DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in C. elegans

Assembly of the CDC45-MCM-GINS helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors not present in yeast. Here, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor, by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome resulting from DONSON mutations.

[1]  Martin A. M. Reijns,et al.  In silico protein interaction screening uncovers DONSON’s role in replication initiation , 2023, Science.

[2]  Hirofumi Tanaka,et al.  Novel role of DONSON in CMG helicase assembly during vertebrate DNA replication initiation , 2023, The EMBO journal.

[3]  Joseph T. P. Yeeles,et al.  How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication , 2023, Molecular cell.

[4]  J. Diffley,et al.  Mechanism of replication origin melting nucleated by CMG helicase assembly , 2022, Nature.

[5]  Joseph T. P. Yeeles,et al.  Fast and efficient DNA replication with purified human proteins , 2022, Nature.

[6]  S. Gygi,et al.  CDC7-independent G1/S transition revealed by targeted protein degradation , 2022, Nature.

[7]  J. Diffley,et al.  The Initiation of Eukaryotic DNA Replication. , 2022, Annual review of biochemistry.

[8]  Kara A. Bernstein,et al.  Role and Regulation of the RECQL4 Family during Genomic Integrity Maintenance , 2021, Genes.

[9]  A. Brazma,et al.  The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences , 2021, Nucleic Acids Res..

[10]  Joseph T. P. Yeeles,et al.  Structure of a human replisome shows the organisation and interactions of a DNA replication machine , 2021, The EMBO journal.

[11]  L. Pearl,et al.  Phosphorylation-dependent assembly of DNA damage response systems and the central roles of TOPBP1 , 2021, DNA repair.

[12]  S. Ovchinnikov,et al.  ColabFold: making protein folding accessible to all , 2022, Nature Methods.

[13]  K. Labib,et al.  TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans , 2021, The EMBO journal.

[14]  Oriol Vinyals,et al.  Highly accurate protein structure prediction with AlphaFold , 2021, Nature.

[15]  Conrad C. Huang,et al.  UCSF ChimeraX: Structure visualization for researchers, educators, and developers , 2020, Protein science : a publication of the Protein Society.

[16]  P. Zegerman,et al.  Identification of the critical replication targets of CDK reveals direct regulation of replication initiation factors by the embryo polarity machinery in C. elegans , 2020, bioRxiv.

[17]  L. Pellegrini,et al.  CryoEM structures of human CMG–ATPγS–DNA and CMG–AND-1 complexes , 2020, Nucleic acids research.

[18]  G. Stewart,et al.  DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain , 2020, Nature Communications.

[19]  G. Cannone,et al.  Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork , 2019, bioRxiv.

[20]  M. O’Donnell,et al.  Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase , 2019, Cell.

[21]  Alessandro Costa,et al.  The mechanism of eukaryotic CMG helicase activation , 2018, Nature.

[22]  Conrad C. Huang,et al.  UCSF ChimeraX: Meeting modern challenges in visualization and analysis , 2018, Protein science : a publication of the Protein Society.

[23]  Michael E. Coulter,et al.  Integrated genome and transcriptome sequencing identifies a noncoding mutation in the genome replication factor DONSON as the cause of microcephaly-micromelia syndrome , 2017, Genome research.

[24]  James M. Dewar,et al.  Mechanisms of DNA replication termination , 2017, Nature Reviews Molecular Cell Biology.

[25]  Sarah K. Nordquist,et al.  Systematic Functional Characterization of Human 21st Chromosome Orthologs in Caenorhabditis elegans , 2017, G3: Genes, Genomes, Genetics.

[26]  Martin A. M. Reijns,et al.  Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism , 2017, Nature Genetics.

[27]  A. Gartner,et al.  CUL-2LRR-1 and UBXN-3/FAF1 drive replisome disassembly during DNA replication termination and mitosis , 2017, Nature Cell Biology.

[28]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[29]  J. Diffley,et al.  Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates , 2017, Molecular cell.

[30]  J. Diffley,et al.  How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication , 2017, Molecular cell.

[31]  L. Pintard,et al.  RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans , 2016, G3: Genes, Genomes, Genetics.

[32]  S. Bell,et al.  Chromosome Duplication in Saccharomyces cerevisiae , 2016, Genetics.

[33]  J. Diffley,et al.  Phosphopeptide binding by Sld3 links Dbf4‐dependent kinase to MCM replicative helicase activation , 2016, The EMBO journal.

[34]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[35]  Ricky D. Edmondson,et al.  A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1 , 2015, Nucleic acids research.

