Advertisement

Identification of a Substrate Recognition Domain in the Replication Stress Response Protein Zinc Finger Ran-binding Domain-containing Protein 3 (ZRANB3)*

  • Akosua Badu-Nkansah
    Footnotes
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
    Search for articles by this author
  • Aaron C. Mason
    Affiliations
    Department of Biological Sciences, Vanderbilt University College of Arts and Sciences, Nashville, Tennessee 37232
    Search for articles by this author
  • Brandt F. Eichman
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

    Department of Biological Sciences, Vanderbilt University College of Arts and Sciences, Nashville, Tennessee 37232
    Search for articles by this author
  • David Cortez
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: (615)-343-0704.
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
    Search for articles by this author
  • Author Footnotes
    * This work was supported by National Institutes of Health Grants R01CA102729-11A1S1, R01CA160432, and P01CA092584 (to D. C.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 Supported by a diversity supplement grant 3R01CA102729.
Open AccessPublished:February 16, 2016DOI:https://doi.org/10.1074/jbc.M115.709733
      DNA damage and other forms of replication stress can cause replication forks to stall. Replication stress response proteins stabilize and resolve stalled forks by mechanisms that include fork remodeling to facilitate repair or bypass of damaged templates. Several enzymes including SMARCAL1, HLTF, and ZRANB3 catalyze these reactions. SMARCAL1 and HLTF utilize structurally distinct accessory domains attached to an ATPase motor domain to facilitate DNA binding and catalysis of fork remodeling reactions. Here we describe a substrate recognition domain within ZRANB3 that is needed for it to recognize forked DNA structures, hydrolyze ATP, catalyze fork remodeling, and act as a structure-specific endonuclease. Thus, substrate recognition domains are a common feature of fork remodeling, SNF2-family, DNA-dependent ATPases, and our study provides further mechanistic understanding of how these enzymes maintain genome integrity during DNA replication.

