Structural basis for recruitment of human flap endonuclease 1 to PCNA
2004; Springer Nature; Volume: 24; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7600519
ISSN1460-2075
AutoresShigeru Sakurai, Ken Kitano, Hiroto Yamaguchi, Keisuke Hamada, Kengo Okada, Kotaro Fukuda, Makiyo Uchida, Eiko Ohtsuka, Hiroshi Morioka, Toshio Hakoshima,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle16 December 2004free access Structural basis for recruitment of human flap endonuclease 1 to PCNA Shigeru Sakurai Shigeru Sakurai Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Ken Kitano Ken Kitano Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Hiroto Yamaguchi Hiroto Yamaguchi CREST, Japan Science and Technology Agency, Takayama, Ikoma, Nara, Japan Search for more papers by this author Keisuke Hamada Keisuke Hamada Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Kengo Okada Kengo Okada Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Kotaro Fukuda Kotaro Fukuda Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan Search for more papers by this author Makiyo Uchida Makiyo Uchida Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan Search for more papers by this author Eiko Ohtsuka Eiko Ohtsuka Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan Search for more papers by this author Hiroshi Morioka Corresponding Author Hiroshi Morioka Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan Search for more papers by this author Toshio Hakoshima Corresponding Author Toshio Hakoshima Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan CREST, Japan Science and Technology Agency, Takayama, Ikoma, Nara, Japan Search for more papers by this author Shigeru Sakurai Shigeru Sakurai Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Ken Kitano Ken Kitano Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Hiroto Yamaguchi Hiroto Yamaguchi CREST, Japan Science and Technology Agency, Takayama, Ikoma, Nara, Japan Search for more papers by this author Keisuke Hamada Keisuke Hamada Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Kengo Okada Kengo Okada Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan Search for more papers by this author Kotaro Fukuda Kotaro Fukuda Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan Search for more papers by this author Makiyo Uchida Makiyo Uchida Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan Search for more papers by this author Eiko Ohtsuka Eiko Ohtsuka Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan Search for more papers by this author Hiroshi Morioka Corresponding Author Hiroshi Morioka Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan Search for more papers by this author Toshio Hakoshima Corresponding Author Toshio Hakoshima Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan CREST, Japan Science and Technology Agency, Takayama, Ikoma, Nara, Japan Search for more papers by this author Author Information Shigeru Sakurai1,‡, Ken Kitano1,‡, Hiroto Yamaguchi2, Keisuke Hamada1, Kengo Okada1, Kotaro Fukuda3, Makiyo Uchida3, Eiko Ohtsuka3, Hiroshi Morioka 3 and Toshio Hakoshima 1,2 1Structural Biology Laboratory, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan 2CREST, Japan Science and Technology Agency, Takayama, Ikoma, Nara, Japan 3Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan ‡These authors contributed equally to this work *Corresponding authors: Structural Biology Laboratory, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Tel.: +81 743 72 5570; Fax: +81 743 72 5579; E-mail: [email protected] School of Pharmaceutical Sciences, Hokkaido University, N12, W6, Kita-ku, Sapporo 060-0812, Japan. Tel.: +81 11 706 3751; Fax: +81 11 706 4989; E-mail: [email protected] The EMBO Journal (2005)24:683-693https://doi.org/10.1038/sj.emboj.7600519 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1–PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms two β-strands connected by a short helix, the βA–αA–βB motif, participating in β–β and hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive 'locked-down' orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation. Introduction DNA replication in eukaryotes is a highly coordinated process involving many proteins that work cooperatively to ensure the accurate and efficient replication of DNA (Waga and Stillman, 1998). In this process, flap endonuclease-1 (FEN1) plays a crucial role in the removal of RNA primers during Okazaki fragment maturation in lagging strand DNA synthesis (Liu et al, 2004). FEN1 belongs to the XPG-like family of structure-specific nucleases that include bacteriophage and bacterial 5′-nucleases. Flap DNA removal by FEN1 