Activation of a human chromosomal replication origin by protein tethering.
Activation of a human chromosomal replication origin by protein tethering.
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DOI：
10.1093/nar/gkt368
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发表时间：
2013-07
影响因子：
14.9
通讯作者：
Leffak M
中科院分区：
文献类型：
--
作者：
Chen X;Liu G;Leffak M
哺乳动物染色体复制起始点的具体情况还不完全清楚。为了分析复制前复合体(Pre-RCS)的组装和激活，我们测试了染色质乙酰转移酶和复制蛋白的拴系结合对含有GAL4结合盒的染色体c-myc起源缺失突变体的影响。GAL4DBD(DNA结合域)与Orc2、CDT1、E2F1或HBO1融合后，可协调MCM7解旋酶亚基、DNA解离元件(DUE)结合蛋白DUE-B和微小染色体维持(MCM)解旋酶激活剂CDC45与复制子的招募，并恢复起始活性。相反，在没有c-myc DNA侧翼的情况下，融合蛋白结合不能刺激复制蛋白结合和起始活性。用GAL4结合位点取代c-myc复制子DUE允许Orc2和MCM7结合，但消除了起始活性，表明DUE对于Pre-RC激活是必不可少的。此外，DUE-B的拴系不足以招募CDC45或激活在缺乏DUE的情况下形成的前RCS。这些结果直接表明在染色体背景下，染色质乙酰化、Orc2或CDT1足以将所有下游复制起始活动招募到预期的起源，而染色体起源的活性需要单一的DNA序列。
The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences.
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影响因子：
14.9
作者：
Dorn, Elizabeth S.;Chastain, Paul D., II;Hall, Jonathan R.;Cook, Jeanette Gowen
通讯作者：
Cook, Jeanette Gowen
影响因子：
7
作者：
Bieda, M;Xu, XQ;Farnham, PJ
通讯作者：
Farnham, PJ
影响因子：
10.5
作者：
Eaton, Matthew L.;Galani, Kyriaki;MacAlpine, David M.
通讯作者：
MacAlpine, David M.
DOI：
10.1073/pnas.0911500106
发表时间：
2009-12-01
影响因子：
11.1
作者：
Evrin, Cecile;Clarke, Pippa;Speck, Christian
通讯作者：
Speck, Christian
影响因子：
4.8
作者：
Burke, TW;Cook, JG;Nevins, JR
通讯作者：
Nevins, JR