Distinguishing the Roles of Topoisomerases I and II in Relief of Transcription-Induced Torsional Stress in Yeast rRNA Genes
Distinguishing the Roles of Topoisomerases I and II in Relief of Transcription-Induced Torsional Stress in Yeast rRNA Genes
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DOI：
10.1128/mcb.00589-10
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发表时间：
2011-02-01
影响因子：
5.3
通讯作者：
Beyer, Ann L.
中科院分区：
文献类型：
--
作者：
French, Sarah L.;Sikes, Martha L.;Beyer, Ann L.
为了更好地了解拓扑异构酶活性在缓解转录诱导的超级卷曲中的作用，在两种主要拓扑异构酶中的一种或两种缺失的细胞中显示了编码rRNA的酵母基因。在缺乏拓扑异构酶I(Top1)和拓扑异构酶II(TOP2)活性的情况下，加工能力严重受损，聚合酶无法通过6.7-kb基因进行转录。Top1的缺失导致显著的rRNA基因亚群中负超螺旋密度的增加(是正常值的2到6倍)，表现为DNA模板融化的区域。观察到的DNA气泡不是R-环，也没有阻止聚合酶的移动，因为DNA模板融化的基因没有显示出延长减慢的证据。然而，TOP2的失活导致rRNA基因伸长放缓的特征迹象，表明TOP2减轻了转录诱导的正超卷曲。总而言之，这些数据表明，转录聚合酶I前后的扭转具有不同的后果和不同的分辨率。聚合酶前面的正扭转导致超级卷曲(扭动)，并在很大程度上被TOP2解决。聚合酶后面的负扭转导致DNA链分离，并在很大程度上被Top1解决。
To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.