Higurashi M, Cole R D
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Biol Chem. 1991 May 5;266(13):8619-25.
To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.
为了研究DNA甲基化和H1组蛋白在调节DNA结合蛋白作用方面的潜力，通过对H1组蛋白与甲基化或非甲基化DNA混合物进行缓慢盐梯度透析，形成了排列有序的复合物。质粒中被甲基化的位点是CCGG。该位点的胞嘧啶甲基化可保护DNA免受HpaII核酸内切酶的切割，但不能免受MspI的切割。然而，当甲基化DNA与H1形成复合物时，它能免受MspI的切割。这种保护仅对MspI限制位点的一个子集有效。H1结合与DNA甲基化相结合对DNA的保护不适用于EcoRI、PstI或BamHI位点，因此似乎不是由于H1组蛋白使DNA聚集所致。凝胶阻滞分析表明，H1对甲基化DNA的亲和力与其对非甲基化DNA的亲和力没有明显差异。可能与H1结合的甲基化DNA处于一种对MspI核酸内切酶有抗性的构象。由DNA甲基化和H1结合诱导的这种构象变化可能会影响其他DNA结合蛋白的作用，也许在染色质以及H1-DNA复合物中也是如此。
