Repair Complexes of FEN1 Endonuclease, DNA, and Rad9-Hus1-Rad1 Are Distinguished from Their PCNA Counterparts by Functionally Important Stability

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

Jordi Querol-Audía,1, Chunli Yanb,1, Xiaojun Xub, Susan E. Tsutakawac, Miaw-Sheue Tsaic, John A. Tainerc,d,e, Priscilla K. Cooperc, Eva Nogalesa,c,f,2, and Ivaylo Ivanovb,2

aDepartment of Molecular and Cell Biology, California Institute for Quantitative Biosciences and fHoward Hughes Medical Institute, University of California, Berkeley, CA 94720; bDepartment of Chemistry, Georgia State University, Atlanta, GA 30302; cLife Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and dDepartment of Molecular Biology and eSkaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037

Edited* by J. Andrew McCammon, University of California at San Diego, La Jolla, CA, and approved April 16, 2012 (received for review December 29, 2011) Processivity clamps such as proliferating cell nuclear antigen (PCNA) of these factors is through attachment to the interdomain con- and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as nector (IDC) loop of PCNA and the PCNA C terminus (2, 13, versatile scaffolds in the coordinated recruitment of proteins in- 16). The trimeric PCNA ring can provide, at most, three binding volved in DNA replication, cell-cycle control, and DNA repair. sites for replication and repair factors. The crystal structure of Association and handoff of DNA-editing enzymes, such as flap human FEN1 with PCNA indeed revealed three FEN1 enzymes endonuclease 1 (FEN1), with sliding clamps are key processes in bound to the sliding clamp in different orientations (16). Addi- biology, which are incompletely understood from a mechanistic tionally, a biochemical study of the Sulfolobus solfataricus proteins point of view. We have used an integrative computational and supported the idea that distinct protein partners such as DNA experimental approach to define the assemblies of FEN1 with polymerase, FEN1, and DNA ligase could simultaneously asso- double-flap DNA substrates and either proliferating cell nuclear ciate with PCNA (17). The competition among proteins to si- antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models multaneously bind to the surface of PCNA as well as to their of these two ternary complexes were developed and refined common DNA substrate has led to the notion of conformational through extensive molecular dynamics simulations to expose their switching and handoffs of repair intermediates (2, 17); these are BIOCHEMISTRY conformational dynamics. Clustering analysis revealed the most key processes in PCNA biology, which are incompletely un- dominant conformations accessible to the complexes. The cluster derstood from a mechanistic perspective. centroids were subsequently used in conjunction with single-parti- In addition to PCNA, FEN1 is known to associate with the cle electron microscopy data to obtain a 3D EM reconstruction of the alternative checkpoint clamp Rad9-Rad1-Hus1 (9-1-1 complex). human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the Whereas PCNA is comprised of three identical subunits, 9-1-1 is structures of the complexes revealed key differences in the orienta- a heterotrimeric sliding clamp (18–20). This fact reflects the tion and interactions of FEN1 and double-flap DNA with the two different protein partners the two clamps engage and the distinct clamps that are consistent with their respective functions in provid- roles these complexes play in coordinating DNA processing. In ing inherent flexibility for lagging strand DNA replication or inher- contrast to PCNA, the 9-1-1 complex is thought to serve as a ent stability for DNA repair. recruitment platform to bring checkpoint effector kinases to sites of DNA damage, thus activating checkpoint control, and also lap endonuclease 1 (FEN1) belongs to a class of essential functions to stabilize stalled replication forks that have encoun- Fnucleases (the FEN1 5′ nuclease superfamily) present in all tered DNA lesions (21–23). It has also been demonstrated that domains of life (1). FEN1 catalyzes the endonucleolytic cleavage 9-1-1 interacts with and stimulates enzymes involved in base of bifurcated DNA or RNA structures known as 5′ flaps. These excision repair (BER), such as NEIL1, MYH, TDG, FEN1, and 5′ flaps are generated during lagging strand DNA synthesis or DNA Ligase I, thus potentially linking BER activities to check- – during long-patch base excision repair. The FEN1 substrates are point coordination (24 27). in fact double-flap DNA (dfDNA) with DNA on the opposite In view of their crucial involvement in replication and repair, side of the 5′ flap, forming a single nucleotide 3′ flap when bound a detailed structural comparison of the ternary PCNA/FEN1/ to the enzyme (2, 3). By removing the 5′ ssDNA or RNA flap DNA and 9-1-1/FEN1/DNA complexes would be of great value. from such substrates, FEN1 produces a single nicked product Though structural snapshots are available for the individual that could be sealed by the subsequent action of a DNA ligase components of such assemblies (PCNA [Protein Data Bank (4). Consistent with its crucial role in DNA replication and re- (PDB) ID code: 1VYM], human 9-1-1 [3GGR]) (Fig. S1) and for pair, FEN1 is highly expressed in all proliferative tissues, and its two binary complexes [FEN1/DNA (3Q8L) and FEN1/PCNA – activity is key for the maintenance of genomic integrity (5). (1UL1)] (3, 16, 18 20, 28), the larger ternary assemblies present FEN1 has been identified as a cancer susceptibility gene, and mutations in it have been linked to a number of genetic diseases, such as EM map myotonic dystrophy, Huntington disease, sev- Author contributions: J.Q.-A., S.E.T., J.A.T., P.K.C., E.N., and I.I. designed research; J.Q.-A., – C.Y., X.X., S.E.T., M.-S.T., and I.I. performed research; M.-S.T. contributed new reagents/ eral ataxias, fragile X syndrome, and cancer (6 10). analytic tools; J.Q.-A., C.Y., X.X., S.E.T., and I.I. analyzed data; and J.Q.-A., C.Y., J.A.T., E.N., The nuclease activity of FEN1 can be stimulated by association and I.I. wrote the paper. with processivity clamps such as proliferating cell nuclear antigen The authors declare no conflict of interest. (PCNA), which encircle DNA at sites of replication and repair – *This Direct Submission article had a prearranged editor. (11 13). PCNA is a recognized master coordinator of cellular Data deposition: The data reported in this paper have been deposited in the Electron responses to DNA damage and interacts with numerous DNA Microscopy Data Bank, www.ebi.ac.uk/pdbe/emdb/ [accession codes: EMD-2029 (FEN1/ repair and cell-cycle control proteins. In this capacity, PCNA DNA/9-1-1 negative-stain EM map) and EMD-2030 (9-1-1 complex) EM map]. serves not only as a mobile platform for the attachment of these 1J.Q.-A. and C.Y. contributed equally to this work. proteins to DNA but, importantly, plays an active role in the re- 2To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. cruitment and release of these crucial participants at the repli- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cation fork (14, 15). The dominant mode of interaction for many 1073/pnas.1121116109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1121116109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 extreme challenges to molecular crystallography (MX). Here we generate the initial model of the 9-1-1 ternary complex we used an report models for the ternary PCNA/FEN1/DNA and 9-1-1/ overlay of the human 9-1-1 structure with truncated Rad9 subunit FEN1/DNA assemblies, which were constructed by combining all (9Δ-1-1; PDB ID code 3GGR) (20) onto the PCNA/FEN1/DNA available high-resolution MX data for the individual components model (Fig. 1B). FEN1 was modeled bound to the Rad1 subunit and subassemblies. The models were refined by multinanosecond based on previous experimental work (20). The final models were atomistic molecular dynamics (MD) simulations. Single-particle selected after pairwise rmsd clustering analysis of the MD tra- electron microscopy (EM) of negatively stained samples in- jectories (31, 32), and the structure closest to the centroid of the dicated that the structure defined by the 9-1-1/FEN1/DNA most populated cluster was chosen as representative for each A B model exists in solution. Subsequently, the computational model ternary complex (Fig. 1 and ). Δ was integrated with the EM data resulting in a 3D reconstruction In both PCNA and 9 -1-1 ternary models, FEN1 occupied an for the ternary assembly determined at 18-Å resolution. Finally, overall upright position on the polymerase binding face of PCNA we present a detailed comparative analysis of the two ternary or on the corresponding face of the checkpoint clamp. The path of fl complexes. the DNA bends 100° at the position of the double ap bound by FEN1, consistent with the structure of FEN1/DNA (3). Sakurai Results and Discussion et al. (16) had postulated that an additional “swing-in” motion of ∼ Overall Structure of the Ternary FEN1 Complexes. To shed light on 50° would be required to move FEN1 (chain Y) into a productive the conformations and structural dynamics of the ternary FEN1 conformation with DNA passing through the PCNA ring. How- complexes, we carried out multinanosecond MD simulations ever, we observe that no such swing-in motion is required due to the 100° bend of the dfDNA by FEN1 and the tilted orientation of (Movies S1 and S2) and were able to refine models of PCNA/ the upstream DNA with respect to the plane of PCNA or 9-1-1. In FEN1/DNA and 9-1-1/FEN1/DNA, which exhibited no structural both models the enzyme was facing the central cavity of the PCNA discrepancies or steric hindrance between the sliding clamps, or 9Δ-1-1 ring, a position that would promote DNA exchange with DNA, and the nuclease (Fig. 1). The initial PCNA ternary com- — other PCNA-bound proteins such as polymerases or ligases. A plex was based on two structures the FEN1/DNA complex (PDB major global difference between PCNA and 9Δ-1-1 ternary com- ID code 3Q8L) (3) and the FEN1/PCNA structure (PDB ID code plexes was a 14-Å shift of the FEN1 core toward Rad1 compared 1UL1) (16). The FEN1/PCNA structure showed three bound with a more upright position found in the PCNA complex. copies of FEN1 (X, Y, and Z chains) in three orientations relative to the PCNA homotrimer. Initial overlays of FEN1 in these two EM Analysis and Computational Flexible Fitting of the 9-1-1/FEN1 structures showed that chain Y orientation was the only one which Binary and Ternary Complexes. Hybrid methods for structural would allow a double-stranded DNA extension on the 3′ flap side analysis constitute an emergent area, which could shed light on (upstream with respect to replication) to pass through the PCNA biological complexes with a high degree of structural plasticity. ring (29). Therefore, only chain Y orientation was used in the The FEN1 ternary assembles are examples of systems, wherein the model. The range of conformations observed in the crystal struc- inherent flexibility has so far precluded crystallographic analysis. ture suggests segmental flexibility whereby a flexibly linked protein As a direct visualization technique, single-particle EM could be (such as FEN1) can adopt several discrete positions (30). To used to investigate such flexible assemblies (33, 34). Binary complex formation for FEN1 and 9-1-1 was assessed by incubating both proteins at 30 °C and subsequently running size- exclusion chromatography (SEC). Analysis of the corresponding fractions by SDS/PAGE showed 9-1-1 and FEN1 eluting at different retention times, thus indicating weak interaction under our experimental conditions (Fig. S2). To increase the stability of the complex, the sample was cross-linked before size-exclusion chromatography. Fractions containing the 9-1-1/FEN1 complex were then visualized by EM. In parallel, a 3D EM reconstruction of the 9-1-1 checkpoint clamp was generated that superimposed well with the 9-1-1 crystal structure (Fig. S3). Due to the pseu- dosymmetry of the 9-1-1 reconstruction at this resolution, it was not possible to definitively discriminate between the 9-1-1 subunits solely based on the EM density. Reference-free 2D class averages for the binary 9-1-1/FEN1 complex showed shapes that could be classified as either six-lobed rings with an additional density in the periphery of the ring or as compact two-lobed rods with an extra density region to one side (Fig. 2A, Top). Comparing the images to the 3D reconstruction of 9-1-1, the six-lobed rings and two-lobed rods were respectively assigned as top and side views of the checkpoint clamp. The position of the extra density adjacent to the 9-1-1 ring was highly variable and corresponded in all cases to a single FEN1 enzyme bound to the clamp. Our findings indicate that only one of the Fig. 1. Dominant structures for the PCNA/FEN1/DNA and 9Δ-1-1/FEN1/DNA 9-1-1 subunits is competent to associate with FEN1. Based on complexes from MD trajectory clustering analysis. The (A) PCNA and (B)9Δ-1-1 previously reported surface plasmon resonance measurements complex are shown as cartoons. PCNA is shown in green, FEN1 in purple, (20) and crystallographic results (19), we assigned Rad1 as the Rad1 in green, Hus1 in yellow, Rad 9 in blue, and DNA in black. The gray FEN1-interacting subunit. Importantly, the side views clearly surfaces are the FEN1/DNA from the original starting models. The surfaces for the two clamps in the starting models were omitted for clarity. (C and D) showed how FEN1 could swing by at least 90° from a position in- Computed B-factors mapped onto the PCNA and 9Δ-1-1 models. Coloring line with the plane of the ring to an upright orientation facing the corresponds to computed B-factor values from high (red) to low (blue). central hole of 9-1-1. This observation is particularly interesting in (Insets) Highlight of the B-factor difference for upstream DNA as it passes light of the multiple FEN1 orientations in the Sakurai et al. (16) through the two clamps. binary PCNA structure. In the structure, chain X corresponded to

