Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases.
Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases.
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DOI：
10.1093/nar/gkt696
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发表时间：
2013-11
影响因子：
14.9
通讯作者：
Fisher LM
中科院分区：
文献类型：
--
作者：
Arnoldi E;Pan XS;Fisher LM
抗菌氟喹诺酮类药物捕获促旋酶和拓扑异构酶（topo）IV的切割复合物，在参与DNA转运的弯曲DNA门内诱导位点特异性DNA断裂。尽管它对药物作用和揭示拓扑异构酶催化的潜在位点很重要，但DNA选择性的机制知之甚少。为了探索其功能基础，我们产生了突变版本的强烈切割的E-网站，并使用了一种新的竞争性测定来检查他们的gemifloxacin介导的DNA断裂肺炎链球菌topo IV和促旋酶。钙诱导切割的平行研究区分'内在识别'的DNA切割位点的拓扑异构酶IV从药物诱导的偏好。分析显示，断裂位点前的−4G、−2A和− 1 T碱基（在−1和+1之间）和−3处的酶独特或简并决定簇，以及+2/+3处的药物特异性偏好和与药物嵌入相关的+1嘌呤具有强烈的酶决定需求。在ColE 1衍生质粒中发现的新V位点处也观察到类似的切割规则。与DNA结合数据相一致，我们的研究结果提供了DNA，酶和药物对DNA切割的贡献在门的功能证据，提出了一种DNA歧视机制，涉及酶诱导的DNA弯曲/螺旋扭曲和切割复合物的稳定性和氟喹诺酮类药物作为重要的切割增强疗法的先进理解。
Antibacterial fluoroquinolones trap a cleavage complex of gyrase and topoisomerase (topo) IV inducing site-specific DNA breakage within a bent DNA gate engaged in DNA transport. Despite its importance for drug action and in revealing potential sites of topoisomerase catalysis, the mechanism of DNA selectivity is poorly understood. To explore its functional basis, we generated mutant versions of the strongly cleaved E-site and used a novel competitive assay to examine their gemifloxacin-mediated DNA breakage by Streptococcus pneumoniae topo IV and gyrase. Parallel studies of Ca2+-induced cleavage distinguished ‘intrinsic recognition’ of DNA cleavage sites by topo IV from drug-induced preferences. Analysis revealed strong enzyme-determined requirements for −4G, −2A and −1T bases preceding the breakage site (between −1 and +1) and enzyme-unique or degenerate determinants at −3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation. Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids. In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advance understanding of fluoroquinolones as important cleavage-enhancing therapeutics.
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DOI：
10.1038/nrc2607
发表时间：
2009-05
期刊：
Nature reviews. Cancer
影响因子：
--
作者：
通讯作者：
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影响因子：
3.7
作者：
Laponogov I;Pan XS;Veselkov DA;McAuley KE;Fisher LM;Sanderson MR
通讯作者：
Sanderson MR
影响因子：
5.6
作者：
LOCKSHON, D;MORRIS, DR
通讯作者：
MORRIS, DR
影响因子：
64.8
作者：
Dong, Ken C.;Berger, James M.
通讯作者：
Berger, James M.
DOI：
10.1038/nrc2608
发表时间：
2009-05
期刊：
Nature reviews. Cancer
影响因子：
--
作者：
通讯作者：
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