Limits...
Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling.

Schneider K, Fuchs C, Dobay A, Rottach A, Qin W, Wolf P, Álvarez-Castro JM, Nalaskowski MM, Kremmer E, Schmid V, Leonhardt H, Schermelleh L - Nucleic Acids Res. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Center for Integrated Protein Science, Ludwig Maximilians University Munich (LMU), 82152 Planegg-Martinsried, Germany.

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.

Show MeSH

Related in: MedlinePlus

Two-loading-platform-model for the cell cycle–dependent targeting of Dnmt1 to RF and pHC. Schematic representation not drawn to scale. Closed and open lollipops indicate methylated and non-methylated CpG sites, respectively; flags indicate heterochromatin specific marker (e.g. H3K9me3). Dnmt1 is depicted in green. The model postulates two auxiliary factors that act as immobilizing platforms under certain conditions: PCNA (red) that assembles as trimeric ring at the replication fork throughout S phase, and a second unspecified factor (e.g. Uhrf1, blue) that binds strongly to hemimethylated postreplicative heterochromatin. Independent of replication, most Dnmt1 molecules (∼80% in G1, >50% in S phase) are freely roaming the nucleoplasm (left column). In addition, a non-specified MC with a pseudo koff,1 is constitutively present throughout interphase, which may be attributed to either non-specific binding to chromatin or transient trapping (‘corralling’) of the large enzyme in the nucleoplasmic environment. When replicating euchromatic sequences in early S phase (upper row) an additional ∼20% fraction of the Dnmt1 pool transiently binds via the PBD to immobilized PCNA rings (red donut) with a mean residence time (1/koff,1) of ∼10 s (1). Targeting to PCNA at RF enhances the efficiency of a small fraction of Dnmt1 to form metastable covalent complexes (koff,1) with hemimethylated CpG substrate sites in close vicinity. This may occur on already assembled nucleosomes, likely involving complex formation with one or several auxiliary factors (2 a), or directly on the naked DNA substrate adjacent to PCNA (2 b) or to nucleosomes (2 c). In late S phase, replication through chromatin with now abundant heterochromatic marks in conjunction with dense CpG methylation triggers the generation of high-affinity binding sites for an auxiliary protein. These may then act as second loading platform (dark blue pentagons) for TS-mediated binding with mean residence time (1 / koff,2) of ∼22 s involving ∼25% of the Dnmt1 pool. Formation of this transient complex with subsequent substrate binding of a small subset of molecules occurs either directly at the replication fork promoted by PBD-mediated targeting, or PCNA independently at already displaced postreplicative heterochromatin chromatin that may have escaped loading in the first instance (3). This second PCNA-independent loading complex may be assembled well into G2 phase, until all hemimethylated Dnmt1 target sites are fully methylated, which finally triggers disassembly of the loading complex and dissociation of Dnmt1 (4). Of note, this conceptual model is based on the differential availability of binding sites and the free interplay of forces. While higher affinity binding sites are occasionally generated also in early S phase, they may be too sparse to constitute a separate MC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643600&req=5

gkt191-F6: Two-loading-platform-model for the cell cycle–dependent targeting of Dnmt1 to RF and pHC. Schematic representation not drawn to scale. Closed and open lollipops indicate methylated and non-methylated CpG sites, respectively; flags indicate heterochromatin specific marker (e.g. H3K9me3). Dnmt1 is depicted in green. The model postulates two auxiliary factors that act as immobilizing platforms under certain conditions: PCNA (red) that assembles as trimeric ring at the replication fork throughout S phase, and a second unspecified factor (e.g. Uhrf1, blue) that binds strongly to hemimethylated postreplicative heterochromatin. Independent of replication, most Dnmt1 molecules (∼80% in G1, >50% in S phase) are freely roaming the nucleoplasm (left column). In addition, a non-specified MC with a pseudo koff,1 is constitutively present throughout interphase, which may be attributed to either non-specific binding to chromatin or transient trapping (‘corralling’) of the large enzyme in the nucleoplasmic environment. When replicating euchromatic sequences in early S phase (upper row) an additional ∼20% fraction of the Dnmt1 pool transiently binds via the PBD to immobilized PCNA rings (red donut) with a mean residence time (1/koff,1) of ∼10 s (1). Targeting to PCNA at RF enhances the efficiency of a small fraction of Dnmt1 to form metastable covalent complexes (koff,1) with hemimethylated CpG substrate sites in close vicinity. This may occur on already assembled nucleosomes, likely involving complex formation with one or several auxiliary factors (2 a), or directly on the naked DNA substrate adjacent to PCNA (2 b) or to nucleosomes (2 c). In late S phase, replication through chromatin with now abundant heterochromatic marks in conjunction with dense CpG methylation triggers the generation of high-affinity binding sites for an auxiliary protein. These may then act as second loading platform (dark blue pentagons) for TS-mediated binding with mean residence time (1 / koff,2) of ∼22 s involving ∼25% of the Dnmt1 pool. Formation of this transient complex with subsequent substrate binding of a small subset of molecules occurs either directly at the replication fork promoted by PBD-mediated targeting, or PCNA independently at already displaced postreplicative heterochromatin chromatin that may have escaped loading in the first instance (3). This second PCNA-independent loading complex may be assembled well into G2 phase, until all hemimethylated Dnmt1 target sites are fully methylated, which finally triggers disassembly of the loading complex and dissociation of Dnmt1 (4). Of note, this conceptual model is based on the differential availability of binding sites and the free interplay of forces. While higher affinity binding sites are occasionally generated also in early S phase, they may be too sparse to constitute a separate MC.