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1121116109 Querol-Audí et al. Downloaded by guest on September 30, 2021 S4A). After cross-linking and SEC (Fig. S4 B and C), samples were visualized by negative-stain EM (Fig. S5A). Representative reference-free 2D class averages of the ternary 9Δ-1-1/FEN1/DNA complex showed that in the presence of DNA, the extra density lobe above the 9Δ-1-1 ring adopted a well-defined fixed position (Fig. 2A, Middle). Addition of DNA locked FEN1 into the upright position facing the central cavity of the 9Δ-1-1 clamp, providing direct experimental support for our computational results. Computationally generated projections of our model (filtered at 20-Å resolution) qualitatively resembled the EM reference-free class averages (Fig. S5B). Thus, we used this filtered structure as an initial model to assign relative orientations to the different experimental views of the complex. We did 3D refinement using iterative projection matching in EMAN2 (35, 36), reaching a final 3D reconstruction of the 9Δ-1- 1/FEN1/DNA complex at an estimated resolution of 18 Å (Fig. 2B). The 3D map contains two principal features—a hexagonal nonsymmetrical ring with an external diameter of ∼85 Å and a central channel of ∼30 Å corresponding to 9Δ-1-1, and a two- lobed extra density ∼70 Å in length, corresponding to FEN1 and connected to the 9Δ-1-1 ring. Although the presence of DNA was suggested by the clear effect in the relative positioning of FEN1 with respect to the 9Δ-1-1 ring and a strong OD260 peak in SEC after cross-linking, the reconstruction did not show clear density for DNA. This finding was not surprising, given the well- known difficulty in visualizing DNA by negative staining. As a final stage of our atomistic model generation and re-

finement, we used flexible fitting into the experimental EM map BIOCHEMISTRY using the molecular dynamics flexible fitting (MDFF) method (37, 38) (Fig. 2C). MDFF involves a MD simulation wherein external forces proportional to the EM density gradient are applied to bias Fig. 2. Single-particle EM analysis shows how FEN1 interacts flexibly with the atoms of the model into the high-density regions of the EM fi Δ 9-1-1 and adopts a xed position on 9 -1-1 in the presence of the DNA map. The DNA substrate was excluded from the MDFF fitting substrate. (A) Representative reference-free 2D class averages (top and side fi Δ views) for the 9-1-1/FEN1 binary complex are compared with those corre- procedure. After the re nement and minimization, the 9 -1-1/ sponding to the 9Δ-1-1/FEN1/DNA ternary complex. Top and side views of FEN1/DNA atomic model exhibited excellent agreement with the the 9-1-1 complex are shown (Bottom) for reference. (B) Side and top views experimental EM map with fewer than 300 atoms found outside of the 9Δ-1-1/FEN1/DNA 3D reconstruction. (C) MDFF flexible fitting of the the EM density envelope (at a conservative density threshold of 9Δ-1-1/FEN1/DNA complex into the 3D map of the ternary complex. 3.6; rigid-body fit for the complex is compared with the flexible fit by MDFF in Fig. S6). All flexible elements, including the surface loops of 9-1-1 and the helical arch of FEN1, were consistent with the in-line (“sideways”) position, whereas chains Y and Z corre- the EM map. Importantly, the EM map supported the observation sponded to the “upright” position. Our results show that the binary in the computational analysis that FEN1 was tilted toward Rad1. FEN1/9-1-1 complex displays the same range of conformational variability as the binary FEN1/PCNA complex, suggesting signif- Structural Determinants of Flexibility for the Ternary Assemblies in icance to this flexibility. The FEN1 positioning in the Sakurai the Simulations. To identify regional points of flexibility with structure could, in principle, be limited by specific crystal contacts functional implications, we plotted the time evolution of rmsd and steric constraints within the crystal. By contrast, our EM values for each ternary assembly and its constituent parts over 100 results reflect the conformational range for the complexes in so- ns of dynamics (Fig. S7 A and B). The most adaptable component lution. Thus, we propose that both the 9-1-1/FEN1 and PCNA/ was the DNA, which underwent the largest displacement in av- FEN1 binary assemblies are flexibly tethered. The functional role erage heavy-atom rmsd [7.14 ± 0.76 Å (SD) for the PCNA and of such flexibility could be to allow initial FEN1 binding in the 6.11 ± 0.54 Å (SD) for the 9Δ-1-1 complex, respectively]. By sideways orientation. In this position FEN1 could stay associated contrast, the core domain