      Introduction

      Genomic replication is a highly challenging task. The DNA replication machinery must precisely duplicate billions of base pairs while tolerating a multitude of obstacles including damaged DNA, collisions with transcriptional machineries, unusual DNA structures, and other difficult to replicate sequences (
      • Zeman M.K.
      • Cimprich K.A.
      Causes and consequences of replication stress.
      ). Many of these obstacles stall replication forks and activate replication stress responses that stabilize and restart persistently stalled forks. These mechanisms include fork remodeling to regress replication forks into a chicken foot DNA structure (
      • Petermann E.
      • Helleday T.
      Pathways of mammalian replication fork restart.
      ,
      • Neelsen K.J.
      • Lopes M.
      Replication fork reversal in eukaryotes: from dead end to dynamic response.
      ). Fork regression may facilitate DNA repair or template switching to bypass the obstruction (
      • Neelsen K.J.
      • Lopes M.
      Replication fork reversal in eukaryotes: from dead end to dynamic response.
      ).
      Several members of the SNF2 family of DNA-dependent ATPases including SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1),
      The abbreviations used are: SMARCAL1
      SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1
      dsDNA
      double-stranded DNA
      HARP
      HepA-related protein
      HIRAN
      HIP116 Rad5p N-terminal
      HLTF
      helicase-like transcription factor
      MT1 mutants
      L760A/D761A/I762A
      MT2 mutants
      W790A/S791A/S792A
      PCNA
      proliferating cell nuclear antigen
      RPA
      replication protein A
      SIOD
      Schimke-Immunoosseous Dysplasia
      SRD
      substrate recognition domain
      ssDNA
      single-stranded DNA
      ZRANB3
      zinc finger Ran-binding domain containing 3.
      HLTF (helicase-like transcription factor) and ZRANB3 (zinc finger Ran-binding domain containing 3) are replication stress response proteins that catalyze fork remodeling including fork regression (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Ciccia A.
      • Nimonkar A.V.
      • Hu Y.
      • Hajdu I.
      • Achar Y.J.
      • Izhar L.
      • Petit S.A.
      • Adamson B.
      • Yoon J.C.
      • Kowalczykowski S.C.
      • Livingston D.M.
      • Haracska L.
      • Elledge S.J.
      The ZRANB3 translocase associates with poly-ubiquitinated PCNA to promote fork restart and limit recombination after replication stress.
      • Blastyák A.
      • Hajdú I.
      • Unk I.
      • Haracska L.
      Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA.
      ). The replication stress response is essential to complete replication accurately. Therefore, defects in this response cause human disease (
      • Zeman M.K.
      • Cimprich K.A.
      Causes and consequences of replication stress.
      ). For example, bi-allelic loss of function mutations in SMARCAL1 cause Schimke-Immunoosseous Dysplasia (SIOD) (
      • Boerkoel C.F.
      • Takashima H.
      • John J.
      • Yan J.
      • Stankiewicz P.
      • Rosenbarker L.
      • André J.L.
      • Bogdanovic R.
      • Burguet A.
      • Cockfield S.
      • Cordeiro I.
      • Fründ S.
      • Illies F.
      • Joseph M.
      • Kaitila I.
      • Lama G.
      • Loirat C.
      • McLeod D.R.
      • Milford D.V.
      • Petty E.M.
      • Rodrigo F.
      • Saraiva J.M.
      • Schmidt B.
      • Smith G.C.
      • Spranger J.
      • Stein A.
      • Thiele H.
      • Tizard J.
      • Weksberg R.
      • Lupski J.R.
      • Stockton D.W.
      Mutant chromatin remodeling protein SMARCAL1 causes Schimke immuno-osseous dysplasia.
      ). SIOD is a developmental disorder characterized by growth defects, immune-deficiency, and renal failure. Recent studies also suggest that SIOD may be a cancer predisposition syndrome (
      • Baradaran-Heravi A.
      • Raams A.
      • Lubieniecka J.
      • Cho K.S.
      • DeHaai K.A.
      • Basiratnia M.
      • Mari P.-O.
      • Xue Y.
      • Rauth M.
      • Olney A.H.
      • Shago M.
      • Choi K.
      • Weksberg R.A.
      • Nowaczyk M.J.M.
      • Wang W.
      • Jaspers N.G.J.
      • Boerkoel C.F.
      SMARCAL1 deficiency predisposes to non-Hodgkin lymphoma and hypersensitivity to genotoxic agents in vivo.
      ,
      • Carroll C.
      • Hunley T.E.
      • Guo Y.
      • Cortez D.
      A novel splice site mutation in SMARCAL1 results in aberrant exon definition in a child with schimke immunoosseous dysplasia.
      • Carroll C.
      • Badu-Nkansah A.
      • Hunley T.
      • Baradaran-Heravi A.
      • Cortez D.
      • Frangoul H.
      Schimke Immunoosseous Dysplasia associated with undifferentiated carcinoma and a novel SMARCAL1 mutation in a child.
      ). HLTF is silenced in colorectal cancer and ZRANB3 is mutated in endometrial cancers suggesting that both may be tumor suppressors (
      • Moinova H.R.
      • Chen W.-D.
      • Shen L.
      • Smiraglia D.
      • Olechnowicz J.
      • Ravi L.
      • Kasturi L.
      • Myeroff L.
      • Plass C.
      • Parsons R.
      • Minna J.
      • Willson J.K.V.
      • Green S.B.
      • Issa J.-P.
      • Markowitz S.D.
      HLTF gene silencing in human colon cancer.
      ,
      • Lawrence M.S.
      • Stojanov P.
      • Mermel C.H.
      • Robinson J.T.
      • Garraway L.A.
      • Golub T. R
      • Meyerson M.
      • Gabriel S.B.
      • Lander E.S.
      • Getz G.
      Discovery and saturation analysis of cancer genes across 21 tumour types.
      ).
      SMARCAL1 localizes to stalled replication forks through an interaction with RPA (
      • Bansbach C.E.
      • Bétous R.
      • Lovejoy C.A.
      • Glick G.G.
      • Cortez D.
      The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks.
      • Postow L.
      • Woo E.M.
      • Chait B.T.
      • Funabiki H.
      Identification of SMARCAL1 as a component of the DNA damage response.
      ,
      • Ciccia A.
      • Bredemeyer A.L.
      • Sowa M.E.
      • Terret M.-E.
      • Jallepalli P.V.
      • Harper J.W.
      • Elledge S.J.
      The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart.
      • Yuan J.
      • Ghosal G.
      • Chen J.
      The annealing helicase HARP protects stalled replication forks.
      ). The RPA interaction also regulates SMARCAL1 enzymatic activity to ensure that it regresses only stalled forks (
      • Bhat K.P.
      • Bétous R.
      • Cortez D.
      High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.
      ,
      • Bétous R.
      • Couch F.B.
      • Mason A.C.
      • Eichman B.F.
      • Manosas M.
      • Cortez D.
      Substrate-selective repair and restart of replication forks by DNA translocases.
      ). Although HLTF is present at replication forks, it is unclear if it is recruited through a protein-protein interaction or simply by its structure-specific DNA binding activity (
      • Kile A.C.
      • Chavez D.A.
      • Bacal J.
      • Eldirany S.
      • Korzhnev D.M.
      • Bezsonova I.
      • Eichman B.F.
      • Cimprich K.A.
      HLTF's ancient HIRAN domain binds 3′ DNA ends to drive replication fork reversal.
      ). ZRANB3 is recruited by binding to poly-ubiquitinated PCNA (
      • Ciccia A.
      • Nimonkar A.V.
      • Hu Y.
      • Hajdu I.
      • Achar Y.J.
      • Izhar L.
      • Petit S.A.
      • Adamson B.
      • Yoon J.C.
      • Kowalczykowski S.C.
      • Livingston D.M.
      • Haracska L.
      • Elledge S.J.
      The ZRANB3 translocase associates with poly-ubiquitinated PCNA to promote fork restart and limit recombination after replication stress.
      ).
      The enzymatic activities of SMARCAL1 and HLTF are dependent on a SNF2 ATPase motor domain and a substrate recognition domain (SRD) that is thought to mediate binding to specific structures at stalled replication forks. The SRD of SMARCAL1 is its HARP2 domain, which is required for SMARCAL1 binding to branched DNA structures as well as DNA-dependent ATPase and fork regression activities (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      ). The HARP domain is structurally related to the damage recognition domain of the XPB helicase and the mismatch recognition domain of MutS (
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      ). The SRD in HLTF is its HIRAN domain, which is unrelated in sequence and structure to the HARP domain and interacts with the exposed 3′ end of small DNA flaps (
      • Kile A.C.
      • Chavez D.A.
      • Bacal J.
      • Eldirany S.
      • Korzhnev D.M.
      • Bezsonova I.
      • Eichman B.F.
      • Cimprich K.A.
      HLTF's ancient HIRAN domain binds 3′ DNA ends to drive replication fork reversal.
      ,
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      • Hishiki A.
      • Hara K.
      • Ikegaya Y.
      • Yokoyama H.
      • Shimizu T.
      • Sato M.
      • Hashimoto H.
      Structure of a Novel DNA-binding domain of helicase-like transcription factor (HLTF) and its functional implication in DNA damage tolerance.
      ). The HIRAN domain is also important for HLTF mediated fork regression activity (
      • Kile A.C.
      • Chavez D.A.
      • Bacal J.
      • Eldirany S.
      • Korzhnev D.M.
      • Bezsonova I.
      • Eichman B.F.
      • Cimprich K.A.
      HLTF's ancient HIRAN domain binds 3′ DNA ends to drive replication fork reversal.
      ,
      • Achar Y.J.
      • Balogh D.
      • Neculai D.
      • Juhasz S.
      • Morocz M.
      • Gali H.
      • Dhe-Paganon S.
      • Venclovas Č.
      • Haracska L.
      Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling.
      ). In both SMARCAL1 and HLTF, mutations in the HARP or HIRAN domains interfere with their ability to bind DNA (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Kile A.C.
      • Chavez D.A.
      • Bacal J.
      • Eldirany S.
      • Korzhnev D.M.
      • Bezsonova I.
      • Eichman B.F.
      • Cimprich K.A.
      HLTF's ancient HIRAN domain binds 3′ DNA ends to drive replication fork reversal.
      ,
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      • Hishiki A.
      • Hara K.
      • Ikegaya Y.
      • Yokoyama H.
      • Shimizu T.
      • Sato M.
      • Hashimoto H.
      Structure of a Novel DNA-binding domain of helicase-like transcription factor (HLTF) and its functional implication in DNA damage tolerance.
      ).
      Yuan et al. reported that ZRANB3 contains a domain similar in sequence to the HARP domains of SMARCAL1 (
      • Yuan J.
      • Chen J.
      The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.
      ). However, they reported that deletion of this putative SRD domain inactivates its strand annealing activity without interfering with DNA binding or DNA-dependent ATPase activity (
      • Yuan J.
      • Chen J.
      The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.
      ). Given the apparent differences in the reported activities of the SMARCAL1 HARP and ZRANB3 HARP-like domains, we revisited the requirements for ZRANB3 to bind DNA, hydrolyze ATP and catalyze fork remodeling. We define a ZRANB3 SRD that is essential for all three functions and define the ZRANB3 minimal enzymatic unit for fork remodeling as containing only the SNF2 ATPase domain and its SRD.

