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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.

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Refined diffusion-coupled compartmental model for three MCs and determination of kdiff from GFP multimer measurements. (A) The nuclear compartment is divided into four main compartments: bleached and unbleached molecules in the bound or free state, respectively. The bound state can be subdivided into three compartments with specific properties. For Dnmt1, we choose two DMCs (DMC1 and DMC2, blue and green frame, respectively) and one CMC (red frame) with a fixed koff. Molecules bind and unbind with association and dissociation rates given by kon,i and koff,i, respectively. In our refined modeling approach, the parameter fbl is experimentally determined for each individual FRAP series. Migration of molecules is implemented in the model by introducing a new diffusion rate constant kdiff. This parameter corrects for the size-dependent exchange of the free molecules between the bleached and unbleached compartment. Parameter and variables entering the sets of differential equations are written in bold; predetermined/fixed values are indicated. (B) Quantitative FRAP evaluation of GFP multimers. The GFP mobility of the dimer and the tetramer decreases stepwise as compared with the monomer. The kdiff factor for the GFP constructs is estimated from the model with no DMC/CMC. From these values, the corresponding kdiff factor for the size of GFP-Dnmt1 is estimated using an exponential regression curve (inset diagram).
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gkt191-F4: Refined diffusion-coupled compartmental model for three MCs and determination of kdiff from GFP multimer measurements. (A) The nuclear compartment is divided into four main compartments: bleached and unbleached molecules in the bound or free state, respectively. The bound state can be subdivided into three compartments with specific properties. For Dnmt1, we choose two DMCs (DMC1 and DMC2, blue and green frame, respectively) and one CMC (red frame) with a fixed koff. Molecules bind and unbind with association and dissociation rates given by kon,i and koff,i, respectively. In our refined modeling approach, the parameter fbl is experimentally determined for each individual FRAP series. Migration of molecules is implemented in the model by introducing a new diffusion rate constant kdiff. This parameter corrects for the size-dependent exchange of the free molecules between the bleached and unbleached compartment. Parameter and variables entering the sets of differential equations are written in bold; predetermined/fixed values are indicated. (B) Quantitative FRAP evaluation of GFP multimers. The GFP mobility of the dimer and the tetramer decreases stepwise as compared with the monomer. The kdiff factor for the GFP constructs is estimated from the model with no DMC/CMC. From these values, the corresponding kdiff factor for the size of GFP-Dnmt1 is estimated using an exponential regression curve (inset diagram).

Mentions: The mathematical models used to statistically infer the kinetic parameters from corrected and normalized FRAP datasets are based on a compartmental approach and biochemical kinetic principles. The model for diffusion-uncoupled FRAP, i.e. for molecules that diffuse much more rapidly than they bind or unbind, has previously been described (22,36). A model for diffusion-coupled FRAP is developed in this work and illustrated in Figure 4A; a similar approach has been taken in (37). The model considers transitions between the bound and the free state of a protein with association rate constant kon and dissociation rate constant koff . As substantiated in the Supplementary Methods, the association and dissociation dynamics can be expressed in terms of linear ordinary differential equations (ODEs) when replacing kon by an effective association rate constant. The ODEs are given below. While bound proteins remain fixed at the respective binding sites, free proteins diffuse through the nucleus, thus changing their locations. Movements between the bleached and the unbleached section are modeled with a diffusion rate constant kdiff. Its value depends on the geometry of the cell and is not immediately eligible for interpretation purposes. See the Supplementary Methods for details on the modeling of the movement of proteins.


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)

Refined diffusion-coupled compartmental model for three MCs and determination of kdiff from GFP multimer measurements. (A) The nuclear compartment is divided into four main compartments: bleached and unbleached molecules in the bound or free state, respectively. The bound state can be subdivided into three compartments with specific properties. For Dnmt1, we choose two DMCs (DMC1 and DMC2, blue and green frame, respectively) and one CMC (red frame) with a fixed koff. Molecules bind and unbind with association and dissociation rates given by kon,i and koff,i, respectively. In our refined modeling approach, the parameter fbl is experimentally determined for each individual FRAP series. Migration of molecules is implemented in the model by introducing a new diffusion rate constant kdiff. This parameter corrects for the size-dependent exchange of the free molecules between the bleached and unbleached compartment. Parameter and variables entering the sets of differential equations are written in bold; predetermined/fixed values are indicated. (B) Quantitative FRAP evaluation of GFP multimers. The GFP mobility of the dimer and the tetramer decreases stepwise as compared with the monomer. The kdiff factor for the GFP constructs is estimated from the model with no DMC/CMC. From these values, the corresponding kdiff factor for the size of GFP-Dnmt1 is estimated using an exponential regression curve (inset diagram).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt191-F4: Refined diffusion-coupled compartmental model for three MCs and determination of kdiff from GFP multimer measurements. (A) The nuclear compartment is divided into four main compartments: bleached and unbleached molecules in the bound or free state, respectively. The bound state can be subdivided into three compartments with specific properties. For Dnmt1, we choose two DMCs (DMC1 and DMC2, blue and green frame, respectively) and one CMC (red frame) with a fixed koff. Molecules bind and unbind with association and dissociation rates given by kon,i and koff,i, respectively. In our refined modeling approach, the parameter fbl is experimentally determined for each individual FRAP series. Migration of molecules is implemented in the model by introducing a new diffusion rate constant kdiff. This parameter corrects for the size-dependent exchange of the free molecules between the bleached and unbleached compartment. Parameter and variables entering the sets of differential equations are written in bold; predetermined/fixed values are indicated. (B) Quantitative FRAP evaluation of GFP multimers. The GFP mobility of the dimer and the tetramer decreases stepwise as compared with the monomer. The kdiff factor for the GFP constructs is estimated from the model with no DMC/CMC. From these values, the corresponding kdiff factor for the size of GFP-Dnmt1 is estimated using an exponential regression curve (inset diagram).
Mentions: The mathematical models used to statistically infer the kinetic parameters from corrected and normalized FRAP datasets are based on a compartmental approach and biochemical kinetic principles. The model for diffusion-uncoupled FRAP, i.e. for molecules that diffuse much more rapidly than they bind or unbind, has previously been described (22,36). A model for diffusion-coupled FRAP is developed in this work and illustrated in Figure 4A; a similar approach has been taken in (37). The model considers transitions between the bound and the free state of a protein with association rate constant kon and dissociation rate constant koff . As substantiated in the Supplementary Methods, the association and dissociation dynamics can be expressed in terms of linear ordinary differential equations (ODEs) when replacing kon by an effective association rate constant. The ODEs are given below. While bound proteins remain fixed at the respective binding sites, free proteins diffuse through the nucleus, thus changing their locations. Movements between the bleached and the unbleached section are modeled with a diffusion rate constant kdiff. Its value depends on the geometry of the cell and is not immediately eligible for interpretation purposes. See the Supplementary Methods for details on the modeling of the movement of proteins.

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