2020同步年報

048 ACTIVITY REPORT 2020 genes and dispersed repeated elements, whereas DNMT3B specializes in methylating pericentric satellite repeats and the gene body of actively transcribed genes. Heterozygous mutations of DNMT3A are associated with acute myeloid leukemia 2 whereas homozygous mutations of DNMT3B contribute to immunodeficiency, centromere instability and the facial anomalies (ICF) syndrome. 3 DNMT3A methylates DNA in a cooperative manner, whereas DNMT3B exhibits a non-cooperative methylation of DNA through catalyzing multiple CpG sites before dissociation. Unlike DNMT3A that prefers T downstream of the CpG sites, DNMT3B favors A at the flanking position. Previously, structural studies of DNMT focused mainly on DNMT3A. 4-6 To determine the mecha- nistic basis of DNMT3B-mediated methylation, a research team led by Hanna S. Yuan (Academia Sinica) solved the structures of apo and DNA-bound forms of DNMT3B. The diffraction data were collected at TPS 05A and TLS 15A1 of NSRRC. 7 Figures 1(a) and 1(b) show the overall structures of the apo and DNA-ribose bound DNMT3B-3L complex. Both these structures reveal a heterotetramer presenting a linear assembly of 3L–3B–3B–3L. The bound DNA is snug- ly enclosed by the catalytic loop and the TRD loop of the DNMT3B dimer. Binding of DNA causes considerable con- formational changes of these two loops ( Figs. 1(c) and 1(d) ). In the catalytic loop, Val657 moves toward the DNA minor groove by ~1.3 Å upon DNA binding ( Fig. 1(c) ), whereas the disordered region (residues Ile781–Asn786) of the TRD loop in the apo form becomes well ordered in the DNA-bound form ( Fig. 1(d) ), as illustrated by the well defined electron density ( Fig. 1(e) ), and interacts with DNA at the major groove. The CpG site is thus clamped by the catalytic loop and TRD loop from two separate sides of the DNA molecule. In the structure of DNMT3B-3L bound with CpGpG DNA, the cytosine is flipped out and inserted into the catalytic pocket of DNMT3B. The conserved Val657 lo- cated in the catalytic loop coordinates DNA bases surround- ing the cytosine and stabilizes guanine G5' at the opposite stand on forming a hydrogen bond between its backbone carbonyl oxygen and the N2 atom of G5'. In the catalytic pocket, the flipped-out cytosine is located near C651 and forms hydrogen bonds with Ser649, Glu697, Arg731 and Arg733 ( Fig. 1(f) ). These particular interactions contribute to the cytosine-specific recognition of CpG sites by DNMT3B. Cys651 presumably represents the catalytic residue me- diating a nucleophilic attack of the cytosine C6 position. As analyzed with a HpaII cleavage assay, substitutions of cysteine and valine at the 651 and 657 positions (C651A and V657G) cause complete and partial loss of enzymatic activity of DNMT3B, respectively ( Fig 1(g) ). Notably, apart from observing all residues and cofactors in the DNA-bound form structure, the authors also identified a proton-wire water channel comprising four water molecules linked via hydrogen bonds, located near the cytosine C5 position in both promoters of the DNMT3B dimer ( Fig. 1(h) ), which are likely to be responsible for the deprotonation from C5 during cytosine methylation. Based on these observations, the authors proposed a complete working mechanism of cytosine methylation by DNMT3B ( Fig. 1(i) ). For guanine recognition in CpG sites by human DNMT3B, residue Asn779 located in the TRD loop specifically interacts with the G6 position of target CpG sites via a weak hydro- gen bond (3.5 Å) between the Nδ atom of Asn779 and the O6 atom of G6 ( Fig. 2(a) ). Asn779 is conserved among DNMT3B and DNMT3A, but in DNMT3A the equivalent res- idue of Asn779 (Asn838) does not contact G6. In contrast, G6 is coordinated with Arg836 via hydrogen bonds and water-mediated hydrogen bonds ( Fig. 2(c) ). 6 Little is known about the structural basis of a flanking sequence preference of DNMT3B. DNMT3B exhibits a preference for TpCpGpG methylation, with a T residue at the −1 position and a G residue at the +1 position of its target CpG sites. Residue Asn779 not only interacts with the guanine (G6) in the CpG site, but also interacts remotely with the flanking guanine at the +1 position at distance 3.9 Å between the Nδ and O6 atoms of G7 ( Fig. 2(a) ). The structure of DNMT3B in a complex with CpGpT DNA reveals that N779 continues to interact with G6 in the CpG site (the O6 atom), but releases its interaction with the flanking T as the distance between the Nδ atom of N779 and the O6 atom of the thymine increases beyond 5.5 Å ( Fig. 2(b) ). Besides N779, residue K777 also makes van der Waals inter- actions with the flanking guanine of the CpGpG site ( Fig. 2(a) ). As analyzed with a methylation activity assay, wild- type DNMT3B exhibited high methyltransferase activity at CpGpG sites (CpGpG ≈ CpGpA > CpGpT). Substitutions of asparagine at position 779 decreases the enzymatic activity of DNMT3B toward the CpGpG site. The substitution of Lys777 or both Asn779 and Lys777 causes a decrease of the enzymatic activity of DNMT3B toward CpGpG site greater than that caused by Asn779 substitution ( Fig. 2(d) ). K777 is hence indicated to play a primary role and N779 a minor role in recognizing the flanking G residue, resulting in a sequence preference for CpGpG sites. The authors found that, compared to that in the apo DNMT3A structure, the TRD loop in the DNMT3B apo structure is mostly visible, indicating that the TRD loop of DNMT3B is more stable than that of DNMT3A ( Fig. 2(e) ). This condition raises a speculation that the TRD loop might contribute to processive methylation by DNMT3B. The TRD loop (residues 772–789) in the DNMT3B-DNA complex is stabilized by residue Gln772, which forms a hydrogen-bond network with Ile774, Ser780, Lys782, Gln783 and Gly784 in both protomers of the DNMT3B dimer ( Fig. 2(f) ). The TRD loop (residues 831–848) of DNMT3A is, however, partial- ly stabilized by residue Arg831, which forms a complete hydrogen-bonded network with Ile833, Ser839, Lys841 and Gln842 of only one of the two protomers ( Fig. 2(g) ). The