of FEN1 and the subunits of PCNA and and move along DNA without affecting the function of proteins the 9Δ-1-1 clamp displayed minimal internal displacements [1.90 ± bound to the front face of the clamp. Upon encountering the 0.14 Å (SD) for the FEN1 core, 2.31 ± 0.18 Å (SD) for PCNA, and correct DNA substrate, FEN1 could swing into the catalytically 3.54 ± 0.24 Å (SD) for 9-1-1]. The most significant internal competent upright orientation and engage in handoffs with pro- motions in the proteins were confined to surface loops of the teins simultaneously attached to the other two clamp subunits. clamps (e.g., P-loop of PCNA, IDC loops of Hus1 and Rad9), the To obtain a ternary 9-1-1/FEN1/DNA complex, we based the extreme C-terminal ends of PCNA, and a few flexible outer helices dfDNA substrate on one from the FEN1/DNA crystal (3), except in FEN1 (α13, α4, and α5, α10, α11, and α12; Fig. S1). Importantly, that 10 additional bases were introduced at the 3′ end (details although motions bringing FEN1 closer to the PCNA central provided in SI Materials and Methods). A band-shift assay was cavity were observed in the simulation (Fig. 1A and Fig. S7 A and performed in the presence of either full-length 9-1-1 or a trun- B), the overall displacement along this mode was rather subtle. cation construct lacking the extra C-terminal tail of Rad9 (9Δ-1-1). The displacement was reflected in the observed rmsd for the No band shift was observed with the full-length 9-1-1 (Fig. S4A), complexes [3.58 ± 0.49 Å (SD) for the PCNA complex and 6.94 ± suggesting that in the absence of the checkpoint clamp loader the 0.38 Å (SD) for the 9Δ-1-1 complex excluding DNA]. The rmsd Rad9 C-terminal tail may interfere with DNA binding. However, result for the DNA is corroborated by the high computed B-factors a band shift was observed when FEN1 and DNA were incubated mapped onto the structures of the two ternary complexes (Fig. 1 C with 9Δ-1-1, indicating formation of a ternary complex (Fig. and D). Not surprisingly, the entire DNA substrate was found to

Querol-Audí et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 be highly flexible; the one exception was the central portion of the upstream DNA, wherein the DNA backbone was found to form extensive contacts with the positive inner surface of the sliding clamps. Notably, the B-factors for this middle region of the upstream DNA are lower in the case of the 9Δ-1-1 complex, indicating tighter association. Other mobile regions included the H2TH DNA binding motif (helices α10, α11, and α12) and the “helical arch” region (helices α4 and α5), which caps the 5′ flap and the active site of FEN1. The helical arch is disordered in the absence of DNA (16) and is likely less stable even in the presence of DNA compared with the rest of the FEN1 core. The mobility of H2TH may have significance for the dsDNA binding mediated by H2TH/K+ (3). A pronounced tilt was observed in the two models between the axis of the upstream DNA and the plane of the PCNA or 9Δ-1-1 ring (Fig. S7E): 17.3 ± 2.9° (SD) for PCNA and 27.2 ± 2.0° (SD) for 9Δ-1-1. Our findings carry strong parallels to previous computational and experimental work. Conventionally, DNA is drawn perpendicular to PCNA in cartoon models. However, a tilted orientation was first suggested by our computational studies on PCNA/DNA binary complex (39). Experimentally, the crystal structure of the bacterial β-clamp/DNA complex (40) in- dicated that DNA passes through the clamp at a sharp angle of 22°. A PCNA crystal structure with a short DNA revealed the DNA at a 40° angle. Because further extension of the DNA would sterically clash with the ring, such a large tilting angle appears unlikely in this case (41). EM analysis of PCNA/Pol-B/DNA and PCNA/ligase/DNA assemblies suggested that DNA was tilted by 13° and 16°, respectively (42, 43). By contrast, in the replication factor C/ PCNA/DNA ternary assembly, the DNA axis was found to be nearly perpendicular to the PCNA ring (44). This versatile mode of association between the sliding clamp and DNA appears to be a universal feature observed in bacterial, archaeal, and eukaryotic Fig. 3. In the ternary complexes PCNA and 9-1-1 engage the upstream DNA clamps, and may allow for distinct functionalities with specific duplex in two distinct binding modes. (A and B) Cartoon representations of PCNA and 9-1-1 binding to dsDNA, colored in blue for Rad9 and PCNA1, protein partners. yellow for Hus1 and PCNA3, and green for Rad1 and PCNA2. The dsDNA phosphodiester groups (gray spheres) and basic residues on the inner surface DNA Is More Tightly Bound in the 9-1-1 than in the PCNA Ternary of PCNA and 9-1-1 (red surfaces) are shown. Below we explicitly