is also essential during long-patch base excision repair (Klungland and Lindahl, 1997). In yeast, FEN1 mutants display severely impaired phenotypes such as UV sensitivity, deficient chromosome segregation, conditional lethality and accumulation in S phase. In mice, haplo-insufficiency of FEN1 leads to rapid tumor progression (Kucherlapati et al, 2002; Henneke et al, 2003). Moreover, FEN1 has been shown to participate in physical and functional interactions with the Werner Syndrome protein WRN, which is a member of the RecQ helicase family (Brosh et al, 2001; Hickson, 2003). Werner Syndrome is a human premature aging disorder well characterized by chromosomal instability. Thus, FEN1 appears to be a key player in maintaining genomic stability by participating in the DNA replication and repair processes, which are the early events that modulate cancer susceptibility and tumorigenesis (Henneke et al, 2003). Despite its pronounced importance in biology and medicine, no three-dimensional structure of eukaryotic FEN1 has been elucidated. In vitro experiments have shown that the FEN1 activity is markedly stimulated by proliferating cell nuclear antigen (PCNA), which is well known as the 'DNA sliding clamp' (Kelman, 1997; Tsurimoto, 1998). This stimulation is induced by direct binding of FEN1 to PCNA, leading to a 10- to 50-fold increase in its nuclease activity (Li et al, 1995; Wu et al, 1996; Tom et al, 2000). The interaction between FEN1 and PCNA is an essential prerequisite that provides for the subsequent FEN1 functionality found in cells. Mutations in FEN1 that disrupt the interaction with PCNA decrease the cleavage efficiency of flap DNA at the replication fork (Stucki et al, 2001), thus leading to the generation of unfavorably long flap DNA strands (Gary et al, 1999). PCNA forms a trimeric ring with three-fold symmetry perpendicular to the ring plane, and possesses a central hole that can be used to clamp DNA (Krishna et al, 1994). Importantly, PCNA binds several DNA-editing enzymes that function in DNA metabolic processes ranging from DNA methylation, base excision repair, nucleotide excision repair, mismatch repair, DNA replication and translesion DNA synthesis (Maga and Hubscher, 2003). It appears that PCNA acts as a platform for these enzymes, including FEN1, and facilitates the efficient functioning of these proteins. To date, crystal structures of complexes between PCNA and peptides from several PCNA-interacting proteins have been reported since the first structure of human PCNA bound to a peptide from the cyclin-dependent protein kinase inhibitor p21CIP1/WAF1 (hereafter referred to as p21) had been determined (Gulbis et al, 1996). However, the precise molecular mechanism by which these entire proteins cooperate with PCNA on the DNA remains unknown. Studying the entire complex would add to previous knowledge of peptide-based interactions. PCNA-binding proteins including p21 possess a short PCNA-binding motif, QXX(I/L/M)XXF(F/Y), located either at the N- or C-terminal region (reviewed in Warbrick, 1998; Tsurimoto, 1999; Matsumoto, 2001), while the crystal structure of human PCNA complexed with the C-terminal peptide of p21 (the PCNA–p21 complex) revealed the presence of extensive interactions with over 18 residues of p21 containing the motif (Gulbis et al, 1996). Interestingly, human FEN1 possesses a C-terminal tail consisting of ∼45 residues containing the short PCNA-binding motif. This long C-terminal tail represents one of the features common to eukaryotic FEN1s. Flanking residues of the motif may be utilized in interactions with DNA (Stucki et al, 2001). We now report on the crystal structure of human FEN1 complexed with PCNA. This is the first structure of eukaryotic FEN1 and the structure of the entire complex providing the first evidence pertaining to the presence of protein–protein interactions within the FEN1–PCNA complex. The structure revealed the FEN1–PCNA interfaces consisting of protein–peptide and protein–protein interactions. The main interaction involves the N-terminal half of the C-terminal tail of each FEN1 molecule, forming the βA–αA–βB motif anchored to one PCNA subunit. The second interaction involves the enzyme core domain of FEN1. This protein–protein interaction maintains the enzyme in an inactive 'locked-down' complex, as implied in the complex between the little finger (LF) domain of the Y-family DNA polymerase Pol IV and the Escherichia coli β-clamp processivity factor (Bunting et al, 2003), and preserves the central hole of PCNA for the purposes of DNA tracking. FEN1 possesses a short linker region containing small residues between the core