Reconstitution and mechanism of the stimulation of de novo methylation by human DNMT3L
Reconstitution and mechanism of the stimulation of de novo methylation by human DNMT3L
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DOI：
10.1074/jbc.m603140200
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发表时间：
2006-09-08
影响因子：
4.8
通讯作者：
Chedin, Frederic
中科院分区：
文献类型：
--
作者：
Kareta, Michael S.;Botello, Zaida M.;Chedin, Frederic
DNMT 3样蛋白DNMT 3L是生殖系DNA甲基化所必需的，尽管它本身作为DNA甲基转移酶是无活性的。先前的研究表明，DNMT 3L与活性从头DNA甲基转移酶DNMT 3A和DNMT 3B物理缔合，并刺激它们在细胞培养系统中的催化活性。然而，DNMT 3L刺激从头甲基化的机制仍不清楚。在这里，我们已经纯化了全长人DNMT 3A 2和DNMT 3L蛋白，并确定了独特的条件，允许适当的重建DNMT 3L的DNMT 3A 2从头甲基转移酶活性的刺激。这些条件包括使用类似生理条件的缓冲液和两种蛋白质的预孵育。在这些条件下，在等摩尔量的DNMT 3L和DNMT 3A 2蛋白质时达到最大刺激，并且DNMT 3A 2的催化效率增加高达20倍。生化分析显示，尽管DNMT 3L本身不显著结合甲基供体S-腺苷-L-甲硫氨酸（SAM），但它强烈增加SAM与DNMT 3A 2的结合。相反，DNA结合没有明显改善。使用尺寸排阻色谱法分析溶液中的DNA甲基转移酶复合物显示，DNMT 3A 2形成异质尺寸的大结构，而DNMT 3L作为单体出现。DNMT 3L与DNMT 3A 2的结合促进了DNMT 3A 2亚基的显著重组，并导致形成具有增强的DNA甲基转移酶活性和增加的SAM结合的特异性复合物。
The DNMT3-like protein, DNMT3L, is required for germ line DNA methylation, although it is inactive as a DNA methyltransferase per se. Previous studies have shown that DNMT3L physically associates with the active de novo DNA methyltransferases, DNMT3A and DNMT3B, and stimulates their catalytic activities in a cell culture system. However, the mechanism by which DNMT3L stimulates de novo methylation remains unclear. Here, we have purified the full-length human DNMT3A2 and DNMT3L proteins and determined unique conditions that allow for the proper reconstitution of the stimulation of DNMT3A2 de novo methyltransferase activity by DNMT3L. These conditions include the use of buffers resembling physiological conditions and the preincubation of the two proteins. Under these conditions, maximal stimulation is reached at equimolar amounts of DNMT3L and DNMT3A2 proteins, and the catalytic efficiency of DNMT3A2 is increased up to 20-fold. Biochemical analysis revealed that whereas DNMT3L on its own does not significantly bind to the methyl group donor, S-adenosyl-L-methionine (SAM), it strongly increases the binding of SAM to DNMT3A2. DNA binding, on the contrary, was not appreciably improved. Analysis of DNA methyltransferase complexes in solution using size exclusion chromatography revealed that DNMT3A2 forms large structures of heterogeneous sizes, whereas DNMT3L appears as a monomer. Binding of DNMT3L to DNMT3A2 promotes a dramatic reorganization of DNMT3A2 subunits and leads to the formation of specific complexes with enhanced DNA methyltransferase activity and increased SAM binding.