, N., Genschel, J., Iyer, R.R., Burgers, P.M., and Mod- lished online October 20, 2010. rich, P. (2004). A defined human system that supports bidirectional mismatch- Matsui, E., Musti, K.V., Abe, J., Yamasaki, K., Matsui, I., and Harata, K. (2002). provoked excision. Mol. Cell 15, 31–41. Molecular structure and novel DNA binding sites located in loops of flap endo- Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular nuclease-1 from Pyrococcus horikoshii. J. Biol. Chem. 277, 37840–37847. graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132. Mimitou, E.P., and Symington, L.S. (2008). Sae2, Exo1 and Sgs1 collaborate in Fang, W.H., and Modrich, P. (1993). Human strand-specific mismatch repair DNA double-strand break processing. Nature 455, 770–774. occurs by a bidirectional mechanism similar to that of the bacterial reaction. Mueser, T.C., Nossal, N.G., and Hyde, C.C. (1996). Structure of bacteriophage J. Biol. Chem. 268, 11838–11844. T4 RNase H, a 50 to 30 RNA-DNA and DNA-DNA exonuclease with sequence Feng, M., Patel, D., Dervan, J.J., Ceska, T., Suck, D., Haq, I., and Sayers, J.R. similarity to the RAD2 family of eukaryotic proteins. Cell 85, 1101–1112. (2004). Roles of divalent metal ions in flap endonuclease-substrate interac- Nielsen, F.C., Ja¨ ger, A.C., Lu¨ tzen, A., Bundgaard, J.R., and Rasmussen, L.J. tions. Nat. Struct. Mol. Biol. 11, 450–456. (2004). Characterization of human exonuclease 1 in complex with mismatch Genschel, J., and Modrich, P. (2003). Mechanism of 50-directed excision in repair proteins, subcellular localization and association with PCNA. Oncogene human mismatch repair. Mol. Cell 12, 1077–1086. 23, 1457–1468. Genschel, J., and Modrich, P. (2009). Functions of MutLalpha, replication Nimonkar, A.V., Ozsoy, A.Z., Genschel, J., Modrich, P., and Kowalczykowski, protein A (RPA), and HMGB1 in 50-directed mismatch repair. J. Biol. Chem. S.C. (2008). Human exonuclease 1 and BLM helicase interact to resect DNA 284, 21536–21544. and initiate DNA repair. Proc. Natl. Acad. Sci. USA 105, 16906–16911. Genschel, J., Bazemore, L.R., and Modrich, P. (2002). Human exonuclease I is Otwinowski, Z. (1998). Denzo and Scalepack programs: An oscillation data required for 50 and 30 mismatch repair. J. Biol. Chem. 277, 13302–13311. processing suite for macromolecular crystallography. Gloor, J.W., Balakrishnan, L., and Bambara, R.A. (2010a). Flap endonuclease 1 Pelletier, H., Sawaya, M.R., Kumar, A., Wilson, S.H., and Kraut, J. (1994). mechanism analysis indicates flap base binding prior to threading. J. Biol. Structures of ternary complexes of rat DNA polymerase beta, a DNA Chem. 285, 34922–34931. template-primer, and ddCTP. Science 264, 1891–1903. Gloor, J.W., Balakrishnan, L., and Bambara, R.A. (2010b). Flap endonuclease Pelletier, H., Sawaya, M.R., Wolfle, W., Wilson, S.H., and Kraut, J. (1996). 1 mechanism analysis indicates flap base binding prior to threading. J. Biol. Crystal structures of human DNA polymerase beta complexed with DNA: Chem. 285, 34922–34931. implications for catalytic mechanism, processivity, and fidelity. Biochemistry Gradia, S., Acharya, S., and Fishel, R. (1997). The human mismatch recogni- 35, 12742–12761. tion complex hMSH2-hMSH6 functions as a novel molecular switch. Cell 91, Peltoma¨ ki, P. (2003). Role of DNA mismatch repair defects in the pathogenesis 995–1005. of human cancer. J. Clin. Oncol. 21, 1174–1179. Gradia, S., Subramanian, D., Wilson, T., Acharya, S., Makhov, A., Griffith, J., Sakurai, S., Kitano, K., Yamaguchi, H., Hamada, K., Okada, K., Fukuda, K., and Fishel, R. (1999). hMSH2-hMSH6 forms a hydrolysis-independent sliding Uchida, M., Ohtsuka, E., Morioka, H., and Hakoshima, T. (2005). Structural clamp on mismatched DNA. Mol. Cell 3, 255–261. basis for recruitment of human flap endonuclease 1 to PCNA. EMBO J. 24, Gravel, S., Chapman, J.R., Magill, C., and Jackson, S.P. (2008). DNA helicases 683–693. Sgs1 and BLM promote DNA double-strand break resection. Genes Dev. 22, Schmutte, C., Marinescu, R.C., Sadoff, M.M., Guerrette, S., Overhauser, J., 2767–2772. and Fishel, R. (1998). Human exonuclease I interacts with the mismatch repair Hartlerode, A.J., and Scully, R. (2009). Mechanisms of double-strand break protein hMSH2. Cancer Res. 58, 4537–4542. repair in somatic mammalian cells. Biochem. J. 423, 157–168. Schmutte, C., Sadoff, M.M., Shim, K.S., Acharya, S., and Fishel, R. (2001). The