domain and the C-terminal tail. This linker region acts as a hinge, which endows FEN1 with a degree of freedom to swing the core domain, and may play a role in switching the enzyme activity. Results Overall structure of the FEN1–PCNA complex The crystal structure of human FEN1 complexed with human PCNA was determined by the molecular replacement method (see Materials and methods and Table I). The structure revealed three FEN1 molecules (Figure 1, molecules X, Y and Z) bound to one PCNA trimer. Human FEN1 and PCNA consist of 380 (42 kDa) and 261 (29 kDa) amino-acid residues, respectively. The total mass (213 kDa) of the complex calculated from the structure agreed with the results from gel-filtration chromatography and dynamic light scattering, both of which yielded a monodisperse peak in solution (Sakurai et al, 2003). Figure 1.A stereo view of the human FEN1–PCNA complex. Three FEN1 molecules are colored in blue (X), red (Y) and green (Z), and the three subunits of the PCNA trimer in yellow (A), cyan (B) and orange (C). The C-termini of FEN1 and PCNA are labeled. Metal ions bound to the active sites of FEN1 (X and Y) are shown in magenta. Proposed catalytic faces of FEN1 are indicated by arrows. Download figure Download PowerPoint Table 1. Crystallographic statistics of the human FEN1–PCNA complex X-ray data Space group P212121 Cell parameters, a, b, c (Å) 82.2, 143.4, 246.7 Resolution (Å)a 50–2.9 (3.0–2.9) Completeness (%)b 85.1 (58.3) I/σI 13.3 (2.9) Rmerge (%) 8.0 (28.0) Refinement Number of residues included FEN1 (X, Y, Z) 313, 312, 349 (of 380) PCNA (A, B, C) 256, 258, 255 (of 261) Number of atoms 13,162 Number of reflections (total/unique) 234,329/55,775 R/Rfree (%)c 22.0/28.4 Average B-factor (Å2) Total 59.2 FEN1 (X, Y, Z) 55.7, 78.8, 85.2 PCNA (A, B, C) 36.1, 40.1, 46.9 R.m.s. bond length (Å), angles (deg) 0.007, 1.4 a Statistics for the outer resolution shell are given in parentheses. b Intensities (I/σI>1.0) were merged and used in the refinement. c R=∑∣∣Fobs∣−∣Fcalc∣∣/∑∣Fobs∣. Rfree is the same as R, but for a 5% subset of all reflections that were never used in the crystallographic refinement. Three PCNA subunits (subunits A, B and C in Figure 1) are tightly associated to form a closed ring. Each subunit contains two topologically identical domains, which are connected by the interdomain connector (IDC) loop. Each domain consists of two β–α–β–β–β motifs that are related by a pseudo two-fold symmetry. In the trimer, six β sheets form a circular outer layer that supports 12 α helices forming the inner surface. This architecture is essentially the same as those previously reported for PCNA (Krishna et al, 1994; Gulbis et al, 1996) and all secondary structural elements are preserved in our PCNA complex. The structures of human PCNAs bound to FEN1 and a p21 peptide are closely superimposable over the core secondary structural elements, with a small root-mean-square (r.m.s.) deviation (0.6 Å) for 247 Cα carbon atom positions in the trimer, while local structural differences are found in the IDC loops, caused by differences in the interactions with FEN1 as described later. Other differences are found in the prominent loop (βD2–βE2) residues 186–193, which are visible in our complex but were invisible in the p21 complex. Each PCNA subunit binds one FEN1 molecule (molecules A, B and C bind X, Y and Z, respectively). Structure of human FEN1 The structure of human FEN1 consists of the nuclease core domain (residues 1–332) and the C-terminal tail (333–380) (Figure 2A). The N-terminal half (333–359) of the C-terminal tail is visible, whereas the C-terminal half (∼20 residues) was not observed in the density map. It has been suggested that the C-terminal half, which is rich in lysine residues, is involved in DNA binding (Stucki et al, 2001) and might be flexible without DNA. The visible C-terminal tail region forms two β-strands and one helical conformation. This βA–αA–βB motif represents the main interacting interface with PCNA. In fact, strand βA and the flanking helix αA are formed by the conserved short PCNA-binding motif QXXLXXFF. Figure 2.Structure of human FEN1. All diagrams of FEN1 molecules are viewed from the same viewpoint as in (A). (A) Ribbon model of the molecular structure of human FEN1 (molecule Z). Two metal ions (M1 and M2) in the active site of molecule X are superimposed and depicted in magenta. (B) A stereo diagram showing superimposition of three human FEN1 molecules (X, Y and Z) including metal ions. Each molecule is shown in the same color as in Figure 1. (C) A stereo diagram showing superimposition of human FEN1 (green), M. jannachii FEN1 (Hwang