Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping.
Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping.
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DOI：
10.1016/j.jsb.2008.08.006
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发表时间：
2008-12
影响因子：
3
通讯作者：
Nogales, Eva
中科院分区：
文献类型：
--
作者：
Clarey, Megan G.;Botchan, Michael;Nogales, Eva
关键词：
细胞周期蛋白依赖性蛋白激酶(CDKs)使果蝇黑腹果蝇起源识别复合体(DmORC)过度磷酸化，允许核苷酸结合，但抑制ORC1的ATPase活性，并阻断ORC与DNA的依赖于ATP的相互作用。在这里，我们介绍了ORC与核苷酸结合的单粒子电子显微镜(EM)研究，包括去磷酸化和高磷酸化状态。三维图像重建显示，核苷酸结合导致类似的构象独立于磷酸化状态。在我们研究中达到的中等分辨率下，ATP促进了沿着复合体环状核心的变化，磷酸化贡献的差异可以忽略不计。因此，DmORC的过度磷酸化不会导致ORC结构的中观重排。为了更好地了解ORC在起源重塑中的作用，我们进行了原子力显微镜(AFM)研究，结果表明，当ORC结合时，688bp线性DNA片段的轮廓长度缩短了相当于~130bp。这一数据，再加上之前的研究表明，ORC结合时环状DNA的连接数发生了变化，表明ORC可能以类似于DNAA的方式包裹DNA。根据现有的数据和我们的结构，我们提出了AAA+和有翼螺旋结构域的亚基排列，并推测了ORC复合体周围133bp的DNA的路径。
Hyperphosphorylation of the Drosophila melanogaster origin recognition complex (DmORC) by cyclin dependent kinases (CDKs) allows nucleotide binding but inhibits the ATPase activity of Orc1, and ablates the ATP-dependent interaction of ORC with DNA. Here we present single particle electron microscopy (EM) studies of ORC bound to nucleotide in both the dephosphorylated and hyper-phosphorylated states. 3D image reconstructions show that nucleotide binding gives rise to an analogous conformation independent of phosphorylation state. At the intermediate resolution achieved in our studies, ATP promotes changes along the toroidal core of the complex with negligible differences contributed by phosphorylation. Thus, hyperphosphorylation of DmORC does not induce meso-scale rearrangement of the ORC structure. To better understand ORC’s role in origin remodeling, we performed atomic force microscopy (AFM) studies that show the contour length of a 688 bp linear DNA fragment shortens by the equivalent of ~130 bp upon ORC binding. This data, coupled with previous studies that showed a linking number change in circular DNA upon ORC binding, suggests that ORC may wrap the DNA in a manner akin to DnaA. Based on existing data and our structures, we propose a subunit arrangement for the AAA+ and winged helix domains, and in addition, speculate on a path of the 133 bp of DNA around the ORC complex.
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DOI：
10.1046/j.1432-1327.1999.00613.x
发表时间：
1999-09-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Findeisen, M;El-Denary, M;Strausfeld, U
通讯作者：
Strausfeld, U
影响因子：
56.9
作者：
GOSSEN, M;PAK, DTS;BOTCHAN, MR
通讯作者：
BOTCHAN, MR
影响因子：
64.8
作者：
Nguyen, VQ;Co, C;Li, JJ
通讯作者：
Li, JJ
DOI：
10.1073/pnas.94.11.5611
发表时间：
1997-05-27
影响因子：
11.1
作者：
Donovan, S;Harwood, J;Diffley, JFX
通讯作者：
Diffley, JFX
影响因子：
56.9
作者：
Dueber, Erin L. Cunningham;Corn, Jacob E.;Berger, James M.
通讯作者：
Berger, James M.