The DNMT3A R882H mutant displays altered flanking sequence preferences.
The DNMT3A R882H mutant displays altered flanking sequence preferences.
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DOI：
10.1093/nar/gky168
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发表时间：
2018-04-06
影响因子：
14.9
通讯作者：
Jeltsch A
中科院分区：
文献类型：
--
作者：
Emperle M;Rajavelu A;Kunert S;Arimondo PB;Reinhardt R;Jurkowska RZ;Jeltsch A
DNMT3A R882H突变常见于急性髓性白血病（AML）。它位于DNMT3A的亚基和DNA结合界面上，据报道会导致活性降低和主要的负面影响。我们研究了R882H突变对DNMT3A的机制后果，显示总体DNA甲基化活性降低了大约40%。生化实验表明，R882H不会改变DNMT3A的DNA结合亲和力、蛋白质稳定性或亚核分布。引人注目的是，DNA甲基化实验显示，DNMT3A-R882H突变体的侧翼序列偏好发生了显著变化。基于这些结果，设计了不同的DNA底物，并选择了不同的侧翼序列，使其对R882H有利或不利。动力学分析表明，与野生型DNMT3A相比，R882H对R882H有利的底物的甲基化率提高了45%，而对R882H不利的底物的甲基化率降低了7倍。我们的数据通过显示CpG位点特异性活性的变化，扩展了R882H突变潜在致癌作用的模型。这一结果表明，R882参与了DNMT3A侧翼序列偏好的间接读取，并可能通过揭示突变特异性效应来解释R882H突变在癌症患者中的特殊富集。
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.
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