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Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels*

Open AccessPublished:March 19, 2012DOI:https://doi.org/10.1074/jbc.M112.346361
      Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

      Introduction

      To counteract the deleterious effects of the large variety of DNA lesions caused by endogenous or environmental factors, living organisms have evolved a multitude of biochemical strategies to maintain genomic integrity. DNA can be repaired either by directly reversing damage or, alternatively, by excision of abnormal DNA elements prior to DNA repair. Removal of these elements requires many nucleases that in a highly controlled fashion resolve irregular DNA structures like flaps, loops, splayed arms, replication forks, and Holliday junctions formed during DNA repair and/or recombination. Many of these abnormal structures contain highly toxic single-stranded regions. Nucleases function either autonomously or in complex with additional proteins, such as DNA polymerases that work in close association with nucleases to increase the fidelity of the replication machinery. One of the most studied nucleases is the RecB protein that is closely associated with the RecBCD complex involved in the recovery of double strand breaks using homologous recombination (
      • Dillingham M.S.
      • Kowalczykowski S.C.
      RecBCD enzyme and the repair of double-stranded DNA breaks.
      ,
      • Singleton M.R.
      • Dillingham M.S.
      • Gaudier M.
      • Kowalczykowski S.C.
      • Wigley D.B.
      Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks.
      ,
      • Wang J.
      • Chen R.
      • Julin D.A.
      A single nuclease active site of the Escherichia coli RecBCD enzyme catalyzes single-stranded DNA degradation in both directions.
      ,
      • Yu M.
      • Souaya J.
      • Julin D.A.
      The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli.
      ). Structural studies (
      • Dillingham M.S.
      • Kowalczykowski S.C.
      RecBCD enzyme and the repair of double-stranded DNA breaks.
      ,
      • Singleton M.R.
      • Dillingham M.S.
      • Gaudier M.
      • Kowalczykowski S.C.
      • Wigley D.B.
      Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks.
      ) indicate that the RecB protein has a helicase activity that separates the two strands of damaged DNA. The resulting “fork” structure is then processed by the RecB nuclease activity (
      • Shevelev I.V.
      • Hübscher U.
      The 3′-5′ exonucleases.
      ).
      Although Archaea appear to lack the RecBCD complex, many archeal genomes nevertheless, encode proteins that have been annotated as “predicted RecB family nuclease” (
      • Kinch L.N.
      • Ginalski K.
      • Rychlewski L.
      • Grishin N.V.
      Identification of novel restriction endonuclease-like fold families among hypothetical proteins.
      ,
      • Aravind L.
      • Makarova K.S.
      • Koonin E.V.
      Survey and summary. Holliday junction resolvases and related nucleases. Identification of new families, phyletic distribution and evolutionary trajectories.
      ). The structure and biochemical characterizations of Pab2263, one of the RecB-like nucleases in Pyrococcus abyssi, have been described (
      • Ren B.
      • Kuhn J.
      • Meslet-Cladiere L.
      • Myllykallio H.
      • Ladenstein R.
      Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi.
      ,
      • Ren B.
      • Kühn J.
      • Meslet-Cladiere L.
      • Briffotaux J.
      • Norais C.
      • Lavigne R.
      • Flament D.
      • Ladenstein R.
      • Myllykallio H.
      Structure and function of a novel endonuclease acting on branched DNA substrates.
      ,
      • Creze C.
      • Lestini R.
      • Kühn J.
      • Ligabue A.
      • Becker H.F.
      • Czjzek M.
      • Flament D.
      • Myllykallio H.
      Structure and function of a novel endonuclease acting on branched DNA substrates.
      ). This protein binds to ssDNA
      The abbreviations used are: ssDNA
      single-strand DNA
      dsDNA
      double-strand DNA
      PCNA
      proliferating cell nuclear antigen
      FEN-1
      flap endonuclease 1
      PIP
      PCNA interaction peptide
      IDCL
      interdomain connecting loop
      SAXS
      small angle x-ray scattering
      TAMRA
      carboxytetramethylrhodamine.
      at nanomolar concentrations and possesses a nuclease activity specific for ssDNA and was thus dubbed “NucS.” Qualitative experiments have indicated that both 3′ and 5′ extremities of ssDNA are cleaved by P. abyssi NucS (PabNucS). Although bipolar endonucleases are not common, human Dna2 endonuclease and Mus81 can cleave both 5′ and 3′ tailed ssDNAs with comparable efficiency (
      • Ehmsen K.T.