      Experimental Procedures

      Recombinant DNA Cloning

      All ZRANB3 expression vectors and amino acid numberings are based on ZRANB3 isoform 2. The 1–501∼720–869 minimal enzymatic unit contains a (GGGGS)3 linker (
      • Chen X.
      • Zaro J.L.
      • Shen W.-C.
      Fusion protein linkers: property, design and functionality.
      ). All vectors and oligonucleotide sequences used for PCR and mutagenesis are available upon request.

      Recombinant Protein Expression and Purification

      FLAG-ZRANB3 proteins (WT (wild type), Δ712–818, K163D (mutation in walker A motif in ATPase domain that impairs ATP hydrolysis), 1–501, 1–650, 1–869, ΔNZF, ΔHNH, ΔAPIM, Δ651–720, Δ712–794, Δ795–859, L760A/D761A/I762A (MT1), W790A/S791A/S792A (MT2)) were purified from HEK293T cells as previously described (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ) with the following modifications: cells were lysed for 40 min on ice in lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm DTT, 0.2 mm PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 0.1% Triton X-100, 0.1 mm EDTA). Cleared cell lysate was incubated with FLAG-M2 beads (Sigma F2426, EZ View Red Anti-FLAG M2 Affinity gel) for 4 h at 4 °C. Beads were washed twice with lysis buffer, once with LiCl buffer (10 mm HEPES, pH 7.9, 0.3 m LiCl, 20% glycerol, 0.2 mm EDTA, 0.1% Triton X-100, 1 mm DTT, 0.2 mm PMSF, 1.5 mm MgCl2, 5 μg/ml aprotinin, 5 μg/ml leupeptin), and twice with the elution buffer (20 mm HEPES, pH 7.9, 0.1 m KCl, 1.5 mm MgCl2, 0.2 mm EDTA, 20% glycerol, 0.01% Nonidet P-40, 1 mm DTT, 0.2 mm PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin). Proteins were eluted using 300 μg/ml FLAG peptide (F3290 Sigma). FLAG-ZRANB3 protein from insect cells was purified using the same procedure as FLAG-SMARCAL1 (
      • Yusufzai T.
      • Kadonaga J.T.
      HARP is an ATP-driven annealing helicase.
      ).
      GST-720–869 and GST-720–869 L760A/D761A/I762A (MT1) were purified from ArcticExpress Escherichia coli (Agilent Technologies). Cells were grown at 37 °C and upon reaching an OD600, protein expression was induced with 1 mm IPTG and grown at 16 °C overnight. The cell pellet was solubilized in lysis buffer (25 mm Tris, pH 8.0, 50 mm NaCl, 0.1 mm EDTA, 5% glycerol, 1 mm DTT, 0.1 mm PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin) and lysed by sonication. Triton X-100 was added to reach a final concentration of 1% and the lysate was incubated on ice for 30 min. Following high-speed centrifugation, the lysate was incubated with GST beads for 4 h at 4 °C. Afterward, the beads were washed three times with lysis buffer containing 1% Triton X-100. Protein was eluted with elution buffer (75 mm Tris, pH 8, 15 mm glutathione, 0.1 mg/ml leupeptin), and dialyzed overnight at 4 °C (dialysis buffer: 20 mm Tris, pH 8.0, 1 mm DTT, 0.1 mm EDTA, 20% glycerol, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 0.2 mm PMSF). Dialyzed samples were applied to a heparin column and eluted with increasing concentration of KCl (50 mm, 75 mm, 150 mm, 300 mm). Fractions containing the desired protein were combined and concentrated using a Millipore 10,000 MWCO protein concentration filter.

      Alignment

      ZRANB3 protein sequence alignments were performed using Clustal Omega. The HARP1 and HARP2 sequences were obtained from the boundaries identified previously (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      ). Secondary structure prediction was performed using POLYVIEW, PSIPRED, and JNETPRED prediction software (
      • Porollo A.A.
      • Adamczak R.
      • Meller J.
      POLYVIEW: a flexible visualization tool for structural and functional annotations of proteins.
      ,
      • Buchan D.W.A.
      • Minneci F.
      • Nugent T.C.O.
      • Bryson K.
      • Jones D.T.
      Scalable web services for the PSIPRED Protein Analysis Workbench.
      • Cole C.
      • Barber J.D.
      • Barton G.J.
      The Jpred 3 secondary structure prediction server.
      ).

