Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by the DNMT3A DNA methyltransferase

Abstract

DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA.

DNA methyltransferase DNMT3A forms interaction networks with the CpG site and flanking sequence elements for efficient methylation

“…3 A ), but the influence of the outer flanks on activity increased with more and more disfavored inner flanks. In general, the effect of 3’ flanks was stronger than of the 5’ flanks, and often A/T bases were preferred at these places in agreement with our previous data ( 17 ). Interestingly, strong 5′ outer flank effects were only observed in the TGCCGTTG substrate which is missing the highly favored T(−2) residue.…”

“…In the previous experiments shown in Figure 2A and 2B, flanking effects on CpG methylation were detectable also at the +4 to +7 positions. Related to this, in a previous study we showed strong effects of these sites on the co-methylation of CpG sites in a distance of 12 base pairs and found that A/T bases were preferred (17). We suspected that effects of these regions, which we now call "outer flanks", might have been partially masked by the stronger effects of the inner -3 to +3 flanks.…”

“…The central DNMT3AC subunits of such homo-or heterotetramers interact via the so-called RD interface and form the DNA binding site of the complex (12). Structures of the DNMT3A/3L complex bound to DNA showed that these subunits interact with two CpG sites in 12 base pair distance and methylate them in opposite DNA strands (15,16) (Figure 1A), but biochemical data showed that other arrangements of co-methylation of CpG sites are also possible (17).…”

“…Structures of the DNMT3A/3L complex bound to DNA showed that these subunits interact with two CpG sites in 12 base pair distance and methylate them in opposite DNA strands ( 15 , 16 ) ( Fig. 1 A ), but biochemical data showed that other arrangements of comethylation of CpG sites are also possible ( 17 ).

Figure 1 Structure and activity of DNMT3A complexes.

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Methylation of recombinant mononucleosomes by DNMT3A demonstrates efficient linker DNA methylation and a role of H3K36me3

“…However, it is interesting to note that the strongest methylation in our experiment was observed in the first CpG sites of our construct located up to 33 bp away from the nucleosome entry/exit site, which was not included in the cryo-EM structure. Different scenarios for the strong methylation of these sites are possible: Binding of the DNMT3A2 tetramer to these loci could occur without an interaction with the nucleosome core, in a manner described earlier for the interaction with free DNA 8 , 9 or by binding of a second tetramer side-by-side with the anchored complex as described recently 56 . On the other hand, it also appears possible for the complex to remain in the nucleosome-bound state and exploit the flexibility of the linker DNA and some hinge movement at the nucleosome binding site and perhaps within the DNMT3A2 tetramer to access the more remote sites.…”