      • Heyer W.D.
      Saccharomyces cerevisiae Mus81-Mms4 is a catalytic, DNA structure-selective endonuclease.
      ,
      • Kim J.H.
      • Kim H.D.
      • Ryu G.H.
      • Kim D.H.
      • Hurwitz J.
      • Seo Y.S.
      Isolation of human Dna2 endonuclease and characterization of its enzymatic properties.
      ). Little is currently known about how the activity of these bipolar nucleases is regulated on their various substrates.
      The crystal structure of PabNucS revealed a self-assembled dimer with a dumbbell-like organization that does not resemble any known protein structures (
      • Ren B.
      • Kuhn J.
      • Meslet-Cladiere L.
      • Myllykallio H.
      • Ladenstein R.
      Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi.
      ,
      • Ren B.
      • Kühn J.
      • Meslet-Cladiere L.
      • Briffotaux J.
      • Norais C.
      • Lavigne R.
      • Flament D.
      • Ladenstein R.
      • Myllykallio H.
      Structure and function of a novel endonuclease acting on branched DNA substrates.
      ). PabNucS is composed of 2 domains. The amino-terminal domain displays a half-closed β-barrel and hosts a noncatalytic ssDNA binding site, whereas the carboxyl-terminal domain carries a minimal RecB-like domain, with its classical α/β structure, and contains the active site with a sequence motif conserved in the family of the RecB-like nucleases (
      • Aravind L.
      • Makarova K.S.
      • Koonin E.V.
      Survey and summary. Holliday junction resolvases and related nucleases. Identification of new families, phyletic distribution and evolutionary trajectories.
      ). Cleavage specificity of PabNucS is modulated by poorly understood interactions with dsDNA and the PCNA replication clamp (
      • Ren B.
      • Kühn J.
      • Meslet-Cladiere L.
      • Briffotaux J.
      • Norais C.
      • Lavigne R.
      • Flament D.
      • Ladenstein R.
      • Myllykallio H.
      Structure and function of a novel endonuclease acting on branched DNA substrates.
      ). In cell-free extracts of P. abyssi, PabNucS forms a high affinity complex with PCNA (KD = 15 nm) (
      • Ren B.
      • Kühn J.
      • Meslet-Cladiere L.
      • Briffotaux J.
      • Norais C.
      • Lavigne R.
      • Flament D.
      • Ladenstein R.
      • Myllykallio H.
      Structure and function of a novel endonuclease acting on branched DNA substrates.
      ). This interaction with the replication clamp is of particular interest as the toroidal sliding clamp encircles DNA and functions as a central coordinator for a large number of DNA replication and repair proteins (
      • Krishna T.S.
      • Kong X.P.
      • Gary S.
      • Burgers P.M.
      • Kuriyan J.
      Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA.
      ,
      • Pan M.
      • Kelman L.M.
      • Kelman Z.
      The archaeal PCNA proteins.
      ). For instance, the processivity of the P. abyssi DNA polymerases is increased by PCNA (
      • Castrec B.
      • Laurent S.
      • Henneke G.
      • Flament D.
      • Raffin J.P.
      The glycine-rich motif of Pyrococcus abyssi DNA polymerase D is critical for protein stability.
      ,
      • Castrec B.
      • Rouillon C.
      • Henneke G.
      • Flament D.
      • Querellou J.
      • Raffin J.P.
      Binding to PCNA in euryarchaeal DNA replication requires two PIP motifs for DNA polymerase D and one PIP motif for DNA polymerase B.
      ,
      • Henneke G.
      • Flament D.
      • Hübscher U.
      • Querellou J.
      • Raffin J.P.
      The hyperthermophilic euryarchaeota Pyrococcus abyssi likely requires the two DNA polymerases D and B for DNA replication.
      ,
      • Henneke G.
      • Gueguen Y.
      • Flament D.
      • Azam P.
      • Querellou J.
      • Dietrich J.
      • Hübscher U.
      • Raffin J.P.
      Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.
      ,
      • Rouillon C.
      • Henneke G.
      • Flament D.
      • Querellou J.
      • Raffin J.P.
      DNA polymerase switching on homotrimeric PCNA at the replication fork of the euryarchaea Pyrococcus abyssi.
      ), which also improves the binding affinity of eukaryotic and archaeal flap endonucleases (FEN-1) toward DNA (
      • Chapados B.R.
      • Hosfield D.J.
      • Han S.
      • Qiu J.
      • Yelent B.
      • Shen B.
      • Tainer J.A.
      Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair.
      ,
      • Gomes X.V.
      • Burgers P.M.
      Two modes of FEN1 binding to PCNA regulated by DNA.
      ,
      • Hutton R.D.
      • Roberts J.A.
      • Penedo J.C.
      • White M.F.
      PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms. Substrate targeting and catalytic step.
      ). Most PCNA-interacting proteins contain short canonical PCNA interaction peptides (PIPs) that bind to the interdomain connecting loop (IDCL), a region linking the two similar domains of a PCNA monomer. The PCNA-binding motif has been identified in a large number of proteins involved in DNA metabolic processes ranging from DNA replication, DNA repair, to cell cycle control (
      • Tsurimoto T.
      PCNA binding protein.
      ,
      • Warbrick E.
      PCNA binding through a conserved motif.
      ). The PIP motif corresponding to the carboxyl terminus of P. abyssi NucS interacts with the replication clamp PCNA in an array screening (
      • Meslet-Cladiére L.
      • Norais C.
      • Kuhn J.
      • Briffotaux J.
      • Sloostra J.W.
      • Ferrari E.
      • Hübscher U.
      • Flament D.
      • Myllykallio H.
      A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen.
      ). As the PIP/IDCL interface is a distinctive feature of the interactions of these enzymes with PCNA, regulatory mechanisms must exist to ensure the spatial and temporal coordination of these fundamentally distinct PCNA-dependent processes. Additional protein-protein interfaces may confer specificity on PCNA interactions and regulate the activity of the associated proteins (
      • Castrec B.
      • Rouillon C.
      • Henneke G.
      • Flament D.
      • Querellou J.
      • Raffin J.P.
      Binding to PCNA in euryarchaeal DNA replication requires two PIP motifs for DNA polymerase D and one PIP motif for DNA polymerase B.
      ,
      • Kiyonari S.
      • Takayama K.
      • Nishida H.
      • Ishino Y.
      Identification of a novel binding motif in Pyrococcus furiosus DNA ligase for the functional interaction with proliferating cell nuclear antigen.
      ,
      • Rolef Ben-Shahar T.
      • Castillo A.G.
      • Osborne M.J.
      • Borden K.L.
      • Kornblatt J.
      • Verreault A.
      Two fundamentally distinct PCNA interaction peptides contribute to chromatin assembly factor 1 function.
      ,
      • Xu H.
      • Zhang P.
      • Liu L.
      • Lee M.Y.
      A novel PCNA-binding motif identified by the panning of a random peptide display library.
      ). PCNA coordinates the handoff of a DNA substrate between different enzymes during DNA replication, repair, or recombination (
      • Chapados B.R.
      • Hosfield D.J.
      • Han S.
      • Qiu J.
      • Yelent B.
      • Shen B.
      • Tainer J.A.
      Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair.
      ). For instance, the replication clamp regulates the activities of DNA pol δ and FEN-1 during lagging strand synthesis (
      • Garg P.
      • Burgers P.M.
      DNA polymerases that propagate the eukaryotic DNA replication fork.
      ), eukaryotic DNA ligase binds to the PCNA trimer and its interaction prevents FEN-1 to interact with PCNA (
      • Subramanian J.
      • Vijayakumar S.
      • Tomkinson A.E.
      • Arnheim N.
      Genetic instability induced by overexpression of DNA ligase I in budding yeast.
      ,
      • Pascal J.M.
      • Tsodikov O.V.
      • Hura G.L.
      • Song W.
      • Cotner E.A.
      • Classen S.
      • Tomkinson A.E.
      • Tainer J.A.
      • Ellenberger T.
      A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA.