      DNA Substrate Assembly and Purification

      The single-stranded (30nt), double-stranded (30nt), splayed arm, replication fork, fork regression, and fork restoration DNA substrates used for ATPase, DNA binding, and fork remodeling reactions were assembled and purified as described previously (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Bhat K.P.
      • Bétous R.
      • Cortez D.
      High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.
      ,
      • Bétous R.
      • Couch F.B.
      • Mason A.C.
      • Eichman B.F.
      • Manosas M.
      • Cortez D.
      Substrate-selective repair and restart of replication forks by DNA translocases.
      ).

      ATPase Assay

      ATPase assays were conducted as previously described (
      • Yusufzai T.
      • Kadonaga J.T.
      HARP is an ATP-driven annealing helicase.
      ).

      DNA Binding Assay

      DNA binding was conducted largely as previously described (
      • Yusufzai T.
      • Kadonaga J.T.
      HARP is an ATP-driven annealing helicase.
      ). Briefly, protein was incubated with 10 nm of 32P-labeled DNA substrate at 30 °C for 30 min. Following incubation, 15% Ficol was added to a final concentration of 2.5%. Samples were resolved on a 5% or 8% polyacrylamide gel in 1×TBE (100 mm Tris, 90 mm boric acid, 2 mm EDTA) at 40 V for 180 min at 4 °C.

      Nuclease Assay

      Nuclease assays were conducted as previously described (
      • Weston R.
      • Peeters H.
      • Ahel D.
      ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response.
      ). Briefly, protein was incubated with 10 nm of 32P-labeled splayed arm substrate for 30 min at 30 °C in nuclease buffer (50 mm Tris, pH 7.5, 50 mm NaCl, 5 mm MgCl2, 2 mm ATP, 0.1 mg/ml BSA). The products were resolved on a 10% polyacrylamide gel in 1×TBE at 80 V for 80 min at room temperature.

      Fork Remodeling Assay

      Fork regression and fork restoration reactions were conducted as previously described (
      • Bhat K.P.
      • Bétous R.
      • Cortez D.
      High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.
      ,
      • Bétous R.
      • Couch F.B.
      • Mason A.C.
      • Eichman B.F.
      • Manosas M.
      • Cortez D.
      Substrate-selective repair and restart of replication forks by DNA translocases.
      ). For the fork regression reaction, 3 nm of protein was incubated with 3 nm of labeled leading strand gap regression substrate for increasing amount of time at 37 °C. For the fork restoration reaction, increasing amount of protein was incubated with 3 nm of the lagging strand gap restoration substrate for 30 min at room temperature. The products were resolved on an 8% polyacrylamide gel in 1×TBE at 80 V for 80 min at room temperature.

