The DNMT3A R882H mutation is frequently observed in acute myeloid leukemia (AML). It is located in the subunit and DNA binding interface of DNMT3A and has been reported to cause a reduction in activity and dominant negative effects. We investigated the mechanistic consequences of the R882H mutation on DNMT3A showing a roughly 40% reduction in overall DNA methylation activity. Biochemical assays demonstrated that R882H does not change DNA binding affinity, protein stability or subnuclear distribution of DNMT3A. Strikingly, DNA methylation experiments revealed pronounced changes in the flanking sequence preference of the DNMT3A-R882H mutant. Based on these results, different DNA substrates with selected flanking sequences were designed to be favored or disfavored by R882H. Kinetic analyses showed that the R882H favored substrate was methylated by R882H with 45% increased rate when compared with wildtype DNMT3A, while methylation of the disfavored substrate was reduced 7-fold. Our data expand the model of the potential carcinogenic effect of the R882H mutation by showing CpG site specific activity changes. This result suggests that R882 is involved in the indirect readout of flanking sequence preferences of DNMT3A and it may explain the particular enrichment of the R882H mutation in cancer patients by revealing mutation specific effects.

中文翻译：

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DNMT3A R882H突变体显示改变的侧翼序列偏好

DNMT3A R882H突变经常在急性髓细胞性白血病（AML）中观察到。它位于DNMT3A的亚基和DNA结合界面，据报道会导致活性降低和显性负作用。我们研究了R882H突变对DNMT3A的机械作用，显示总体DNA甲基化活性降低了约40％。生化分析表明，R882H不会改变DNMT3A的DNA结合亲和力，蛋白质稳定性或亚核分布。令人惊讶的是，DNA甲基化实验揭示了DNMT3A-R882H突变体侧翼序列偏好的明显变化。基于这些结果，设计了具有选定侧翼序列的不同DNA底物，以使其受到R882H的支持或不利。动力学分析表明，与野生型DNMT3A相比，R882H对R882H有利的底物进行了甲基化，增加了45％，而不利的底物的甲基化减少了7倍。我们的数据通过显示CpG位点比活性变化，扩展了R882H突变潜在致癌作用的模型。该结果表明R882参与了DNMT3A侧翼序列偏好的间接读出，并且可以通过揭示突变特异性作用来解释癌症患者中R882H突变的特别富集。