et al, 1998) (violet) and P. furiosus FEN1 (Hosfield et al, 1998) (gold). The insertion sites at helix α0 and loop α10–α11 in human FEN1 are indicated by blue arrows and the deletion site at loop β6–β7 is shown as a red arrow. (D) Electrostatic molecular surfaces of human and M. jannachii FEN1s. Each surface was colored in the range <−10 to >+10kBT, where kB is the Boltzman constant and T is the temperature. The positive potential is shown in blue and the negative potential in red. (E) A closed-up stereo view of the active sites of human (green, molecule Y) and P. furiosus (gold) FEN1s. The orientation viewed is approximately that of a 90° rotation from (A) along the horizontal axis. The omit Fo–Fc electron density maps for the two metals (M1 and M2) are shown in blue (contoured at 5σ). The metal ions and side chain of Asp34 in P. furiosus FEN1 have not been deposited in PDB. Download figure Download PowerPoint The core domain is folded into an α/β structure with a groove formed by a twisted seven-stranded mixed β-sheet in the topology β1–β7–β6–β5–β2–β3–β4 and two helical regions: one formed by helices α0 and α5–α11, and the other by helices α1–α4, α12 and α13 (Figure 2A). These two helical regions are located on both sides of the β-sheet, which forms a central groove. The region between strand β3 and helix α4 contains 46 residues that project from the main body of the domain. In the electron density map, 29 (residues 100–128), 31 (103–133) and six (114–119) residues of this large looping-out region were not observed for molecules X, Y and Z, respectively. This region, containing many charged and hydrophobic residues, is thought to thread along the single strand of the DNA substrate (Tom et al, 2000; Storici et al, 2002) and hereafter is referred to as the 'clamp region'. A superimposition of the three FEN1 molecules in the crystal (Figure 2B) shows the diverse conformations of the clamp regions. The loop between helices α2 and α3 (loop α2–α3) also seems to be flexible. Indeed, those for molecules X and Y are disordered. Finally, the C-terminal tails of the three FEN1 molecules project from the core domains in different directions. Excluding these flexible regions, the core domains of the three molecules are superimposed with an r.m.s. deviation of 0.8 Å (for 265 Cα). Comparison with archaeal FEN1s The structure of human FEN1 (molecule Z) was compared with those of two FEN1s from the hyperthermophilic archaea, Methanococcus jannaschii (Hwang et al, 1998) and Pyrococcus furiosus (Hosfield et al, 1998) (Figure 2C). These archaeal FEN1s exhibit 34 and 38% sequence identity to human FEN1, respectively (Figure 3). From archaea to humans, the primary structures of the clamp regions are mostly conserved without any deletion or insertion. In archaeal FEN1 and related bacteriophage enzymes, the clamp regions have been referred to as a 'helical arch' in T5 5′-exonuclease (Ceska et al, 1996), or a 'L1 loop' or 'helical clamp' in M. jannaschii (Hwang et al, 1998) and P. furiosus (Hosfield et al, 1998) FEN1s, respectively. In our crystal, this region contains only two short helices, αa and αb. Even single amino-acid substitutions in this clamp region of human FEN1 have been reported to significantly decrease the nuclease activity (Storici et al, 2002). Deletion of the region in M. jannaschii FEN1 completely abolished the nuclease activity (Hwang et al, 1998). However, no common structure was observed in the FEN1 structures (Figure 2C). We believe that the clamp region of FEN1s would be intrinsically flexible, which could allow for effective tracking of flap DNA. Figure 3.Aligned amino-acid sequences of human and two archaeal FEN1s. The alignment was adjusted according to the three-dimensional structures. Secondary structure elements of human FEN1 (as in Figure 2A) are shown at the top, and residues whose electron densities were not observed in any of the three molecules are shown as dashed lines. Identical residues between human and archaeal FEN1s are shown in blue letters. The active-site residues which bind metal ions (pink) and the hinge region (red) are highlighted. The clamp region is boxed with black lines, and helices observed in the structures of two archaeal FEN1s (Hosfield et al, 1998; Hwang et al, 1998) are highlighted in light-blue. The PCNA-binding region of human FEN1 is boxed with blue lines, and those of human p21 have been aligned at the bottom. The X subscript indicates the number of followed core-domain residues. Download figure Download PowerPoint Compared with the archaeal FEN1s, human FEN1 possesses two major and one minor insertion sites. One major insertion is