      Results

      Region 720–869 Is Highly Conserved and Necessary for ZRANB3 ATPase Activity

      ZRANB3 is a DNA-dependent ATPase in the same SNF2 family as SMARCAL1 and HLTF (
      • Flaus A.
      • Martin D.M.A.
      • Barton G.J.
      • Owen-Hughes T.
      Identification of multiple distinct Snf2 subfamilies with conserved structural motifs.
      ). SMARCAL1 and ZRANB3 are both annealing helicases that re-anneal complementary DNA strands (
      • Yusufzai T.
      • Kadonaga J.T.
      HARP is an ATP-driven annealing helicase.
      ,
      • Yusufzai T.
      • Kadonaga J.T.
      Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif.
      ) and catalyze replication fork remodeling reactions (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Ciccia A.
      • Nimonkar A.V.
      • Hu Y.
      • Hajdu I.
      • Achar Y.J.
      • Izhar L.
      • Petit S.A.
      • Adamson B.
      • Yoon J.C.
      • Kowalczykowski S.C.
      • Livingston D.M.
      • Haracska L.
      • Elledge S.J.
      The ZRANB3 translocase associates with poly-ubiquitinated PCNA to promote fork restart and limit recombination after replication stress.
      ). The SMARCAL1 HARP2 domain is required for SMARCAL1 to bind DNA, hydrolyze ATP, anneal DNA, and remodel replication forks (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      ).
      A previous study concluded that a region encompassing amino acids 712–818 in ZRANB3 contains a HARP-like domain that is required for ZRANB3 annealing helicase activity (
      • Yuan J.
      • Chen J.
      The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.
      ). However, unlike the SMARCAL1 HARP domain, the HARP-like domain was reported to be dispensable for DNA binding and ATPase activity. Fork remodeling was not tested. Due to the striking functional differences between the HARP and HARP-like domains, we revisited whether the HARP-like domain of ZRANB3 really shares similar functional properties to the SMARCAL1 HARP domains. We purified wild type (WT) and Δ712–818 ZRANB3 (Fig. 1A) and tested their ability to bind a splayed arm DNA substrate and hydrolyze ATP. In contrast to the previously published findings, purified Δ712–818 ZRANB3, which lacks the HARP-like domain, failed to bind a splayed arm DNA substrate (Fig. 1B). It also lacked DNA-stimulated ATPase activity (Fig. 1C). In contrast, WT ZRANB3 displayed both DNA-binding and DNA-dependent ATPase activity.
      Figure thumbnail gr1
      FIGURE 1Δ712–818 ZRANB3 does not bind DNA and cannot hydrolyze ATP. A, Coomassie-stained SDS-PAGE gel of purified wild-type (WT) and Δ712–818 ZRANB3. B, Δ712–818 ZRANB3 and WT ZRANB3 were incubated with a splayed arm DNA substrate. To assess DNA binding, samples were resolved on a polyacrylamide gel and visualized by autoradiography. A representative experiment is shown. C, Δ712–818 ZRANB3 and WT ZRANB3 were incubated with a splayed arm substrate, and ATPase activity was measured. The mean and standard deviation from three experiments are shown. In most cases the standard deviation is smaller than the symbol size. The inset is an anti-FLAG immunoblot of the purified proteins.
      It is unclear whether deleting amino acids 712–818 generates a protein that is properly folded. Since important amino acids in the SMARCAL1 HARP domain have already been identified (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      ), we attempted to generate a sequence alignment between the SMARCAL1 HARP domains and the ZRANB3 HARP-like domain to identify critical amino acids for mutagenesis. However, we were unable to find sufficient sequence similarity to generate a useful alignment. Therefore, using evolutionarily conserved regions of ZRANB3 as a guide, we designed and tested various deletion, truncation, and point mutants to determine regions within the protein that are necessary for DNA-dependent ATPase activity (Fig. 2A). Deletion of the NZF and APIM motifs, which bind polyubiquitinated PCNA (
      • Ciccia A.
      • Nimonkar A.V.
      • Hu Y.
      • Hajdu I.
      • Achar Y.J.
      • Izhar L.
      • Petit S.A.
      • Adamson B.
      • Yoon J.C.
      • Kowalczykowski S.C.
      • Livingston D.M.
      • Haracska L.
      • Elledge S.J.
      The ZRANB3 translocase associates with poly-ubiquitinated PCNA to promote fork restart and limit recombination after replication stress.
      ), did not impair ATPase activity (Fig. 2B). Deletion of the HNH nuclease domain caused a modest but reproducible decrease in activity (Fig. 2B). Deletion of amino acids 651–720 also yielded an active enzyme (Fig. 2C). In contrast, ZRANB3 Δ712–794 and ZRANB3 Δ795–859 were both inactive (Fig. 2D).
      Figure thumbnail gr2
      FIGURE 2Amino acids 721–869 are necessary for ZRANB3 DNA-dependent ATPase activity. A, schematic and summary of results for the various deletion, truncation, and point mutants. Purified (B) ΔNZF, ΔHNH, ΔAPIM motif deletion mutants; C, Δ651–720 deletion mutant; D, Δ712–794 and Δ795–859 deletion mutants; E, 1–501 and 1–650 truncation mutants; F, 1–869 truncation mutant; and G, triple mutants L760A/D761A/I762A (MT1) and W790A/S791A/S792A (MT2) were incubated with a splayed arm DNA substrate and ATPase activity was measured. A representative experiment (of at least two replicates) is shown for each mutant.
      Likewise, C-terminal deletion constructs containing only the ATPase domain (ZRANB3 1–501) or the ATPase domain, PIP and NZF motifs (ZRANB3 1–650) were also inactive (Fig. 2E). However, a protein consisting of amino acids 1–869 was as active as the wild type protein (Fig. 2F). In all cases, the active proteins required DNA for ATP hydrolysis. Thus, the ZRANB3 ATPase domain requires an accessory domain that likely includes amino acids 721–869 for activity.
      Amino acids 720–869 contain most, but not all, of the residues previously described to make-up the HARP-like domain. It is relatively highly evolutionarily conserved compared with flanking regions of ZRANB3 (Fig. 3). However, our sequence alignment failed to detect significant similarity with the HARP domains of SMARCAL1. We also compared the known secondary structure of the HARP domain to the predicted secondary structure of this ZRANB3 region. While there is some similarity, the ZRANB3 domain contains a large insertion that is predicted to be α-helical. Mutations in highly conserved amino acids within this helix and in other highly conserved amino acids in this region (MT1: L760A/D761A/I762A and MT2: W790A/S791A/S792A) inactivate the protein (Fig. 2G). These data confirm that this region is necessary for DNA-dependent ATPase activity. Based on this data as well as additional information (see below) we designate amino acids 720–869 of ZRANB3 as a substrate recognition domain (SRD).
      Figure thumbnail gr3
      FIGURE 3ZRANB3 amino acids 720–869 are highly conserved but have minimal similarity to the HARP domains of SMARCAL1. Amino acid sequence numbering corresponds to human ZRANB3 isoform 2. Secondary structure of mouse HARP1 (based on PDB ID 4O66), and predicted secondary structure of ZRANB3 generated from POLYVIEW, PSIPRED, and JNETPRED are depicted. MTI (L760A/D761A/I762A) residues are indicated as stars and MT2 (W790A/S791A/S792A) residues are indicated as squares.

      The ZRANB3 SRD Is Sufficient to Impart DNA Binding, ATPase, and Fork Remodeling Activities to the ATPase Domain

      Since the ZRANB3 ATPase domain by itself is not active, we tested whether addition of the SRD via a flexible linker (Fig. 4A) is sufficient to impart DNA-dependent activity. Indeed, ZRANB3 1–501∼720–869 is active in the presence of DNA although its activity is modestly decreased compared with wild-type ZRANB3 (Fig. 4, B and C). Consistent with its DNA-dependent ATPase activity, ZRANB3 1–501∼720–869 is capable of binding complex DNA substrates that mimic a replication fork (Fig. 4D). Similar to WT ZRANB3, ZRANB3 1–501∼720–869 also catalyzes fork regression and fork restoration reactions, whereas an ATPase-deficient mutant (K163D) is inactive in these assays (Fig. 4, E and F).
      Figure thumbnail gr4
      FIGURE 4ZRANB3 1–501∼720–869 represents a minimal enzymatic unit that retains DNA binding, ATPase, and fork remodeling activities. A, schematic of ZRANB3 1–501∼720–869. B, Coomassie-stained SDS-PAGE gel of WT (lane 1), K163D (lane 2), and 1–501∼720–869 (lane 3) ZRANB3. C, ATPase activity of ZRANB3 1–501∼720–869 to a splayed arm DNA substrate. The mean and standard deviation for three experiments are shown. In most cases the standard deviation is smaller than the symbol size. D, DNA binding activity of ZRANB3 1–501∼720–869 to a replication fork mimicking DNA substrate. Samples were resolved on a polyacrylamide gel and visualized by autoradiography. The inset is an anti-FLAG immunoblot of the purified proteins. E, purified WT ZRANB3, K163D and ZRANB3 1–501∼720–869 were incubated with a model stalled fork DNA substrate for increasing times. Reaction products were separated by gel electrophoresis and visualized by autoradiography. F, increasing amounts of purified WT ZRANB3, K163D and ZRANB3 1–501∼720–869 were incubated with a model fork restoration substrate for 30 min at room temperature. Reaction products were separated by gel electrophoresis and visualized by autoradiography.
      Incorporation of the MT1 mutations into this minimal enzymatic unit (1–501∼720–869-MT1) inactivates its ATPase, DNA binding, and fork remodeling activities (Fig. 5). Thus, we conclude that amino acids 720–869 of ZRANB3 encode a SRD that is necessary and sufficient to impart DNA binding, ATPase and in vitro fork remodeling activities onto the ZRANB3 motor domain. Furthermore, this analysis defines the minimal enzymatic unit of ZRANB3 capable of catalyzing fork remodeling as containing amino acids 1–501 and 720–869.
      Figure thumbnail gr5
      FIGURE 5The substrate recognition domain is necessary and sufficient to impart DNA binding, ATPase, and fork remodeling activities to the ZRANB3 ATPase domain. A, Coomassie-stained SDS-PAGE gel of purified 1–501∼720–869 (lane 1) and 1–501∼720–869 containing MT1 mutations (lane 2). B, ATPase activity of ZRANB3 1–501∼720–869 and 1–501∼720–869-MT1. The mean and standard deviation for three experiments are shown. C, DNA binding of 1–501∼720–869, 1–501∼720–869-MT1, and 1–501 to a replication fork mimicking DNA substrate. Products were separated by gel electrophoresis and visualized by autoradiography. D, fork regression activity of 1–501∼720–869 and 1–501∼720–869-MT1 to a model stalled fork DNA substrate for increasing times. Reaction products were separated by gel electrophoresis and visualized by autoradiography.