list all Complex. Intriguingly, the mode of association with DNA was persistent DNA contacts (observed in more than 50% of the frames in the strikingly different for PCNA vs. 9Δ-1-1 ternary complex (Fig. 3). MD trajectories) for the PCNA and the 9-1-1 complex, respectively. (C) Av- The dynamic association of the upstream DNA to the inner cavities eraged structures for the upstream DNA duplex from FEN1/DNA (Left), of PCNA and 9Δ-1-1 was extensively sampled in the simulations. PCNA/FEN1/DNA (Center), and 9Δ-1-1/FEN1/DNA (Right) simulations. The Despite similarity in overall architecture, PCNA and 9-1-1 present DNA is shown in ribbons representation. The DNA axis was computed with distinct inner surfaces to DNA that are characterized by different the program Curves+ and is shown in red. The gray lines are representative shapes, electrostatics, hydrophobic and polar patches, and sec- of the widths of the major and minor grooves of DNA. ondary structure elements. A highly asymmetric mode of association was observed in the PCNA complex, with only one of the persistent in the MD trajectories compared with the PCNA PCNA subunits forming the majority of stable contacts with the complex (Fig. S7 C and D). The distinct distribution of basic res- DNA backbone (K14, K20, K217, R146, and R149). The second idue contacts in the two complexes likely determined the overall subunit was contributing three stable contacts (K20, K80, and positioning of the DNA (Fig. S7E). K217), and the third subunit made no persistent contacts with We also observed differences in the DNA bend between FEN1 DNA at all. Of these, K20, K80, and R149 were found in yeast and the clamps (Fig. 3C). We averaged the structure of DNA over studies to be functionally important for DNA-mediated replication 80 ns (excluding the first 20 ns as equilibration) from three simu- factor C association, and K14, R146, and R149 were shown to lations: (i) FEN1/DNA, (ii) PCNA/FEN1/DNA, and (iii)the9Δ-1- make contact with DNA in the crystal structure (41). On the DNA side, the Arg and Lys residues making salt bridge interactions were 1/FEN1/DNA. The axis of the upstream duplex was computed with evenly distributed between the two DNA strands. the program Curves+ (45). In the absence of either clamp, the By contrast, basic residues in the 9Δ-1-1 central hole track along upstream DNA axis is essentially straight and conforms closely to the template strand. The extended interface formed between the the expected canonical B-form geometry. By contrast, the asym- template strand and 9Δ-1-1 contains 10 persistent contacts. Three metric mode of association observed for the PCNA/FEN1/DNA ∼ additional contacts (K229 from Rad1, K25 from Hus1, and K92 complex results in a moderate bending of the DNA axis by 20°. from Rad9) are made with the opposite strand and are positioned Such structural distortion may introduce strain in the DNA sub- roughly one helical turn apart. Consistent with the previously strate between the 5′ flap and the clamp-binding region, which noted differences in the computed B-factors, 9Δ-1-1 displays sig- could assist the dissociation of the substrate after flap removal. nificantly tighter association with DNA, forming 13 stable contacts Consistent with the more symmetric contact distribution, the 9-1-1 with the DNA backbone (compared with nine contacts in the complex exhibits no such tendency for DNA bending. However, the PCNA complex). Unlike PCNA, the set of contacts with 9-1-1 are DNA is not entirely free from structural distortion—we observe more evenly distributed among the three subunits (five contacts contraction of the minor groove at the level of the 9-1-1 interface with Hus1, three with Rad1, and five with Rad9) and are also more accompanied by major groove widening above and below the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1121116109 Querol-Audí et al. Downloaded by guest on September 30, 2021 interface. The electrostatic properties of 9-1-1 appear to be evo- lutionarily functional. An analysis of processivity clamps from a variety of organisms showed that the electrostatic properties of Hus1 and, to a lesser degree, Rad9, but not of Rad1, were more similar to nonring viral processivity factors than to the eukaryotic PCNA (46). These viral processivity factors (e.g., UL42, UL44) possess an increased positive charge on the DNA-binding surface compared with the ring-forming clamps (PCNA, PolIII β subunit) and bind to the DNA directly rather than rely on a topological connection to DNA. Our results are consistent with this view that elevated positive potentials on the inner surface of 9-1-1 (speciﬁ- cally for Hus1 and Rad9; Fig. S1) lead to tighter association with DNA.

