Christman J K, Schneiderman N, Acs G
J Biol Chem. 1985 Apr 10;260(7):4059-68.
Incubation of 5-azacytosine-substituted DNA ([5-aza-C]DNA) with nuclear proteins leads to the formation of highly stable DNA . protein complexes which remain intact in the presence of 1 M NaCl and/or 0.6% Sarkosyl. The proteins involved in binding double-stranded [5-aza-C]DNA in these stable complexes comprise a specific subset of non-histone nuclear proteins that includes DNA methyltransferase. Complex formation does not require S-adenosylmethionine and does not involve covalent linkage of protein to DNA or modification of 5-azacytosine residues. Non-histone nuclear proteins do not form complexes with double-stranded unsubstituted DNA that are resistant to dissociation with NaCl and Sarkosyl but are capable of forming such complexes with single-stranded DNA regardless of whether it contains 5-azacytosine residues or not. However, it can be demonstrated 1) that single-stranded regions do not account for stable binding of proteins to native [5-aza-C]DNA and 2) that many nuclear proteins which form stable complexes with single-stranded DNA are incapable of forming such complexes with double-stranded [5-aza-C]DNA. Synthesis of [5-aza-C]DNA by cells growing in the presence of either 5-azacytidine or 5-aza-2'-deoxycytidine leads to rapid loss of extractable DNA methyltransferase (Creusot, F., Acs, G., and Christman, J.K. (1982) J. Biol. Chem. 257, 2041-2048). Analogous depletion of non-histone nuclear proteins capable of forming stable complexes with [5-aza-C]DNA in vitro is observed, suggesting that the same proteins can form highly stable complexes with [5-aza-C]DNA in vitro and in vivo. Formation of stable complexes between non-histone nuclear proteins and [5-aza-C]DNA could potentially affect not only the activity of DNA methyltransferase but the action of other regulatory proteins or enzymes that interact with DNA. Such interactions could explain effects of 5-azacytidine on gene expression that cannot be directly linked to loss of methyl groups from DNA.
5-氮杂胞嘧啶取代的DNA（[5-氮杂-C]DNA）与核蛋白一起温育会导致形成高度稳定的DNA-蛋白质复合物，这些复合物在1M氯化钠和/或0.6%十二烷基肌氨酸钠存在的情况下仍保持完整。参与在这些稳定复合物中结合双链[5-氮杂-C]DNA的蛋白质包括非组蛋白核蛋白的一个特定子集，其中包括DNA甲基转移酶。复合物的形成不需要S-腺苷甲硫氨酸，也不涉及蛋白质与DNA的共价连接或5-氮杂胞嘧啶残基的修饰。非组蛋白核蛋白不会与双链未取代的DNA形成对氯化钠和十二烷基肌氨酸钠解离有抗性的复合物，但无论其是否含有5-氮杂胞嘧啶残基，都能够与单链DNA形成这样的复合物。然而，可以证明：1）单链区域不能解释蛋白质与天然[5-氮杂-C]DNA的稳定结合；2）许多与单链DNA形成稳定复合物的核蛋白不能与双链[5-氮杂-C]DNA形成这样的复合物。在5-氮杂胞苷或5-氮杂-2'-脱氧胞苷存在下生长的细胞合成[5-氮杂-C]DNA会导致可提取的DNA甲基转移酶迅速丧失（克勒索，F.，阿克斯，G.，和克里斯曼，J.K.（1982年）《生物化学杂志》257，2041 - 2048）。观察到在体外能够与[5-氮杂-C]DNA形成稳定复合物的非组蛋白核蛋白有类似的消耗，这表明相同的蛋白质在体外和体内都能与[5-氮杂-C]DNA形成高度稳定的复合物。非组蛋白核蛋白与[5-氮杂-C]DNA之间稳定复合物的形成不仅可能影响DNA甲基转移酶的活性，还可能影响与DNA相互作用的其他调节蛋白或酶的作用。这样的相互作用可以解释5-氮杂胞苷对基因表达的影响，而这些影响不能直接与DNA甲基基团的丧失联系起来。