      The ZRANB3 SRD Is Required for Structure-specific Endonuclease Activity

      In addition to catalyzing fork remodeling reactions, ZRANB3 was reported to act as an ATP-dependent, structure-specific endonuclease that nicks the duplex DNA of a splayed arm substrate (
      • Weston R.
      • Peeters H.
      • Ahel D.
      ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response.
      ). Endonuclease activity required both the HNH and ATPase domains (
      • Weston R.
      • Peeters H.
      • Ahel D.
      ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response.
      ). Thus, we hypothesized that the SRD domain of ZRANB3 may also be required for its nuclease activity. Indeed, mutations in the SRD inactivate nuclease activity (Fig. 6).
      Figure thumbnail gr6
      FIGURE 6The substrate recognition domain is necessary for ZRANB3 nuclease activity. Nuclease assay of WT, MT1, and ΔHNH ZRANB3 proteins to a splayed arm substrate. Overhang DNA (lane 5) and ssDNA (lane 6) were used as size markers. Products were separated by gel electrophoresis and visualized by autoradiography.

      The ZRANB3 SRD Binds DNA

      To test if amino acids 720–869 in ZRANB3 actually contain a DNA binding domain, we purified recombinant GST-720–869 from E. coli (Fig. 7A). Like full-length ZRANB3, GST-720–869 is not capable of binding either single-stranded or double-stranded DNA (Fig. 7B). However it can bind a splayed arm substrate, albeit with reduced affinity compared with full-length ZRANB3 (Fig. 7, C and D). Incorporating the MT1 mutations into either GST-720–869 or full-length ZRANB3 greatly reduced their ability to bind the splayed arm DNA substrate (Fig. 7, C and D).
      Figure thumbnail gr7
      FIGURE 7ZRANB3 amino acids 720–869 encode a structure-specific DNA binding domain. A, Coomassie-stained SDS-PAGE gel of GST-720–869-MT1 (lane 1) and GST-720–869 (lane 2). B and C, DNA binding of GST-720–869, GST-720–869-MT1, and GST proteins to (B) single or double-stranded DNA or a (C) splayed arm DNA substrate was assessed by a electrophoretic mobility shift assay. D, DNA binding assay of full-length WT and MT1 proteins to a splayed arm DNA substrate. Representative experiments are shown.
      Overall these results indicate that ZRANB3 amino acids 720–869 contains a domain that is both necessary and sufficient to impart substrate-selective DNA binding and enzymatic activity to the ZRANB3 ATPase domain. Thus, it acts as a SRD similar to the HARP domain of SMARCAL1 and the HIRAN domain of HLTF.

