Norvil Allison B, Petell Christopher J, Alabdi Lama, Wu Lanchen, Rossie Sandra, Gowher Humaira
Department of Biochemistry, Purdue University Center for Cancer Research , Purdue University , West Lafayette , Indiana 47907 , United States.
Biochemistry. 2018 Jul 24;57(29):4312-4324. doi: 10.1021/acs.biochem.6b00964. Epub 2016 Nov 4.
The catalytic domains of the de novo DNA methyltransferases Dnmt3a-C and Dnmt3b-C are highly homologous. However, their unique biochemical properties could potentially contribute to differences in the substrate preferences or biological functions of these enzymes. Dnmt3a-C forms tetramers through interactions at the dimer interface, which also promote multimerization on DNA and cooperativity. Similar to the case for processive enzymes, cooperativity allows Dnmt3a-C to methylate multiple sites on the same DNA molecule; however, it is unclear whether Dnmt3b-C methylates DNA by a cooperative or processive mechanism. The importance of the tetramer structure and cooperative mechanism is emphasized by the observation that the R882H mutation in the dimer interface of DNMT3A is highly prevalent in acute myeloid leukemia and leads to a substantial loss of its activity. Under conditions that distinguish between cooperativity and processivity, we show that in contrast to that of Dnmt3a-C, the activity of Dnmt3b-C is not cooperative and confirm the processivity of Dnmt3b-C and the full length Dnmt3b enzyme. Whereas the R878H mutation (mouse homologue of R882H) led to the loss of cooperativity of Dnmt3a-C, the activity and processivity of the analogous Dnmt3b-C R829H variant were comparable to those of the wild-type enzyme. Additionally, buffer acidification that attenuates the dimer interface interactions of Dnmt3a-C had no effect on Dnmt3b-C activity. Taken together, these results demonstrate an important mechanistic difference between Dnmt3b and Dnmt3a and suggest that interactions at the dimer interface may play a limited role in regulating Dnmt3b-C activity. These new insights have potential implications for the distinct biological roles of Dnmt3a and Dnmt3b.
从头合成DNA甲基转移酶Dnmt3a-C和Dnmt3b-C的催化结构域高度同源。然而，它们独特的生化特性可能导致这些酶在底物偏好或生物学功能上存在差异。Dnmt3a-C通过二聚体界面处的相互作用形成四聚体，这也促进了在DNA上的多聚化和协同作用。与持续性酶的情况类似，协同作用使Dnmt3a-C能够甲基化同一DNA分子上的多个位点；然而，尚不清楚Dnmt3b-C是否通过协同或持续性机制甲基化DNA。DNMT3A二聚体界面处的R882H突变在急性髓系白血病中高度常见，并导致其活性大幅丧失，这一观察结果强调了四聚体结构和协同机制的重要性。在区分协同作用和持续性的条件下，我们发现与Dnmt3a-C不同，Dnmt3b-C的活性不具有协同性，并证实了Dnmt3b-C和全长Dnmt3b酶的持续性。虽然R878H突变（R882H的小鼠同源物）导致Dnmt3a-C失去协同性，但类似的Dnmt3b-C R829H变体的活性和持续性与野生型酶相当。此外，缓冲液酸化减弱了Dnmt3a-C的二聚体界面相互作用，但对Dnmt3b-C活性没有影响。综上所述，这些结果证明了Dnmt3b和Dnmt3a之间存在重要的机制差异，并表明二聚体界面处的相互作用在调节Dnmt3b-C活性方面可能起有限作用。这些新见解对Dnmt3a和Dnmt3b不同的生物学作用具有潜在影响。