      ,
      • Pascal J.M.
      • O'Brien P.J.
      • Tomkinson A.E.
      • Ellenberger T.
      Human DNA ligase I completely encircles and partially unwinds nicked DNA.
      ,
      • Levin D.S.
      • Bai W.
      • Yao N.
      • O'Donnell M.
      • Tomkinson A.E.
      An interaction between DNA ligase I and proliferating cell nuclear antigen. Implications for Okazaki fragment synthesis and joining.
      ). Moreover, in the absence of DNA ligase the trimer of PCNA can simultaneously bind 3 FEN-1 molecules (
      • Sakurai S.
      • Kitano K.
      • Yamaguchi H.
      • Hamada K.
      • Okada K.
      • Fukuda K.
      • Uchida M.
      • Ohtsuka E.
      • Morioka H.
      • Hakoshima T.
      Structural basis for recruitment of human flap endonuclease 1 to PCNA.
      ).
      In crenarchaeal organisms, the heterotrimeric PCNA protein is composed of subunits that display distinct client specificities. This feature has been shown, in Sulfolobus solfataricus, to allow the recruitment, in vitro, of different interacting partners on the same ring to form a large macromolecular complex composed of PCNA, DNA polymerase, Fen1, and DNA ligase. This arrangement may allow coordination of sequential activities of these enzymes during the Okazaki fragment maturation process (
      • Dionne I.
      • Nookala R.K.
      • Jackson S.P.
      • Doherty A.J.
      • Bell S.D.
      A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solfataricus.
      ). In euryarchaeal organisms, as in Eucaryotes, the picture is different; the PCNA ring is homotrimeric and it is unclear how PCNA temporally regulates sequential interactions of these distinct partners with DNA.
      What is the overall structural organization of the NucS-PCNA complex? How does PCNA influence the affinity and activity of NucS toward DNA substrates? To answer these questions, we present SAXS, fluorescence resonance energy transfer (FRET), and anisotropy analyses of the P. abyssi NucS and homotrimeric PCNA (PabPCNA) using both, the individual proteins and their complexes. We found that the PabNucS solution structure is modulated through interactions with ssDNA and that the distinct NucS-PCNA complexes are assembled on the 5′ and 3′ flap substrates. Altogether our studies reveal that the structural flexibility of the NucS-PCNA complex mediated by the presence of one major contact point between the two proteins not only facilitates conformational flexibility during DNA binding, but also regulates the cleavage specificity of NucS proteins.

      DISCUSSION

      In a previous study (
      • Ren B.
      • Kühn J.
      • Meslet-Cladiere L.
      • Briffotaux J.
      • Norais C.
      • Lavigne R.
      • Flament D.
      • Ladenstein R.
      • Myllykallio H.
      Structure and function of a novel endonuclease acting on branched DNA substrates.
      ), we have reported the biochemical characterization and crystallographic structure of PabNucS, the prototype of a new family of structure-specific DNA endonucleases. Up to date, the physiological function of bacterial and archaeal NucS orthologs remains unclear because we have not been able to identify a phenotype for Haloferax volcanii strains deleted for nucS. However, the enzyme tightly associates with the replication clamp PabPCNA and can process both 3′ and 5′ ssDNA extremities of branched DNA structures. To get further insight into the structural assembly of the PabPCNA-NucS complex in the presence or absence of ssDNA, we have performed SAXS analyses of the complex and its different constitutive components.
      The solution structure of PabNucS revealed that the C-terminal domains of the two subunits of the dimer, encompassing the nuclease active sites, are found in different conformations in solution and crystal structures. Upon binding with the ssDNA ligand, the proteins do not undergo drastic conformational changes, but adopt a rigid and elongated conformation, where the individuals are similar to that observed in the crystal, which is consistent with the fact that movements in enzymes upon substrate binding are generally small (
      • Gutteridge A.
      • Thornton J.
      Conformational changes observed in enzyme crystal structures upon substrate binding.
      ). These fluctuations of the protein in the ligand-free state could allow it to reach a conformation close to that of the ligand-bound state. As the ligand spans both the N-terminal binding site and the C-terminal active site, another possibility could be that the reduced flexibility of the ligand-bound state induces a distortion of the ssDNA prior to cleavage.
