Disease-Associated Mutation A554V Disrupts Normal Autoinhibition of DNMT1.
Disease-Associated Mutation A554V Disrupts Normal Autoinhibition of DNMT1.
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DOI：
10.3390/dna3030010
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发表时间：
2023-09
期刊：
影响因子：
--
通讯作者：
Carroll, Clara F
中科院分区：
文献类型：
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作者：
Switzer, Rebecca L;Hartman, Zach J;Hewett, Geoffrey R;Carroll, Clara F
DNA甲基转移酶1（DNMT 1）是主要负责细胞中甲基化模式传播的酶。DNMT 1的突变与成人发病的神经退行性疾病的发展有关;这些疾病相关的突变发生在蛋白质的调节复制焦点靶向序列（RFTS）结构域中。RFTS结构域是DNMT 1活性的内源性抑制剂，其结合活性位点并阻止DNA结合。在这里，我们研究了疾病相关突变A554 V对正常RFTS介导的DNMT 1抑制的影响。表达野生型和突变体蛋白并纯化至均一性用于生物化学表征。该突变使DNA结合亲和力增加约8倍。此外，突变酶表现出增加的DNA甲基化活性。圆二色性（CD）光谱显示，突变不显着影响二级结构或相对热稳定性的分离的RFTS域。然而，突变导致在较大的蛋白质的背景下，在CD光谱的变化，也观察到相对热稳定性下降。总的来说，这些证据表明，A554 V破坏了正常RFTS介导的DNMT 1的自抑制，导致过度活跃的突变酶。虽然疾病相关突变不会显著影响分离的RFTS结构域，但突变导致结构域间稳定相互作用的减弱，从而产生DNMT 1的更开放的活性构象。过度活跃的突变体DNMT 1可能是受影响个体中观察到的DNA甲基化增加的原因。
DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals.