Stepwise assembly of chromatin during DNA replication in vitro.
Stepwise assembly of chromatin during DNA replication in vitro.
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DOI：
10.1002/j.1460-2075.1991.tb08031.x
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发表时间：
1991-04
期刊：
影响因子：
--
通讯作者：
Susan Smith;B. Stillman
中科院分区：
文献类型：
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作者：
Susan Smith;B. Stillman
一种支持依赖复制的染色质组装的无细胞系统已被用来确定DNA复制过程中组蛋白沉积的机制。CAF-I是一种人类细胞核因子，在胞浆复制粗提物存在的情况下，促进染色质在复制SV40 DNA上的组装。胞浆提取物的生化分级使染色质组装反应分成两个步骤。在第一步中，CAF-I将新合成的组蛋白H3和H4沉积到复制的DNA上。这种反应依赖于DNA复制，并与DNA复制结合在一起，并利用新合成的组蛋白H3和H4，与染色质中发现的块状组蛋白不同，组蛋白本身不与DNA结合。H3/H4复制的DNA复合体是一种稳定的中间体，具有微球菌核酸酶抗性结构，可通过蔗糖梯度沉淀法分离。在第二步中，复制的前体通过在DNA复制后发生的反应中加入组蛋白H_2A和H_2B来转化为成熟的染色质。对CaF-I的需求至少在反应的第一步表明了这一基本过程的细胞控制水平。
A cell free system that supports replication‐dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication. CAF‐I, a human cell nuclear factor, promotes chromatin assembly on replicating SV40 DNA in the presence of a crude cytosol replication extract. Biochemical fractionation of the cytosol extract has allowed separation of the chromatin assembly reaction into two steps. During the first step, CAF‐I targets the deposition of newly synthesized histones H3 and H4 to the replicating DNA. This reaction is dependent upon and coupled with DNA replication, and utilizes the newly synthesized forms of histones H3 and H4, which unlike bulk histone found in chromatin, do not bind to DNA by themselves. The H3/H4‐replicated DNA complex is a stable intermediate which exhibits a micrococcal nuclease resistant structure and can be isolated by sucrose gradient sedimentation. In the second step, this replicated precursor is converted to mature chromatin by the addition of histones H2A and H2B in a reaction that can occur after DNA replication. The requirement for CAF‐I in at least the first step of the reaction suggests a level of cellular control for this fundamental process.