Mentions: In light of our data we propose a conceptual two-loading-platform model (outlined in Figure 6). According to this, the kinetic balance would shift from predominant PCNA/PBD binding in early S phase, toward TS-mediated binding in later S phase stages when replicating densely methylated heterochromatic sequences. This shift would be triggered by the strongly increased appearance of hemimethylated CpG sites in conjunction with heterochromatic marks (e.g., H3K9me3). These would then offer the target for the formation of a stable complex (involving e.g. Uhrf1) that acts as a second Dnmt1 loading platform on postreplicative chromatin sites. PCNA-independent loading complexes may persist also beyond S phase, until all hemimethylated Dnmt1 target sites are fully methylated, which in turn triggers complex disassembly and gradual loss of TS-mediated binding in G2 phase. Such a mechanism would thus safeguard faithful maintenance of dense methylation at constitutive heterochromatin important for genome stability (3), against the backdrop of a rather slow and inefficient catalytic reaction (40).Figure 6.


Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling.

Schneider K, Fuchs C, Dobay A, Rottach A, Qin W, Wolf P, Álvarez-Castro JM, Nalaskowski MM, Kremmer E, Schmid V, Leonhardt H, Schermelleh L - Nucleic Acids Res. (2013)

Two-loading-platform-model for the cell cycle–dependent targeting of Dnmt1 to RF and pHC. Schematic representation not drawn to scale. Closed and open lollipops indicate methylated and non-methylated CpG sites, respectively; flags indicate heterochromatin specific marker (e.g. H3K9me3). Dnmt1 is depicted in green. The model postulates two auxiliary factors that act as immobilizing platforms under certain conditions: PCNA (red) that assembles as trimeric ring at the replication fork throughout S phase, and a second unspecified factor (e.g. Uhrf1, blue) that binds strongly to hemimethylated postreplicative heterochromatin. Independent of replication, most Dnmt1 molecules (∼80% in G1, >50% in S phase) are freely roaming the nucleoplasm (left column). In addition, a non-specified MC with a pseudo koff,1 is constitutively present throughout interphase, which may be attributed to either non-specific binding to chromatin or transient trapping (‘corralling’) of the large enzyme in the nucleoplasmic environment. When replicating euchromatic sequences in early S phase (upper row) an additional ∼20% fraction of the Dnmt1 pool transiently binds via the PBD to immobilized PCNA rings (red donut) with a mean residence time (1/koff,1) of ∼10 s (1). Targeting to PCNA at RF enhances the efficiency of a small fraction of Dnmt1 to form metastable covalent complexes (koff,1) with hemimethylated CpG substrate sites in close vicinity. This may occur on already assembled nucleosomes, likely involving complex formation with one or several auxiliary factors (2 a), or directly on the naked DNA substrate adjacent to PCNA (2 b) or to nucleosomes (2 c). In late S phase, replication through chromatin with now abundant heterochromatic marks in conjunction with dense CpG methylation triggers the generation of high-affinity binding sites for an auxiliary protein. These may then act as second loading platform (dark blue pentagons) for TS-mediated binding with mean residence time (1 / koff,2) of ∼22 s involving ∼25% of the Dnmt1 pool. Formation of this transient complex with subsequent substrate binding of a small subset of molecules occurs either directly at the replication fork promoted by PBD-mediated targeting, or PCNA independently at already displaced postreplicative heterochromatin chromatin that may have escaped loading in the first instance (3). This second PCNA-independent loading complex may be assembled well into G2 phase, until all hemimethylated Dnmt1 target sites are fully methylated, which finally triggers disassembly of the loading complex and dissociation of Dnmt1 (4). Of note, this conceptual model is based on the differential availability of binding sites and the free interplay of forces. While higher affinity binding sites are occasionally generated also in early S phase, they may be too sparse to constitute a separate MC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643600&req=5