Specific Contacts with the Clamp Surface Determine the FEN1 Orientation in the Complexes. The computational models reveal Fig. 4. Functionally important differences between the PCNA/FEN1/DNA multiple direct interactions between FEN1 and the surface of the and 9-1-1/FEN1/DNA complexes. PCNA and 9-1-1 serve different functions two clamps that go beyond the canonical PCNA-interacting and, as a consequence, display different modes of association to FEN1 and DNA. The DNA and FEN1 are more tilted in 9Δ-1-1 complex than in the protein motif (PIP) motif (Fig. S8). Notably, we have taken care corresponding PCNA complex. The 9Δ-1-1 ring shows more interactions with to average along the simulation trajectories and, based on the upstream duplex and stabilizes DNA passing through the ring. Such a geometric criterion for each interaction type, to exclude con- differences at the structural level may translate into functional differences in tacts occurring only transiently. Thus, all contacts shown are the ways these complexes engage at the replication fork. PCNA/DNA inter- stable and persistent above a characteristic threshold level. actions are linked to processive and mobile sliding, whereas the tighter 9-1-1 Specifically, we note that the long C-terminal tail of FEN1 makes interactions would be consistent with a scaffolding role for 9-1-1. extensive contacts with Rad1 or PCNA along the IDC loop region. Although the FEN1 C-terminal tail is required for stimu- fl lation of FEN1 activity by either PCNA or the checkpoint clamp, segmental exibility, in the DNA-free complexes FEN1 is able to

intriguingly, the exact C-terminal residues responsible for stim- occupy multiple positions. In the ternary complexes the DNA BIOCHEMISTRY ulation by the two clamps appear to be distinct (47). The residues substrate locks FEN1 into an upright position producing a single within the PIP box are critical for stimulation by PCNA. By incision-capable state. The DNA passes through the rings at a tilt contrast, residues at the extreme C-terminal end (last 21 resi- and the tilt angle is larger by 9° in the 9-1-1 complex. The com- dues) are important for stimulation by 9-1-1. In our two models, putational analyses also suggest that 9-1-1, which has more pos- the FEN1/PCNA interface features more hydrophobic contacts, itive inner surface than PCNA, has almost 50% more charged displaying two distinct hydrophobic pockets [(i) P234, V233, interactions with the DNA. It is interesting to note that the more A208, I128, L47, and V45, and (ii) M119, L118, A96, M68, A67, symmetrical PCNA ring shows a biased DNA interaction toward and L66]. Residues from these pockets interact directly with the one side of the ring, whereas the 9-1-1 interactions involve all FEN1 PIP box (βA–αA motif). In contrast, the Rad1 surface three subunits. The distinct DNA interactions are consistent with appears less hydrophobic in this region, lacking both hydropho- different roles for the two clamps in DNA replication and repair. bic pockets (Fig. S8). Suggestively, the closer proximity of the During replication, PCNA must be mobile and slide on DNA tilted FEN1 catalytic core to Rad1 might allow the FEN1 C along with replicative polymerases. However, the checkpoint clamp terminus to come back up to interact with the FEN1 catalytic 9-1-1 acts as a temporary scaffold during repair of a section of core in the 9-1-1 complex, providing a rationale for the per- DNA. Such functional differences arguably dictate corresponding plexing data that the extreme C terminus is required for 9-1-1 differences in the interactions and the mode of association of the clamps in their respective replication/repair complexes. The teth- stimulation but not for stimulation by PCNA. fl Besides the canonical PIP box/IDC loop interaction, the ered exibility observed in the binary complexes of PCNA/FEN1 PCNA C terminus is important for PCNA stimulation of FEN1, and 9-1-1/FEN1 would allow FEN1 to swing from an out-of-the- way position on the outside of the ring to the upright functional although the PCNA C terminus is not required for recruitment fl (2, 13). In an archaeal system, the FEN1 and PCNA C termini position. This exibility is consistent with the rapid interchange form a structured antiparallel β-sheet (2). Accordingly, we ob- between PCNA-bound polymerase and FEN1 