      Discussion

      In this study, we identified a structure recognition domain (SRD) in ZRANB3 that binds branched DNA substrates and confers DNA-dependent ATPase and fork remodeling activity to its SNF2-type motor domain. The SRD is also required for structure-specific endonuclease activity. A minimal enzymatic unit, containing only the SRD and the SNF2 ATPase domains, retains similar fork remodeling activities as the full-length protein. Thus, these data suggest that ZRANB3 shares a similar mechanism of action as SMARCAL1 and HLTF and support the idea that structure recognition domains impart fork remodeling activities onto the motor domains of these proteins.
      Why cells express several different fork remodeling proteins that catalyze similar reactions in vitro is unknown. The exact DNA structure that is formed at a stalled replication fork in a cell is also unknown. Presumably, the stalled fork adopts a multitude of structures dependent upon the nature of the obstacle. This heterogeneity may warrant the need for several fork remodeling enzymes with substrate recognition domains that interact and bind to different DNA forms present at a stalled and/or regressed fork.
      The high-resolution structures of the SRDs of HLTF and SMARCAL1 (HIRAN and HARP domains respectively) have been determined by x-ray crystallography (
      • Kile A.C.
      • Chavez D.A.
      • Bacal J.
      • Eldirany S.
      • Korzhnev D.M.
      • Bezsonova I.
      • Eichman B.F.
      • Cimprich K.A.
      HLTF's ancient HIRAN domain binds 3′ DNA ends to drive replication fork reversal.
      ,
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      • Hishiki A.
      • Hara K.
      • Ikegaya Y.
      • Yokoyama H.
      • Shimizu T.
      • Sato M.
      • Hashimoto H.
      Structure of a Novel DNA-binding domain of helicase-like transcription factor (HLTF) and its functional implication in DNA damage tolerance.
      ). The HIRAN domain structure includes DNA, and explains its binding preference for duplex DNA with a short 3′ single-stranded DNA overhang (
      • Kile A.C.
      • Chavez D.A.
      • Bacal J.
      • Eldirany S.
      • Korzhnev D.M.
      • Bezsonova I.
      • Eichman B.F.
      • Cimprich K.A.
      HLTF's ancient HIRAN domain binds 3′ DNA ends to drive replication fork reversal.
      ). The HARP domain structure did not include DNA, but it resembles domains in other proteins that bind distorted DNA structures (
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      ). SMARCAL1 prefers to bind DNA structures that contain at least five nucleotides of ssDNA (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ), and point mutants in the HARP2 domain impair the ability of the SMARCAL1 catalytic domain to bind branched DNA structures (
      • Bétous R.
      • Mason A.C.
      • Rambo R.P.
      • Bansbach C.E.
      • Badu-Nkansah A.
      • Sirbu B.M.
      • Eichman B.F.
      • Cortez D.
      SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
      ,
      • Mason A.C.
      • Rambo R.P.
      • Greer B.
      • Pritchett M.
      • Tainer J.A.
      • Cortez D.
      • Eichman B.F.
      A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
      ). Whether the HARP domain recognizes the fork junction itself is unknown. Our data indicate that the ZRANB3 SRD does bind forked DNA on its own although with significantly lower binding affinity than when it is attached to the motor domain. Most likely the SRD recognizes the fork structure and the motor binds the duplex DNA. As it hydrolyzes ATP, it can act to displace the nascent strands while re-annealing the parental strands. The SRD may act at the junction to facilitate this reaction.
      Our data are inconsistent with the results from Yuan et al. (
      • Yuan J.
      • Chen J.
      The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.
      ). While the SRD we identified overlaps their HARP-like domain, we find that ZRANB3 is unable to bind forked DNA structures without this region and also lacks DNA-dependent ATPase activity. Also, we found that mutations in the SRD inactivated ZRANB3 endonuclease activity as would be predicted if the SRD were required for DNA binding. We do not know why Yuan and colleagues were able to observe both DNA binding and ATPase activity in their mutant protein; however, we note that other mammalian DNA-dependent ATPases could have contaminated their protein purifications.
      This study extends our understanding of how ZRANB3 operates as a fork remodeling enzyme and determines the necessary components to carry out its enzymatic activities. Future high-resolution structural analyses of the ZRANB3 and SMARCAL1 proteins bound to DNA will be useful to better understand how their SRDs provide specificity to their fork remodeling activities.

      Author Contributions

      A. B. N. and D. C. designed the study and interpreted results. A. B. N. performed most experiments and A. C. M. and B. F. E. assisted with the DNA binding studies of the ZRANB3 SRD. A. B. N. and D. C. wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Miaw-Sheue Tsai for the production of baculovirus-infected insect cells to purify ZRANB3 proteins.