      Using fluorescence anisotropy, we investigated DNA binding properties of the PabNucS-PCNA complex. These studies indicate that the PabPCNA increases the binding affinity of PabNucS toward ssDNA as well as branched DNA substrates carrying either 5′ or 3′ flaps (Fig. 3B). The obtained results clearly indicate that PabPCNA is required for optimal loading of PabNucS on the 5′ and 3′ flaps and is able to increase the cleavage specificity of PabNucS proteins toward ss/ds junctions on 5′ and 3′ flaps (Fig. 6). To our knowledge, this is the first study demonstrating that PCNA is capable of loading an endonuclease to the 3′ and 5′ flaps and prevents nonspecific cleavage on both substrates. Similar experiments using salt concentrations in the range from 150 to 400 mm NaCl resulted in increased KD values at higher ionic strength (Fig. 3C). A plot of log KD as a function of ionic strength (log I) was linear with a slope of 4.4 between 250 and 400 mm NaCl, indicating the formation of many (up to 6) salt bridges between DNA and the PabNucS-PCNA complex (
      • Lohman T.M.
      • Mascotti D.P.
      Thermodynamics of ligand-nucleic acid interactions.
      ,
      • Record Jr., M.T.
      • Lohman M.L.
      • De Haseth P.
      Ion effects on ligand-nucleic acid interactions.
      ). This observation is in agreement with our earlier structural data indicating that the ssDNA binding cleft of the PabNucS protein is rather basic (
      • Ren B.
      • Kühn J.
      • Meslet-Cladiere L.
      • Briffotaux J.
      • Norais C.
      • Lavigne R.
      • Flament D.
      • Ladenstein R.
      • Myllykallio H.
      Structure and function of a novel endonuclease acting on branched DNA substrates.
      ).
      Our characterization of the solution structure of PabPCNA raises the question of whether PabPCNA adopts both or only one of the open and solid ring conformations. When compared with the bacterial DNA polymerase III β-subunit and eukaryotic PCNA, the trimeric form of P. furiosus PCNA is mainly stabilized by a network of ion pairs at the molecular interface rather than by main chain hydrogen bonds and hydrophobic contacts (
      • Matsumiya S.
      • Ishino Y.
      • Morikawa K.
      Crystal structure of an archaeal DNA sliding clamp. Proliferating cell nuclear antigen from Pyrococcus furiosus.
      ,
      • Matsumiya S.
      • Ishino S.
      • Ishino Y.
      • Morikawa K.
      Intermolecular ion pairs maintain the toroidal structure of Pyrococcus furiosus PCNA.
      ). This difference, which is conserved at the primary sequence level among PCNAs from thermophilic archaea, could reflect a weaker intermolecular interaction between the subunits of archaeal thermophilic PCNAs and account for the self-loading process observed for homotrimeric euryarchaeal PCNA (
      • Castrec B.
      • Rouillon C.
      • Henneke G.
      • Flament D.
      • Querellou J.
      • Raffin J.P.
      Binding to PCNA in euryarchaeal DNA replication requires two PIP motifs for DNA polymerase D and one PIP motif for DNA polymerase B.
      ,
      • Henneke G.
      • Flament D.
      • Hübscher U.
      • Querellou J.
      • Raffin J.P.
      The hyperthermophilic euryarchaeota Pyrococcus abyssi likely requires the two DNA polymerases D and B for DNA replication.
      ,
      • Henneke G.
      • Gueguen Y.
      • Flament D.
      • Azam P.
      • Querellou J.
      • Dietrich J.
      • Hübscher U.
      • Raffin J.P.
      Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.
      ,
      • Rouillon C.
      • Henneke G.
      • Flament D.
      • Querellou J.
      • Raffin J.P.
      DNA polymerase switching on homotrimeric PCNA at the replication fork of the euryarchaea Pyrococcus abyssi.
      ). In addition, a recent molecular dynamic study of the role of the eukaryotic replication factor C at the initial step of the clamp loading cycle has raised the hypothesis that the clamp loader could trap and stabilize the open conformation of PCNA (
      • Tainer J.A.
      • McCammon J.A.
      • Ivanov I.