during Okazaki serve that the acidic C termini of both PCNA (A252–E258) and fragment maturation. – Rad1 (E275 S382) interact with a basic region in the FEN1 Materials and Methods catalytic core in our models. Importantly for FEN1 specificity for Rad1, of all three 9-1-1 subunits, only Rad1 has an acidic C Sample Preparation, EM Data Collection, and Processing. Human 9-1-1/FEN1 binary complex was formed by incubating 9-1-1 with a 3-M excess of FEN1 at terminus similar in charge and length to the one on PCNA. Rad1 room temperature for 15 min in 20 mM Hepes (pH 7.6), 80 mM KCl. The also presents a similar acidic surface complementary to the basic complex was cross-linked and purified by SEC through Superdex 200. surface of the FEN1 catalytic core (Fig. S1). 9Δ-1-1/FEN1/DNA ternary complex was cross-linked and further processed as for the binary complex. To visualize the complexes by negative-stain Concluding Remarks. The role of sliding clamps in increasing pro- EM, the purified complexes were loaded onto continuous carbon grids and cessivity for DNA-editing enzymes has been long appreciated. stained either with 2% (wt/vol) phosphotungstic acid (9-1-1/FEN1 binary Whether these clamps simply act as tethers or play a more active complex) or 2% (wt/vol) uranyl acetate (9Δ-1-1/FEN1/DNA ternary complex mechanistic role in DNA replication and repair remains contro- and 9-1-1 alone). Images were collected using the Leginon data collection software (48) on a Fei Tecnai F20 microscope using a 80,000× magnifica- versial. Our combined EM and computational results provide − 2 × insights into the ternary complexes PCNA/FEN1/DNA and 9-1-1/ tion (1.5 Å/pixel) in low-dose mode (20e /Å )onaGatan4K 4K pixel CCD camera (15-μm pixel size). The 2D data were processed using the Appion FEN1/DNA (Fig. 4) and imply an active role for the clamps. The package (49). observed FEN1 interactions in the models are consistent with distinct functions for the complexes. Our models show the DNA Computational Models and Protocols. Initial models were constructed based substrate is functionally bound by FEN1 with the upstream duplex on structures from the Protein Data Bank (accession codes 1UL1 and 3Q8L) (3, passing through the inner hole of the ringed clamps. Due to 16). The sequences of the DNA substrate (in the EM experiments and the

Querol-Audí et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 simulation) and the protein sequences for the 9Δ-1-1 subunits are shown in Hopper II, a Cray XE6 system at the National Energy Research Scientific Com- Fig. S9. The FEN1/DNA complex was superimposed onto the PCNA/FEN1 puting Center. A detailed description of the experimental procedures and the structure by optimally aligning FEN1 to the Y chain in the 1UL1 complex, and computational modeling protocols is given in SI Materials and Methods. the 3′ end of the dfDNA was extended so that it could pass through the PCNA/9-1-1 ring. Hydrogen atoms, counterions (Na+), an additional 100 mM ACKNOWLEDGMENTS. The authors thank Dr. Gabriel Lander, Ernesto Arias, NaCl concentration, and TIP3P solvent (50) were introduced. The systems and Patricia Grob for advice during EM data collection and processing. were then minimized and equilibrated. Production runs were carried out in Computational resources were provided in part by a National Science the isothermal–isobaric ensemble (1 atm and 300 K) for 120 ns for the PCNA/ Foundation (NSF) Teragrid allocation (CHE110042) and through an allocation at the National Energy Research Scientific Computing Center supported by FEN1/DNA and 100 ns for the 9Δ-1-1/ FEN1/DNA complex. The simulations the Department of Energy (Contract No. DE-AC02-05CH11231). Work on the used smooth particle mesh Ewald electrostatics, 10-Å cutoff for short-range project is supported by Georgia State University (I.I.), a Cleon C. Arrington nonbonded interactions, and 2-fs integration time step. All of the simulations research initiation grant (to I.I.), NSF Grant MCB-1149521 (to I.I.), and were performed using the NAMD 2.7 code (51, 52) with the AMBER Parm99SB National Cancer Institute Grants P01 CA092584 and R01 CA081967 (to J.A.T). parameter set (53) with modified nucleic acid parameters (BSC0) (54) on E.N. is a Howard Hughes Medical Institute Investigator.

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