      References

        • Zeman M.K.
        • Cimprich K.A.
        Causes and consequences of replication stress.
        Nat. Cell Biol. 2014; 16: 2-9
        • Petermann E.
        • Helleday T.
        Pathways of mammalian replication fork restart.
        Nat. Rev. Mol. Cell Biol. 2010; 11: 683-687
        • Neelsen K.J.
        • Lopes M.
        Replication fork reversal in eukaryotes: from dead end to dynamic response.
        Nat. Rev. Mol. Cell Biol. 2015; 16: 207-220
        • Bétous R.
        • Mason A.C.
        • Rambo R.P.
        • Bansbach C.E.
        • Badu-Nkansah A.
        • Sirbu B.M.
        • Eichman B.F.
        • Cortez D.
        SMARCAL1 catalyzes fork regression and Holliday junction migration to maintain genome stability during DNA replication.
        Genes Dev. 2012; 26: 151-162
        • Ciccia A.
        • Nimonkar A.V.
        • Hu Y.
        • Hajdu I.
        • Achar Y.J.
        • Izhar L.
        • Petit S.A.
        • Adamson B.
        • Yoon J.C.
        • Kowalczykowski S.C.
        • Livingston D.M.
        • Haracska L.
        • Elledge S.J.
        The ZRANB3 translocase associates with poly-ubiquitinated PCNA to promote fork restart and limit recombination after replication stress.
        Mol. Cell. 2012; 47: 396-409
        • Blastyák A.
        • Hajdú I.
        • Unk I.
        • Haracska L.
        Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA.
        Mol. Cell Biol. 2010; 30: 684-693
        • Boerkoel C.F.
        • Takashima H.
        • John J.
        • Yan J.
        • Stankiewicz P.
        • Rosenbarker L.
        • André J.L.
        • Bogdanovic R.
        • Burguet A.
        • Cockfield S.
        • Cordeiro I.
        • Fründ S.
        • Illies F.
        • Joseph M.
        • Kaitila I.
        • Lama G.
        • Loirat C.
        • McLeod D.R.
        • Milford D.V.
        • Petty E.M.
        • Rodrigo F.
        • Saraiva J.M.
        • Schmidt B.
        • Smith G.C.
        • Spranger J.
        • Stein A.
        • Thiele H.
        • Tizard J.
        • Weksberg R.
        • Lupski J.R.
        • Stockton D.W.
        Mutant chromatin remodeling protein SMARCAL1 causes Schimke immuno-osseous dysplasia.
        Nat. Genet. 2002; 30: 215-220
        • Baradaran-Heravi A.
        • Raams A.
        • Lubieniecka J.
        • Cho K.S.
        • DeHaai K.A.
        • Basiratnia M.
        • Mari P.-O.
        • Xue Y.
        • Rauth M.
        • Olney A.H.
        • Shago M.
        • Choi K.
        • Weksberg R.A.
        • Nowaczyk M.J.M.
        • Wang W.
        • Jaspers N.G.J.
        • Boerkoel C.F.
        SMARCAL1 deficiency predisposes to non-Hodgkin lymphoma and hypersensitivity to genotoxic agents in vivo.
        Am. J. Med. Genet. A. 2012; 158A: 2204-2213
        • Carroll C.
        • Hunley T.E.
        • Guo Y.
        • Cortez D.
        A novel splice site mutation in SMARCAL1 results in aberrant exon definition in a child with schimke immunoosseous dysplasia.
        Am. J. Med. Genet. 2015; 167: 2260-2264
        • Carroll C.
        • Badu-Nkansah A.
        • Hunley T.
        • Baradaran-Heravi A.
        • Cortez D.
        • Frangoul H.
        Schimke Immunoosseous Dysplasia associated with undifferentiated carcinoma and a novel SMARCAL1 mutation in a child.
        Pediatric Blood Cancer. 2013; 60: E88-E90
        • Moinova H.R.
        • Chen W.-D.
        • Shen L.
        • Smiraglia D.
        • Olechnowicz J.
        • Ravi L.
        • Kasturi L.
        • Myeroff L.
        • Plass C.
        • Parsons R.
        • Minna J.
        • Willson J.K.V.
        • Green S.B.
        • Issa J.-P.
        • Markowitz S.D.
        HLTF gene silencing in human colon cancer.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 4562-4567
        • Lawrence M.S.
        • Stojanov P.
        • Mermel C.H.
        • Robinson J.T.
        • Garraway L.A.
        • Golub T. R
        • Meyerson M.
        • Gabriel S.B.
        • Lander E.S.
        • Getz G.
        Discovery and saturation analysis of cancer genes across 21 tumour types.
        Nature. 2014; 505: 495-501
        • Bansbach C.E.
        • Bétous R.
        • Lovejoy C.A.
        • Glick G.G.
        • Cortez D.
        The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks.
        Genes Dev. 2009; 23: 2405-2414
        • Postow L.
        • Woo E.M.
        • Chait B.T.
        • Funabiki H.
        Identification of SMARCAL1 as a component of the DNA damage response.
        J. Biol. Chem. 2009; 284: 35951-35961
        • Ciccia A.
        • Bredemeyer A.L.
        • Sowa M.E.
        • Terret M.-E.
        • Jallepalli P.V.
        • Harper J.W.
        • Elledge S.J.
        The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart.
        Genes Dev. 2009; 23: 2415-2425
        • Yuan J.
        • Ghosal G.
        • Chen J.
        The annealing helicase HARP protects stalled replication forks.
        Genes Dev. 2009; 23: 2394-2399
        • Bhat K.P.
        • Bétous R.
        • Cortez D.
        High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.
        J. Biol. Chem. 2015; 290: 4110-4117
        • Bétous R.
        • Couch F.B.
        • Mason A.C.
        • Eichman B.F.
        • Manosas M.
        • Cortez D.
        Substrate-selective repair and restart of replication forks by DNA translocases.
        Cell Rep. 2013; 3: 1958-1969
        • Kile A.C.
        • Chavez D.A.
        • Bacal J.
        • Eldirany S.
        • Korzhnev D.M.
        • Bezsonova I.
        • Eichman B.F.
        • Cimprich K.A.
        HLTF's ancient HIRAN domain binds 3′ DNA ends to drive replication fork reversal.
        Mol. Cell. 2015; 58: 1090-1100
        • Mason A.C.
        • Rambo R.P.
        • Greer B.
        • Pritchett M.
        • Tainer J.A.
        • Cortez D.
        • Eichman B.F.
        A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
        Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 7618-7623
        • Hishiki A.
        • Hara K.
        • Ikegaya Y.
        • Yokoyama H.
        • Shimizu T.
        • Sato M.
        • Hashimoto H.
        Structure of a Novel DNA-binding domain of helicase-like transcription factor (HLTF) and its functional implication in DNA damage tolerance.
        J. Biol. Chem. 2015; 290: 13215-13223
        • Achar Y.J.
        • Balogh D.
        • Neculai D.
        • Juhasz S.
        • Morocz M.
        • Gali H.
        • Dhe-Paganon S.
        • Venclovas Č.
        • Haracska L.
        Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling.
        Nucleic Acids Res. 2015; 43: 10277-10291
        • Yuan J.
        • Chen J.
        The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.
        Mol. Cell. 2012; 10: 410-421
        • Chen X.
        • Zaro J.L.
        • Shen W.-C.
        Fusion protein linkers: property, design and functionality.
        Adv. Drug Deliv. Rev. 2013; 65: 1357-1369
        • Yusufzai T.
        • Kadonaga J.T.
        HARP is an ATP-driven annealing helicase.
        Science. 2008; 322: 748-750
        • Porollo A.A.
        • Adamczak R.
        • Meller J.
        POLYVIEW: a flexible visualization tool for structural and functional annotations of proteins.
        Bioinformatics. 2004; 20: 2460-2462
        • Buchan D.W.A.
        • Minneci F.
        • Nugent T.C.O.
        • Bryson K.
        • Jones D.T.
        Scalable web services for the PSIPRED Protein Analysis Workbench.
        Nucleic Acids Res. 2013; 41: W349-W357
        • Cole C.
        • Barber J.D.
        • Barton G.J.
        The Jpred 3 secondary structure prediction server.
        Nucleic Acids Res. 2008; 36: W197-W201
        • Weston R.
        • Peeters H.
        • Ahel D.
        ZRANB3 is a structure-specific ATP-dependent endonuclease involved in replication stress response.
        Genes Dev. 2012; 26: 1558-1572
        • Flaus A.
        • Martin D.M.A.
        • Barton G.J.
        • Owen-Hughes T.
        Identification of multiple distinct Snf2 subfamilies with conserved structural motifs.
        Nucleic Acids Res. 2006; 34: 2887-2905
        • Yusufzai T.
        • Kadonaga J.T.
        Annealing helicase 2 (AH2), a DNA-rewinding motor with an HNH motif.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 20970-20973