      Recognition of the ring-opened state of proliferating cell nuclear antigen by replication factor C promotes eukaryotic clamp-loading.
      ), thus strengthening the idea that the open ring conformation of PCNA also exists in solution. This might be explained by the weak interface between monomers of Pyroccocus PCNA, considering that it contains fewer hydrogen bonds than eukaryotic ones, and that hydrogen bonds are weakened at the optimal growth temperature of the organism (95 °C).
      The combination of SAXS and SPR data allows us to characterize, for the first time, the general three-dimensional organization of the PabPCNA-NucS complex and indicates that one molecule of PabNucS dimer binds to the PabPCNA homotrimer, forming a stable 1:1 complex. We stress that formation of this complex has direct functional consequences, because PabPCNA is able to increase the cleavage specificity of PabNucS proteins toward ss/ds junctions on 5′ and 3′ flaps. Furthermore, the PIP/IDCL contact is critical for the interaction. This single major contact point between the carboxyl terminus PIP motif of PabNucS and the IDCL of PabPCNA likely facilitates conformational flexibility of the protein-DNA substrate complexes. A similar structural organization was observed for the archaeal PCNA-DNA ligase complex without DNA (
      • Pascal J.M.
      • Tsodikov O.V.
      • Hura G.L.
      • Song W.
      • Cotner E.A.
      • Classen S.
      • Tomkinson A.E.
      • Tainer J.A.
      • Ellenberger T.
      A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA.
      ), where only one molecule of DNA ligase extends from the PCNA ring in the same plane, and the interaction is mediated by the PIP-IDCL interaction. This particular binding stoichiometry results from the distinct specificity displayed by the different subunits of the heterotrimeric crenarchaeal PCNA (
      • Pascal J.M.
      • Tsodikov O.V.
      • Hura G.L.
      • Song W.
      • Cotner E.A.
      • Classen S.
      • Tomkinson A.E.
      • Tainer J.A.
      • Ellenberger T.
      A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA.
      ,
      • Dionne I.
      • Nookala R.K.
      • Jackson S.P.
      • Doherty A.J.
      • Bell S.D.
      A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solfataricus.
      ). The organization for the PCNA from P. abyssi is different, it is composed of three identical subunits and the three binding sites are thus considered to be equivalent. In this respect, it might be possible that the fixation of one molecule of PabNucS could lead to spatial rearrangements on the PabPCNA preventing subsequent binding events or at least decreasing the kinetics of additional NucS/PCNA binding events. It is known that PCNA may provide more protein-protein interfaces, in addition to the general PIP-IDCL site, which confer different specificities to PCNA and regulate the diverse activities of associated enzymes (
      • Chapados B.R.
      • Hosfield D.J.
      • Han S.
      • Qiu J.
      • Yelent B.
      • Shen B.
      • Tainer J.A.
      Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair.
      ). These contact sites reside on subunits distinct from that engaged in the IDCL-PIP interaction. In particular, these additional contacts may even block access of other active clients to PCNA (
      • Mayanagi K.
      • Kiyonari S.
      • Nishida H.
      • Saito M.
      • Kohda D.
      • Ishino Y.
      • Shirai T.
      • Morikawa K.
      Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex.
      ). The SPR data indicate that the IDCL/PIP PabNucS contact alone is not sufficient to generate long range effects affecting the PabNucS-PCNA complex stoichiometry, but rather additional contacts, close to this main interaction surface and implicating residues located on the same or adjacent PabPCNA subunits. It is also possible that DNA bending induced by the PabNucS-PCNA complex plays a key role in regulating the cleavage specificity of NucS proteins. In this respect, the interaction between PabNucS and PabPCNA resembles that of SsoFen1 with SsoPCNA in that both PabNucS and SsoFen1 display a PIP box motif at the extreme C-terminal of their sequences, thus preventing the formation of an extended β-sheet with the IDCL. Doré and co-workers (
      • Doré A.S.
      • Kilkenny M.L.
      • Jones S.A.
      • Oliver A.W.
      • Roe S.M.
      • Bell S.D.
      • Pearl L.H.
      Structure of an archaeal PCNA1-PCNA2-FEN1 complex. Elucidating PCNA subunit and client enzyme specificity.
