Function and disruption of DNA Methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

Abstract

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein–DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.

Cooperative DNA Binding and Protein/DNA Fiber Formation Increases the Activity of the Dnmt3a DNA Methyltransferase

“…In contrast, as shown previously, binding to a 60-mer substrate was cooperative, as indicated by the fact that the binding curve was sigmoidal and could only be fitted with a Hill coefficient of 2.2 ( Fig. 2C) (14). Based on these results, we conclude that binding of Dnmt3a-C to the 29-mer DNA shows only a very weak cooperativity, indicating that an oligonucleotide of this size can be used as a reference substrate to study Dnmt3a-C methylation The in Vitro Catalytic Turnover Rate of Dnmt3a-C Is Increased on a Long DNA Substrate-Having shown that DNA binding to the short oligonucleotides shows very low cooperativity, we next determined the enzymatic rate of DNA methylation using two biotinylated substrates of different length, a 30-bp oligonucleotide containing one CpG site (at 250 nM) (that has been used as "standard" substrate in many of our studies before) and a 509-bp long substrate that corresponds to a part of the CpG island upstream of the human SUHW1 gene and contains 58 CpG sites (at 100 nM).…”

“…Therefore, we aimed to determine the DNA methylation rates on a long substrate that allows cooperative DNA binding and protein/ DNA fiber formation and compare it with the methylation rate on a short substrate that does not support fiber formation. Previous biochemical and structural data indicated that one Dnmt3a-C complex (consisting of four or more Dnmt3a-C molecules) contacts the DNA with its inner two subunits and methylates cytosine residues in the upper and lower DNA strand located at a distance of 8 -10 bp (10,11,14). DNA binding of these Dnmt3a complexes occurs in a tilted fashion such that adjacent complexes can bind the upper and lower cytosine of one CpG site.…”

“…To date two very different views on the mode of action of Dnmt3a were described in the literature. On one hand, using a variety of techniques, including gel shift DNA binding experiments (10,11), fluorescence polarization equilibrium DNA binding experiments (14), atomic force microscopy experiments (14), and catalytic studies on a multimeric substrate (11,14), our group observed that Dnmt3a binds to DNA in a cooperative manner and forms stable protein/DNA fibers. On the other hand, the group of Reich and coworkers (25)(26)(27) published that Dnmt3a methylates DNA in a processive reaction.…”

“…DNA Binding Experiments-Equilibrium binding of Dnmt3a to Cy5-labeled oligonucleotide substrates was studied by fluorescence anisotropy basically as described (14). Two Cy5-labeled 29-mer DNA substrates were used for the DNA binding studies, one containing two CpG sites (5Ј-ACT TGC AAC GGT CCT AAC CGT CAC CTC TT-3Ј) and one without CpG sites (5Ј-ACT TGC AAC AGT CCT AAC ATT CAC CTC TT-3Ј).…”

“…For Dnmt3a complexes, cooperative DNA binding was observed in equilibrium DNA binding experiments, and the formation of large protein-DNA fibers was observed in gel retardation experiments and by direct scanning force microscopy imaging (10 -12, 14). Furthermore, the protein-protein interface between adjacent Dnmt3a complexes bound to the DNA that is needed for cooperative DNA binding was identified, and mutations in this region significantly reduced the cooperativity (14). However, the functional relevance of Dnmt3a multimerization on DNA has not been addressed so far.…”