gkt191-F6: Two-loading-platform-model for the cell cycle–dependent targeting of Dnmt1 to RF and pHC. Schematic representation not drawn to scale. Closed and open lollipops indicate methylated and non-methylated CpG sites, respectively; flags indicate heterochromatin specific marker (e.g. H3K9me3). Dnmt1 is depicted in green. The model postulates two auxiliary factors that act as immobilizing platforms under certain conditions: PCNA (red) that assembles as trimeric ring at the replication fork throughout S phase, and a second unspecified factor (e.g. Uhrf1, blue) that binds strongly to hemimethylated postreplicative heterochromatin. Independent of replication, most Dnmt1 molecules (∼80% in G1, >50% in S phase) are freely roaming the nucleoplasm (left column). In addition, a non-specified MC with a pseudo koff,1 is constitutively present throughout interphase, which may be attributed to either non-specific binding to chromatin or transient trapping (‘corralling’) of the large enzyme in the nucleoplasmic environment. When replicating euchromatic sequences in early S phase (upper row) an additional ∼20% fraction of the Dnmt1 pool transiently binds via the PBD to immobilized PCNA rings (red donut) with a mean residence time (1/koff,1) of ∼10 s (1). Targeting to PCNA at RF enhances the efficiency of a small fraction of Dnmt1 to form metastable covalent complexes (koff,1) with hemimethylated CpG substrate sites in close vicinity. This may occur on already assembled nucleosomes, likely involving complex formation with one or several auxiliary factors (2 a), or directly on the naked DNA substrate adjacent to PCNA (2 b) or to nucleosomes (2 c). In late S phase, replication through chromatin with now abundant heterochromatic marks in conjunction with dense CpG methylation triggers the generation of high-affinity binding sites for an auxiliary protein. These may then act as second loading platform (dark blue pentagons) for TS-mediated binding with mean residence time (1 / koff,2) of ∼22 s involving ∼25% of the Dnmt1 pool. Formation of this transient complex with subsequent substrate binding of a small subset of molecules occurs either directly at the replication fork promoted by PBD-mediated targeting, or PCNA independently at already displaced postreplicative heterochromatin chromatin that may have escaped loading in the first instance (3). This second PCNA-independent loading complex may be assembled well into G2 phase, until all hemimethylated Dnmt1 target sites are fully methylated, which finally triggers disassembly of the loading complex and dissociation of Dnmt1 (4). Of note, this conceptual model is based on the differential availability of binding sites and the free interplay of forces. While higher affinity binding sites are occasionally generated also in early S phase, they may be too sparse to constitute a separate MC.
Mentions: In light of our data we propose a conceptual two-loading-platform model (outlined in Figure 6). According to this, the kinetic balance would shift from predominant PCNA/PBD binding in early S phase, toward TS-mediated binding in later S phase stages when replicating densely methylated heterochromatic sequences. This shift would be triggered by the strongly increased appearance of hemimethylated CpG sites in conjunction with heterochromatic marks (e.g., H3K9me3). These would then offer the target for the formation of a stable complex (involving e.g. Uhrf1) that acts as a second Dnmt1 loading platform on postreplicative chromatin sites. PCNA-independent loading complexes may persist also beyond S phase, until all hemimethylated Dnmt1 target sites are fully methylated, which in turn triggers complex disassembly and gradual loss of TS-mediated binding in G2 phase. Such a mechanism would thus safeguard faithful maintenance of dense methylation at constitutive heterochromatin important for genome stability (3), against the backdrop of a rather slow and inefficient catalytic reaction (40).Figure 6.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Center for Integrated Protein Science, Ludwig Maximilians University Munich (LMU), 82152 Planegg-Martinsried, Germany.

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.

Show MeSH
Related in: MedlinePlus