The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1*

Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.

[1]  S. Baylin,et al.  DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci , 2000, Nature Genetics.

[2]  H. Leonhardt,et al.  A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei , 1992, Cell.

[3]  Alun D. Hughes,et al.  The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression , 2008, Oncogene.

[4]  S. Jacobsen,et al.  UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells , 2007, Science.

[5]  C. Arrowsmith,et al.  Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1 , 2008, Nature.

[6]  D. Patel,et al.  Structure of DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation , 2011, Science.

[7]  Daniel C. Scott,et al.  Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation , 2008, Cell.

[8]  Albert Jeltsch,et al.  On the Enzymatic Properties of Dnmt1: Specificity, Processivity, Mechanism of Linear Diffusion and Allosteric Regulation of the Enzyme , 2006, Epigenetics.

[9]  A. Bird,et al.  DNA methylation landscapes: provocative insights from epigenomics , 2008, Nature Reviews Genetics.

[10]  K. Robertson,et al.  Preferential Methylation of Unmethylated DNA by Mammaliande Novo DNA Methyltransferase Dnmt3a* , 2002, The Journal of Biological Chemistry.

[11]  K. Mitsuya,et al.  The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA , 2007, Nature.

[12]  J. Bacik,et al.  Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP , 2010, The Journal of Biological Chemistry.

[13]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[14]  T. Bestor,et al.  Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. , 1992, The EMBO journal.

[15]  김삼묘,et al.  “Bioinformatics” 특집을 내면서 , 2000 .

[16]  Fei Long,et al.  REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. , 2004, Acta crystallographica. Section D, Biological crystallography.

[17]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[18]  N. Reich,et al.  DNA cytosine C5 methyltransferase Dnmt1: catalysis-dependent release of allosteric inhibition. , 2005, Biochemistry.

[19]  K Henrick,et al.  Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. , 2004, Acta crystallographica. Section D, Biological crystallography.

[20]  Yusuke Nakamura,et al.  Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism , 2008, Nature.

[21]  M. Saraste,et al.  FEBS Lett , 2000 .

[22]  J. Mornon,et al.  The BAH (bromo‐adjacent homology) domain: a link between DNA methylation, replication and transcriptional regulation , 1999, FEBS letters.

[23]  Avram Goldstein,et al.  THE MECHANISM OF ENZYME-INHIBITOR-SUBSTRATE REACTIONS : ILLUSTRATED BY THE CHOLINESTERASE-PHYSOSTIGMINE-ACETYLCHOLINE SYSTEM. , 1944 .

[24]  Thomas C. Terwilliger,et al.  Electronic Reprint Biological Crystallography Maximum-likelihood Density Modification , 2022 .

[25]  S. Pradhan,et al.  CXXC domain of human DNMT1 is essential for enzymatic activity. , 2008, Biochemistry.

[26]  S. Jacobsen,et al.  The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix , 2008, Nature.

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

[28]  P. Roach,et al.  A real-time assay for CpG-specific cytosine-C5 methyltransferase activity , 2010, Nucleic acids research.

[29]  Liisa Holm,et al.  Searching protein structure databases with DaliLite v.3 , 2008, Bioinform..

[30]  S. Zukin,et al.  Epigenetics , 2009, Alzheimer's & Dementia.

[31]  P. N. Lewis,et al.  Histone H3 thiol reactivity and acetyltransferases in chicken erythrocyte nuclei. , 1988, The Journal of biological chemistry.

[32]  S. Cha.  Kinetic behavior at high enzyme concentrations. Magnitude of errors of Michelis-Menten and other approximations. , 1970, The Journal of biological chemistry.

[33]  H. Ng,et al.  Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. , 1997, Science.