located at the N-terminus (residues 9–15) and the other (residues 267–274) at the loop region between helices α10 and α11 (Figure 3). The former insertion forms an additional helix, α0, which packs against helices α5–α8 and a strand β7. The latter insertion induces re-orientation of helix α10 (Figure 2C). Helix α0 induces significant changes in the shape of the helical region on the left side of the central β-sheet, narrowing the central groove (Figure 2D). A minor insertion (two residues) is located at loop α11–α12. Human FEN1 has three one-residue deletion sites at loop α2–α3, loop α8–α9 and loop α13–βA. In P. furiosus FEN1, insertion residues between β6 and β7 form an antiparallel β ribbon that restricts the active site groove, whereas no insertion is present in human or M. jannaschii FEN1s. The electrostatic potential surface of human FEN1 is comparable to that of M. jannaschii FEN1 (Figure 2D). The bottom of the clamp region forming the central cleft is dominated by negatively charged residues for the active site containing metal ions. Both sides of the central cleft contain basic residues, which provide complementarity to the negative charges of the DNA substrate. In human FEN1, these basic residues are located at helix α0, loop α8–α9 and loop α10–α11 on one side and helix α3, loop β6–β7 and helix α13 on the other side. Compared with M. jannaschii FEN1, the negatively charged cleft of human FEN1 is more closed, partly due to the additional helix α0. On the other hand, human FEN1 possesses a more positively charged groove at the helical region on the right side of the cleft. This region corresponds to the binding site for the upstream DNA strand containing 3′-flap in the crystal structure of Archaeoglobus fulgidus FEN1 bound to a 3′-flap DNA fragment (Chapados et al, 2004). Active site of human FEN1 The active site of human FEN1 is located at the central cleft with two possible metal ions (Figure 2A) and formed by two clusters of conserved acidic residues (Figure 2E). Four residues (Asp34, Asp86, Glu158 and Glu160) form the first metal ion-binding site (M1). Three residues (Asp179, Asp181 and Asp233) form the second metal ion-binding site (M2). These acidic residues are conserved in all known FEN1 enzymes, and the prevailing catalytic mechanism is thought to be universal. In the case of FEN1 molecules X and Y in our crystal, the electron densities for two metal ions were clearly observed in the difference Fourier map (Figure 2E). The density height for M1 is over 5σ in both X and Y, and that for M2 is 3σ and 5σ in X and Y, respectively. In the case of archaeal FEN1s, like our human FEN1, M2 appeared only at a lower electron density level (Hosfield et al, 1998; Hwang et al, 1998). Since no bivalent cations were added during the purification or crystallization procedures, two metal ions would have been tightly bound to FEN1 during these steps. In the present structure, these metal ions were assigned to Mg2+. M1 is known to play an important role in catalysis, probably in the nucleophilic attack of the phosphodiester bonds of DNA, while it has been suggested that M2 is involved in DNA binding (Shen et al, 1997). In P. furiosus FEN1, the conserved Tyr234 (numbered for human) was also involved in the active site via a hydrogen bond to Glu158 and a water-mediated contact with Asp181 (Hosfield et al, 1998). In the present structure, this Tyr residue was observed at the same position; however, the density for the water molecule was not identified, probably due to insufficient resolution. FEN1–PCNA interactions The interactions between FEN1 and PCNA are schematically summarized in Figure 4A. Overall, the binding of each FEN1 to PCNA buries large molecular surface area (∼3580 Å2 for molecules X and A). The interface between FEN1 and PCNA consists of a peptide-protein interaction involving the extended C-terminal tail of FEN1 (residues 336–356) and a channel on the surface of PCNA, and also a protein–protein interaction involving the FEN1 core domain. The latter interaction is mainly mediated by the PCNA-bound FEN1 tail and the edge of each PCNA monomer. This bipartite interaction is reminiscent of that observed in the crystal structure of the complex between the LF domain of E. coli Pol IV and the β-clamp (Bunting et al, 2003), while the protein–protein interaction in the Pol IV–LF–β-clamp complex involves two β-clamp monomers. Figure 4.Interactions between FEN1 and PCNA. (A) Schematic depiction of interactions between FEN1 (green) and PCNA (yellow). The FEN1 hinge region is colored in red. Residues shown in blue participate in protein–protein interactions in one or two FEN1 molecules