DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding
DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding
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DOI：
10.1038/sj.emboj.7600077
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发表时间：
2004-02-25
期刊：
影响因子：
11.4
通讯作者：
Botchan, MR
中科院分区：
文献类型：
--
作者：
Remus, D;Beall, EL;Botchan, MR
果蝇的起源识别复合体（ORC）定位于染色体上的特定位置，卵泡细胞中的绒毛膜基因扩增位点是研究得很好的例子。然而，特异性定位的机制尚不清楚。我们研究了DmORC的DNA结合，以探讨DmORC与DNA相互作用的顺式要求。在体外实验中，DmORC与来自第三绒毛膜位点的DNA和随机片段的相对亲和力最多有6倍的差异，化学探测和DNase1保护实验没有在这些片段上发现DmORC的离散结合位点。因此，DmORC固有的dna结合特异性不足以将DmORC靶向到体内复制的起点。然而，DNA的拓扑状态显著影响DmORC对DNA的亲和力。我们发现DmORC对负超螺旋DNA的亲和力比对松弛或线性DNA的亲和力高约30倍。这些数据提供了生物化学证据，证明后生动物的起源规范可能涉及复制子-启动子相互作用之外的其他机制，并且体内其他蛋白质必须决定ORC的定位。
Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction. DmORC displays at best six-fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA-binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30-fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator-initiator interactions and that in vivo other proteins must determine ORC's localization.