Crystal Structure of hFen1 in apo form

Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5' flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein-protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5' flap prior to its threading into the active site for cleavage. We also found that the β-pin region is rigidified into a short helix in R192F hFEN1-DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the β-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.

[1]  Dustin E. Schones,et al.  JMJD1B Demethylates H4R3me2s and H3K9me2 to Facilitate Gene Expression for Development of Hematopoietic Stem and Progenitor Cells , 2018, Cell reports.

[2]  S. Hamdan,et al.  Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway , 2018, Nucleic acids research.

[3]  J. Tainer,et al.  Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability , 2017, Nature Communications.

[4]  P. Gaillard,et al.  Control of structure-specific endonucleases to maintain genome stability , 2017, Nature Reviews Molecular Cell Biology.

[5]  Bing Tian,et al.  Lysine Acetylation and Succinylation in HeLa Cells and their Essential Roles in Response to UV-induced Stress , 2016, Scientific Reports.

[6]  Lingfeng He,et al.  The FEN1 L209P mutation interferes with long-patch base excision repair and induces cellular transformation , 2016, Oncogene.

[7]  J. Debreczeni,et al.  Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site , 2016, Nature chemical biology.

[8]  T. Ceska,et al.  Direct observation of DNA threading in flap endonuclease complexes , 2016, Nature Structural &Molecular Biology.

[9]  Timothy D. Craggs,et al.  DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction* , 2016, The Journal of Biological Chemistry.

[10]  F. Chien,et al.  Wuho Is a New Member in Maintaining Genome Stability through its Interaction with Flap Endonuclease 1 , 2016, PLoS biology.

[11]  Qiang Xu,et al.  Structural insights into catalysis and dimerization enhanced exonuclease activity of RNase J , 2015, Nucleic acids research.

[12]  S. Stewart,et al.  Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand* , 2015, The Journal of Biological Chemistry.

[13]  J. Tainer,et al.  The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once. , 2014, DNA repair.

[14]  Anita C Jones,et al.  Observation of unpaired substrate DNA in the flap endonuclease-1 active site , 2013, Nucleic acids research.

[15]  R. Bambara,et al.  Flap endonuclease 1. , 2013, Annual review of biochemistry.

[16]  M. Gregory,et al.  Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η , 2013, Proceedings of the National Academy of Sciences.

[17]  Na Liu,et al.  Sequential posttranslational modifications program FEN1 degradation during cell-cycle progression. , 2012, Molecular cell.

[18]  E. Nogales,et al.  Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability , 2012, Proceedings of the National Academy of Sciences.

[19]  J. Tainer,et al.  Flap endonucleases pass 5′-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5′-ends , 2012, Nucleic acids research.

[20]  J. Tainer,et al.  Human Flap Endonuclease Structures, DNA Double-Base Flipping, and a Unified Understanding of the FEN1 Superfamily , 2011, Cell.

[21]  Qin M. Chen,et al.  Methylation of FEN1 suppresses nearby phosphorylation and facilitates PCNA binding , 2010, Nature chemical biology.

[22]  S. Stewart,et al.  FEN1 Ensures Telomere Stability by Facilitating Replication Fork Re-initiation* , 2010, The Journal of Biological Chemistry.

[23]  R. Bambara,et al.  Acetylation of Dna2 Endonuclease/Helicase and Flap Endonuclease 1 by p300 Promotes DNA Stability by Creating Long Flap Intermediates* , 2009, The Journal of Biological Chemistry.

[24]  C. A. Theimer,et al.  The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis* , 2009, The Journal of Biological Chemistry.

[25]  S. Clarke,et al.  Protein arginine methylation in mammals: who, what, and why. , 2009, Molecular cell.

[26]  Zhigang Guo,et al.  Nucleolar Localization and Dynamic Roles of Flap Endonuclease 1 in Ribosomal DNA Replication and Damage Repair , 2008, Molecular and Cellular Biology.

[27]  D. Lin,et al.  Fen1 mutations result in autoimmunity, chronic inflammation and cancers , 2007, Nature Medicine.

[28]  S. Khan,et al.  Lysine Trimethylation of Retinoic Acid Receptor-α , 2007, Molecular & Cellular Proteomics.

[29]  B. Shen,et al.  Concerted Action of Exonuclease and Gap-dependent Endonuclease Activities of FEN-1 Contributes to the Resolution of Triplet Repeat Sequences (CTG)n- and (GAA)n-derived Secondary Structures Formed during Maturation of Okazaki Fragments* , 2006, Journal of Biological Chemistry.

[30]  Pawan Gupta,et al.  Suppression of receptor interacting protein 140 repressive activity by protein arginine methylation , 2006, The EMBO journal.

[31]  M. Waters,et al.  Arginine methylation in a β-hairpin peptide: Implications for Arg-π interactions, ΔCp°, and the cold denatured state , 2006 .

[32]  S. Khorasanizadeh,et al.  Double chromodomains cooperate to recognize the methylated histone H3 tail , 2005, Nature.

[33]  R. Gary,et al.  The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA , 2005, Nucleic acids research.

[34]  U. Hübscher,et al.  The two DNA clamps Rad9/Rad1/Hus1 complex and proliferating cell nuclear antigen differentially regulate flap endonuclease 1 activity. , 2005, Journal of molecular biology.

[35]  T. Hakoshima,et al.  Structural basis for recruitment of human flap endonuclease 1 to PCNA , 2005, The EMBO journal.

[36]  Jay Z. Parrish,et al.  Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks , 2005, EMBO reports.

[37]  Y. Shamoo,et al.  Structural and thermodynamic analysis of human PCNA with peptides derived from DNA polymerase-delta p66 subunit and flap endonuclease-1. , 2004, Structure.

[38]  A. Sancar,et al.  The human Rad9-Rad1-Hus1 checkpoint complex stimulates flap endonuclease 1. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Tainer,et al.  Structural Basis for FEN-1 Substrate Specificity and PCNA-Mediated Activation in DNA Replication and Repair , 2004, Cell.

[40]  G. Dianov,et al.  WRN helicase and FEN-1 form a complex upon replication arrest and together process branchmigrating DNA structures associated with the replication fork. , 2003, Molecular biology of the cell.

[41]  U. Hübscher,et al.  Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation , 2003, Oncogene.

[42]  G. Dianov,et al.  Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity , 2001, The EMBO journal.

[43]  U. Hübscher,et al.  Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300. , 2001, Molecular cell.

[44]  J. Tainer,et al.  Structure of the DNA Repair and Replication Endonuclease and Exonuclease FEN-1 Coupling DNA and PCNA Binding to FEN-1 Activity , 1998, Cell.

[45]  L. Sklar,et al.  Essential Amino Acids for Substrate Binding and Catalysis of Human Flap Endonuclease 1 (*) , 1996, The Journal of Biological Chemistry.

[46]  M. Lieber,et al.  Lagging Strand DNA Synthesis at the Eukaryotic Replication Fork Involves Binding and Stimulation of FEN-1 by Proliferating Cell Nuclear Antigen (*) , 1995, The Journal of Biological Chemistry.

[47]  Y. Hua,et al.  Figures and figure supplements Structural basis for DNA 5 ́-end resection by RecJ Kaiying , 2016 .

[48]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[49]  D. V. van Aalten,et al.  Acta Crystallographica Section D Biological , 2003 .