DNA sequence bias during Tn5 transposition
DNA sequence bias during Tn5 transposition
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DOI：
10.1016/j.jmb.2003.11.039
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发表时间：
2004-01-30
影响因子：
5.6
通讯作者：
Reznikoff, WS
中科院分区：
文献类型：
--
作者：
Ason, B;Reznikoff, WS
转座是导致基因组不稳定的主要机制之一。这种现象与其他DNA重排如V（D）J重组和逆转录病毒DNA整合有关。在Tn 5系统中，只有一种蛋白质，即转座酶（Tnp），是转座子运动中所有催化步骤所必需的。在一个活性位点内移动多条DNA链的复杂性表明，除了Tnp与其识别序列之间保持的特异性接触外，Tnp还与侧翼DNA序列相互作用。在这里，我们证明了Tnp与供体DNA区域相互作用。Tnp保护供体DNA免受DNA酶I消化，表明Tnp在突触期间与供体DNA接触或以其他方式扭曲供体DNA。此外，供体DNA序列在这一区域内的变化改变了Tnp对DNA的亲和力，在突触过程中增加了8倍。在体外选择更稳定的突触复合体揭示了该地区的A/T序列的偏见。我们进一步表明，某些捐助者的DNA序列，有利于突触，也似乎作为热点链转移。TTATA供体序列代表最佳位点。最令人惊讶的事实是，该序列在Tnp识别序列内被发现。优先插入Tnp识别序列内的位点将有效地将元件的一个拷贝插入并形成Tn 5转座子的簇。此外，事实上，几个供体DNA序列，有利于突触，似乎作为转座子插入的热点表明，类似的标准可能存在的Tnp-供体DNA和Tnp-靶DNA的相互作用。(C)2003 Elsevier Ltd.保留所有权利。
Transposition is one of the primary mechanisms causing genome instability. This phenomenon is mechanistically related to other DNA rearrangements such as V(D)J recombination and retroviral DNA integration. In the Tn5 system, only one protein, the transposase (Tnp), is required for all of the catalytic steps involved in transposon movement. The complexity involved in moving multiple DNA strands within one active site suggests that, in addition to the specific contacts maintained between Tnp and its recognition sequence, Tnp also interacts with the flanking DNA sequence. Here, we demonstrate that Tnp interacts with the donor DNA region. Tnp protects the donor DNA from DNase I digestion, suggesting that Tnp is in contact with, or otherwise distorts, the donor DNA during synapsis. In addition, changes in the donor DNA sequence within this region alter the affinity of Tnp for DNA by eightfold during synapsis. In vitro selection for more stable synaptic complexes reveals an A/T sequence bias for this region. We further show that certain donor DNA sequences, which favor synapsis, also appear to serve as hot spots for strand transfer. The TTATA donor sequence represents the best site. Most surprising is the fact that this sequence is found within the Tnp recognition sequence. Preference for insertion into a site within the Tnp recognition sequence would effectively inactivate one copy of the element and form clusters of the Tn5 transposon. In addition, the fact that several donor DNA sequences, which favor synapsis, appear to serve as hot spots for transposon insertion suggest that similar criteria may exist for Tnp-donor DNA and Tnp-target DNA interactions. (C) 2003 Elsevier Ltd. All rights reserved.