      ) have shown that SsoFen1 engages extra contacts with the C-terminal part of PCNA, most likely to compensate for the C-terminal truncation of the enzyme. The same might also hold true for PabNucS, this still open question awaits more resolutive crystallographic data of the PCNA-NucS complex.
      These potential rearrangements regulating the binding stoichiometry of PabNucS could allow or even promote other binding partners to be recruited by the PabPCNA and reflect the requirement of temporal and spatial coordination of their respective activities. In this regard, it is worth noting that PabNucS was found to be associated with Hef nuclease in pull-down experiments (
      • Ren B.
      • Kühn J.
      • Meslet-Cladiere L.
      • Briffotaux J.
      • Norais C.
      • Lavigne R.
      • Flament D.
      • Ladenstein R.
      • Myllykallio H.
      Structure and function of a novel endonuclease acting on branched DNA substrates.
      ). Recent genetic analyses have shown that Hef is involved in numerous DNA repair pathways in the archaeal cells, including resolution of stalled replication forks, interstrand cross-links, and NER (
      • Fujikane R.
      • Ishino S.
      • Ishino Y.
      • Forterre P.
      Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis.
      ,
      • Lestini R.
      • Duan Z.
      • Allers T.
      The archaeal Xpf/Mus81/FANCM homolog Hef and the Holliday junction resolvase Hjc define alternative pathways that are essential for cell viability in Haloferax volcanii.
      ). Up to now, there is no evidence of direct interaction between Hef and PCNA, however, crenarchaeal XPF, the shorter form of Hef lacking the helicase segment, was shown to be dependent on the PCNA for its nuclease activity, activating the catalytic step by 4 orders of magnitude (
      • Hutton R.D.
      • Roberts J.A.
      • Penedo J.C.
      • White M.F.
      PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms. Substrate targeting and catalytic step.
      ,
      • Roberts J.A.
      • Bell S.D.
      • White M.F.
      An archaeal XPF repair endonuclease dependent on a heterotrimeric PCNA.
      ,
      • Rouillon C.
      • White M.F.
      The evolution and mechanisms of nucleotide excision repair proteins.
      ). It is thus tempting to postulate that the activities of NucS and Hef could be coordinated by PCNA in a branched DNA repair pathway.
      In summary, we have shown that the sliding clamp PCNA of P. abyssii can modulate the activity of PabNucS. Our data confirm that PabPCNA increases the binding affinity of PabNucS toward ssDNA as well as branched DNA substrates carrying either 5′ or 3′ flaps and that PabPCNA is required for optimal loading of PabNucS on these substrates. In addition, PabPCNA appeared to direct PabNucS activity toward the ss/dsDNA junction, thus inhibiting nonspecific cleavage outside of the junction. This choreography between PabPCNA and PabNucS can be viewed as a form of regulation of PabNucS activities, which will only take place on the appropriate substrate where PabPCNA is already loaded, thus preventing the potentially deleterious nonspecific cleavage activity of PabNucS on the chromatin. At the structural level, our data also indicate that the PabNucS-PCNA complex distorts DNA by inducing a kink located in the vicinity of the ssDNA/dsDNA junction and that PabNucS binds downstream of the flap on the 5′ flap substrate and upstream of the flap on the 3′ flap substrate. Finally, the solution structure of the PabPCNA-NucS complex indicates that one molecule of PabNucS dimer binds to the PabPCNA homotrimer, and that the PIP/IDCL contact is critical for the interaction. The regulation of this particular stoichiometry could be triggered by additional contacts between PabNucS and PabPCNA inducing long range conformational changes on PabPCNA that in turn could favor the recruitment of additional partners of PabNucS acting in the same DNA repair pathways. More resolutive crystallographic data of the PabPCNA-NucS complex and in vivo characterization of the function of PabNucs will help answer these still open questions.

      Acknowledgments

      We thank the staff on the SAXS beamlines (ID14-EH3, ESRF, Grenoble; SWING, SOLEIL, Saint Aubin; and X33, DESY, Hamburg) for their warm welcome, and especially Clement Blanchet (EMBL-Hamburg) for valuable help during data collection. We also thank members of our laboratories for thoughtful discussions.

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