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In Process Citation

机译：过程中引用

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Human topoisomerase I-B (Top1) efficiently relaxes DNA supercoils during basic cellular processes, and can be transformed into a DNA-damaging agent by antitumour drugs, enzyme mutations and DNA lesions. Here, we describe Gal4-Top1 chimeric proteins (GalTop) with an N-terminal truncation of Top1, and mutations of the Gal4 Zn-cluster and/or Top1 domains that impair their respective DNA-binding activities. Expression levels of chimeras were similar in yeast cells, however, GalTop conferred an increased CPT sensitivity to RAD52(-) yeast cells as compared to a GalTop with mutations of the Gal4 domain, showing that a functional Gal4 domain can alter in vivo functions of Top1. In vitro enzyme activity was tested with a DNA relaxation assay using negatively supercoiled plasmids with 0 to 5 Gal4 consensus motifs. Only GalTop with a functional Gal4 domain could direct DNA relaxation activity of Top1 specifically to DNA molecules containing Gal4 motifs. By using a substrate competition assay, we could demonstratethat the Gal4-anchored Top1 remains functional and efficiently relax DNA substrates in cis. The enhanced CPT sensitivity of GalTop in yeast cells may then be due to alterations of the chromatin-binding activity of Top1. The GalTop chimeras may indeed mimic a normal mechanism by which Top1 is recruited to chromatin sites in living cells. Such hybrid Top1s may be helpful in further dissecting enzyme functions, and constitute a prototype of a site-specific DNA cutter endowed with high cell lethality.
机译：人类拓扑异构酶I-B（Top1）在基本细胞过程中有效地放松了DNA超螺旋，并且可以通过抗肿瘤药，酶突变和DNA损伤转化为DNA破坏剂。在这里，我们描述了具有Top1的N端截断的Gal4-Top1嵌合蛋白（GalTop），以及损害其各自的DNA结合活性的Gal4 Zn簇和/或Top1域的突变。嵌合体在酵母细胞中的表达水平相似，但是，与具有Gal4结构域突变的GalTop相比，GalTop赋予RAD52（-）酵母细胞更高的CPT敏感性，表明功能性Gal4结构域可以改变Top1的体内功能。使用具有0至5个Gal4共有基序的负超螺旋质粒，通过DNA弛豫测定法测试了体外酶活性。只有具有功能性Gal4结构域的GalTop才能将Top1的DNA松弛活性直接导向含有Gal4图案的DNA分子。通过使用底物竞争测定，我们可以证明Gal4锚定的Top1保持功能性并有效地松弛顺式DNA底物。然后，酵母细胞中GalTop的CPT敏感性增强可能是由于Top1染色质结合活性的改变。 GalTop嵌合体确实可以模仿正常机制，通过该机制将Top1募集到活细胞的染色质位点。这种杂合的Top1可能有助于进一步分解酶的功能，并构成具有高细胞杀伤力的位点特异性DNA切割物的原型。

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