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

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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 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. 8528–8533 | PNAS | May 29, 2012 | vol. 109 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1121116109 Downloaded by guest on September 28, 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.
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