out of three: Ser25 in Y and Z, Arg29 in Z and X, and Lys30, Lys80 and Trp298 in X (see text). A tentative water molecule observed between molecules Z and C is indicated by a blue circle. (B) Human FEN1 endonuclease activity enhanced by human wild type (WT) and deletion mutant, Δ8 (254–261), Δ7 (255–261) and Δ5 (257–261), PCNAs. The mobility of the top band is 33 nucleotides (for the downstream primer) and that of the cleaved product is 20 nucleotides (for the flap DNA from the downstream primer). The size markers show 33 nucleotides, as indicated by an arrowhead with S and 20 nucleotides with P. The SF substrate DNA (0.5 pmol) was added into a solution containing FEN1 (0.3 pmol) and PCNA (0, 1, 2, 5, 10 pmol). Control experiments showed little detectable nuclease activity in the absence of PCNA. (C) The bar graph documents the cleavage (%) quantified from the gel with 10 pmol wild-type or mutant PCNA. Each cleavage was calculated from the results of three independent experiments. (D) Pull-down assays of wild-type and mutant recombinant PCNAs by FEN1(CHis). The presence of both FEN1(CHis) and PCNA bands in a single lane indicates the formation of an in vitro complex of the two proteins. The bar graph documents the relative binding by quantifying the protein amounts and normalizing with the beads-bound FEN1 amount. Each binding was calculated from the results of three independent experiments. Download figure Download PowerPoint Peptide-binding interface The interactions that are mediated by the βA–αA–βB motif located at the C-terminal tail of FEN1 bury ∼2500 Å2 of total surface area at the interface and can be divided into three parts. While the interaction mode resembles that found in the PCNA–p21 complex (Gulbis et al, 1996), many differences are found. The short FEN1 strand βA (residues 336TQG338) binds the PCNA C-terminal residues (Ala252–Ile255) in the form of antiparallel β–β-like interactions (βA–C-term interactions). The interactions involve the side chain of FEN1 Gln337, which is the key residue in the consensus PCNA-binding motif and is conserved in other PCNA-binding proteins including p21 (Figure 3). The hydrogen bond from the main chain of PCNA Pro253 to the main chain of FEN1 Gly338 in our complex is replaced by a hydrogen bond to the side chain of p21 Thr145 in the PCNA–p21 complex. Three PCNA residues (Leu251, Ala252 and Pro253) are strictly conserved from archaea to humans. Among these residues, Pro253 might be involved in coordinating two oxygen atoms in the backbone of Ala252 and Pro253 toward βA of FEN1. In fact, a double mutation of P252A and K253A (P253 and K254 in human) in yeast PCNA affected the stimulation of FEN1 activity (Gomes and Burgers, 2000). Mutational studies showed that the βA–C-term interactions, especially the main-chain interactions, are important for enhancing the nuclease activity of human FEN1 (Figures 4B and C). Deletion of PCNA C-terminal residues Δ8 (254–261) resulted in a substantial reduction in the stimulation of FEN1 nuclease activity without a significant reduction in the binding affinity to FEN1 (Figure 4D). The β-like structure of the PCNA C-terminal residues, 252APKI255, could act as a rigid joint that directs the FEN1 core domain toward the DNA substrate. In contrast, deletion of Δ5 (257–261) caused no significant changes in the binding affinity or nuclease activity of FEN1. The extreme C-terminal residues (two, three and six residues for molecules A, B and C, respectively) of this region were disordered in our crystal. Nonpolar contacts are found between FEN1 helix αA (339RLDDFF344) and a large hydrophobic pocket within PCNA. The six FEN1 residues adopt a mixture of 310 and α helical conformations at the present resolution. In the Y FEN1 molecule, the helix is stabilized by intramolecular interactions, where the side chain of Asp342 accepts hydrogen bonds from both the main chain and side chain of Arg339. Three nonpolar FEN1 residues (Leu340, Phe343 and Phe344) located on one side of the helix dock into the hydrophobic pocket of the PCNA molecular surface. These interactions are similar to those in the PCNA–p21 complex. Interestingly, on the opposite side of the helix, FEN1 has an acidic residue, Asp341, which forms a salt bridge with PCNA His44 in our complex. The position corresponding to Asp341 is replaced by threonine in p21, but is restricted to an acidic residue (Asp or Glu) in all FEN1s. The position corresponding to His44 in human PCNA is restricted to a basic residue (His or Arg) in PCNAs, suggesting that salt bridge formation would represent a characteri
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