title: Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling.
abstract: DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle-dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤ 10 s). In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.
authors.affiliation: Department of Biology and Center for Integrated Protein Science, Ludwig Maximilians University Munich (LMU), 82152 Planegg-Martinsried, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Schneider
authors.forename: Christiane
authors.initials: C
authors.lastname: Fuchs
authors.forename: Akos
authors.initials: A
authors.lastname: Dobay
authors.forename: Andrea
authors.initials: A
authors.lastname: Rottach
authors.forename: Weihua
authors.initials: W
authors.lastname: Qin
authors.forename: Patricia
authors.initials: P
authors.lastname: Wolf
authors.forename: José M
authors.initials: JM
authors.lastname: Álvarez-Castro
authors.forename: Marcus M
authors.initials: MM
authors.lastname: Nalaskowski
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kremmer
authors.forename: Volker
authors.initials: V
authors.lastname: Schmid
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.forename: Lothar
authors.initials: L
authors.lastname: Schermelleh
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000378:metabolism*; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D006570:Heterochromatin / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D016196:S Phase
chemical_list: D006570:Heterochromatin; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619381:Dnmt1 protein, mouse
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