Hendrickson, W.A., Horton, J.R., and LeMaster, D.M. (1990). Selenomethionyl interaction of DNA mismatch repair proteins with human exonuclease I. J. Biol. proteins produced for analysis by multiwavelength anomalous diffraction Chem. 276, 33011–33018. (MAD): a vehicle for direct determination of three-dimensional structure. Schro¨ der, G.F., Brunger, A.T., and Levitt, M. (2007). Combining efficient EMBO J. 9, 1665–1672. conformational sampling with a deformable elastic network model facilitates Hohl, M., Dunand-Sauthier, I., Staresincic, L., Jaquier-Gubler, P., Thorel, F., structure refinement at low resolution. Structure 15, 1630–1641. Modesti, M., Clarkson, S.G., and Scha¨ rer, O.D. (2007). Domain swapping Sengerova´ , B., Tomlinson, C., Atack, J.M., Williams, R., Sayers, J.R., Williams, between FEN-1 and XPG defines regions in XPG that mediate nucleotide exci- N.H., and Grasby, J.A. (2010). Brønsted analysis and rate-limiting steps for the sion repair activity and substrate specificity. Nucleic Acids Res. 35, 3053– T5 flap endonuclease catalyzed hydrolysis of exonucleolytic substrates. 3063. Biochemistry 49, 8085–8093. Hosfield, D.J., Mol, C.D., Shen, B., and Tainer, J.A. (1998). Structure of the Sharma, S., Sommers, J.A., Driscoll, H.C., Uzdilla, L., Wilson, T.M., and Brosh, DNA repair and replication endonuclease and exonuclease FEN-1: coupling R.M., Jr. (2003). The exonucleolytic and endonucleolytic cleavage activities of DNA and PCNA binding to FEN-1 activity. Cell 95, 135–146. human exonuclease 1 are stimulated by an interaction with the carboxyl- Hwang, K.Y., Baek, K., Kim, H.Y., and Cho, Y. (1998). The crystal structure of terminal region of the Werner syndrome protein. J. Biol. Chem. 278, 23487– flap endonuclease-1 from Methanococcus jannaschii. Nat. Struct. Biol. 5, 23496. 707–713. Shen, B., Nolan, J.P., Sklar, L.A., and Park, M.S. (1996). Essential amino acids Kolodner, R.D. (1995). Mismatch repair: mechanisms and relationship to for substrate binding and catalysis of human flap endonuclease 1. J. Biol. cancer susceptibility. Trends Biochem. Sci. 20, 397–401. Chem. 271, 9173–9176. Lee, B.I., and Wilson, D.M., III. (1999). The RAD2 domain of human exonu- Shen, B., Nolan, J.P., Sklar, L.A., and Park, M.S. (1997). Functional analysis of clease 1 exhibits 50 to 30 exonuclease and flap structure-specific endonu- point mutations in human flap endonuclease-1 active site. Nucleic Acids Res. clease activities. J. Biol. Chem. 274, 37763–37769. 25, 3332–3338.

222 Cell 145, 212–223, April 15, 2011 ª2011 Elsevier Inc. Steitz, T.A., and Steitz, J.A. (1993). A general two-metal-ion mechanism for Wei, K., Clark, A.B., Wong, E., Kane, M.F., Mazur, D.J., Parris, T., Kolas, N.K., catalytic RNA. Proc. Natl. Acad. Sci. USA 90, 6498–6502. Russell, R., Hou, H., Jr., Kneitz, B., et al. (2003). Inactivation of Exonuclease 1 Storoni, L.C., McCoy, A.J., and Read, R.J. (2004). Likelihood-enhanced fast in mice results in DNA mismatch repair defects, increased cancer suscepti- rotation functions. Acta Crystallogr. D Biol. Crystallogr. 60, 432–438. bility, and male and female sterility. Genes Dev. 17, 603–614. Syson, K., Tomlinson, C., Chapados, B.R., Sayers, J.R., Tainer, J.A., Williams, Winn, M.D., Murshudov, G.N., and Papiz, M.Z. (2003). Macromolecular TLS N.H., and Grasby, J.A. (2008). Three metal ions participate in the reaction cata- refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, lyzed by T5 flap endonuclease. J. Biol. Chem. 283, 28741–28746. 300–321. Tomlinson, C.G., Atack, J.M., Chapados, B., Tainer, J.A., and Grasby, J.A. (2010). Substrate recognition and catalysis by flap endonucleases and related Zhang, Y., Yuan, F., Presnell, S.R., Tian, K., Gao, Y., Tomkinson, A.E., Gu, L., enzymes. Biochem. Soc. Trans. 38, 433–437. 0 and Li, G.M. (2005). Reconstitution of 5 -directed human mismatch repair in Vallur, A.C., and Maizels, N. (2010a). Complementary roles for exonuclease 1 a purified system. Cell 122, 693–705. and Flap endonuclease 1 in maintenance of triplet repeats. J. Biol. Chem. 285, 28514–28519. Zhu, Z., Chung, W.H., Shim, E.Y., Lee, S.E., and Ira, G. (2008). Sgs1 helicase Vallur, A.C., and Maizels, N. (2010b). Distinct activities of exonuclease 1 and and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell flap endonuclease 1 at telomeric g4 DNA. PLoS ONE 5, e8908. 134, 981–994.

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