[36]  A. Gartner,et al.  Both Chromosome Decondensation and Condensation Are Dependent on DNA Replication in C. elegans Embryos , 2015, Cell reports.

[37]  J. Diffley,et al.  Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins , 2015, Nature.

[38]  Thanos D Halazonetis,et al.  DNA replication stress as a hallmark of cancer. , 2015, Annual review of pathology.

[39]  Y. Shirakihara,et al.  Crystal structure of the homology domain of the eukaryotic DNA replication proteins Sld3/Treslin. , 2014, Structure.

[40]  J. Diffley,et al.  DNA Replication and Oncogene-Induced Replicative Stress , 2014, Current Biology.

[41]  J. Ahringer,et al.  CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans , 2014, The Journal of cell biology.

[42]  J. Diffley,et al.  ATPase-Dependent Quality Control of DNA Replication Origin Licensing , 2013, Nature.

[43]  S. Cotterill,et al.  Drosophila RecQ4 Is Directly Involved in Both DNA Replication and the Response to UV Damage in S2 Cells , 2012, PloS one.

[44]  K. Labib,et al.  Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation , 2012, The EMBO journal.

[45]  Masato T. Kanemaki,et al.  Mcm10 Plays a Role in Functioning of the Eukaryotic Replicative DNA Helicase, Cdc45-Mcm-GINS , 2012, Current Biology.

[46]  S. Bell,et al.  Eukaryotic Origin-Dependent DNA Replication In Vitro Reveals Sequential Action of DDK and S-CDK Kinases , 2011, Cell.

[47]  M. Seki,et al.  The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells. Role of the N-terminal region of RECQL4 in cells. , 2011, Biochimica et biophysica acta.

[48]  C. Ponting,et al.  Homology explains the functional similarities of Treslin/Ticrr and Sld3 , 2010, Current Biology.

[49]  H. Araki,et al.  CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon}, and GINS in budding yeast. , 2010, Genes & development.

[50]  A. Shevchenko,et al.  Treslin Collaborates with TopBP1 in Triggering the Initiation of DNA Replication , 2010, Cell.

[51]  Jingchuan Sun,et al.  A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication , 2009, Proceedings of the National Academy of Sciences.

[52]  J. Diffley,et al.  Concerted Loading of Mcm2–7 Double Hexamers around DNA during DNA Replication Origin Licensing , 2009, Cell.

[53]  S. Bell,et al.  Incorporation into the prereplicative complex activates the Mcm2-7 helicase for Cdc7-Dbf4 phosphorylation. , 2009, Genes & development.

[54]  Hiroyuki Araki,et al.  CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast , 2007, Nature.

[55]  J. Diffley,et al.  Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast , 2007, Nature.

[56]  M. Botchan,et al.  Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase , 2006, Proceedings of the National Academy of Sciences.

[57]  Ricky D. Edmondson,et al.  GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks , 2006, Nature Cell Biology.

[58]  A. Venkitaraman,et al.  Initiation of DNA Replication Requires the RECQL4 Protein Mutated in Rothmund-Thomson Syndrome , 2005, Cell.

[59]  M. Botchan,et al.  humpty dumpty Is Required for Developmental DNA Amplification and Cell Proliferation in Drosophila , 2005, Current Biology.

[60]  A. Holway,et al.  Systematic, RNA-Interference-Mediated Identification of mus-101 Modifier Genes in Caenorhabditis elegans , 2005, Genetics.

[61]  J M Carazo,et al.  XMIPP: a new generation of an open-source image processing package for electron microscopy. , 2004, Journal of structural biology.

[62]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[63]  Haruki Nakamura,et al.  Announcing the worldwide Protein Data Bank , 2003, Nature Structural Biology.

[64]  Y. Dong,et al.  Systematic functional analysis of the Caenorhabditis elegans genome using RNAi , 2003, Nature.

[65]  H. Araki,et al.  Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae , 2001, The EMBO journal.

[66]  Robert W. Miller,et al.  Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome , 1999, Nature Genetics.

[67]  N. Munakata.  [Genetics of Caenorhabditis elegans]. , 1989, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[68]  OUP accepted manuscript , 2021, Nucleic Acids Research.

[69]  Christopher J. Williams,et al.  MolProbity: More and better reference data for improved all‐atom structure validation , 2018, Protein science : a publication of the Protein Society.

[70]  Wen Jiang,et al.  EMAN2: an extensible image processing suite for electron microscopy. , 2007, Journal of structural biology.