PKD1 intron 21: triplex DNA formation and effect on replication.
PKD1 intron 21: triplex DNA formation and effect on replication.
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PKD1 内含子 21：三链体 DNA 形成及其对复制的影响。
DOI：
10.1093/nar/gkh312
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
Bissler,JohnJ
中科院分区：
文献类型：
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作者：
Patel,HirenP;Lu,Lu;Blaszak,RichardT;Bissler,JohnJ
虽然常染色体显性遗传性多囊肾病是以常染色体显性遗传方式传播的，但有证据表明，囊肿发生的病理生理学涉及肾小管细胞内遗传性种系突变叠加的第二次击中体细胞突变。PKD 1内含子21的多嘌呤·多嘧啶（Pu·Py）序列可能在促进体细胞突变中起作用。为了更好地表征该片段并评估其参与诱变的潜力，我们采用二维凝胶电泳技术研究了PKD1内含子21的15个Pu·Py镜像重复片段形成分子内三链体的热力学。我们证明，分子内三链体的形式与适度的超螺旋张力检查所有的道。引物延伸研究表明Pu·Py束内的聚合酶仅在一个复制方向上显著停滞。我们发现聚合停滞和潜在的长度的三链体和超螺旋张力的分子内三链体形成之间的相关性。使用HeLa细胞提取物进行的SV40体外复制试验显示，Pu·Py束的存在也导致复制阻断和双链断裂。在DNA复制过程中，PKD1 Pu·Py片段的富含G的模板可能与新生链形成三链体结构，从而阻断复制并可能导致重组和突变。
Although autosomal dominant polycystic kidney disease is transmitted in an autosomal dominant fashion, there is evidence that the pathophysiology of cystogenesis involves a second hit somatic mutation superimposed upon the inherited germline mutation within the renal tubule cells. The polypurine·polypyrimidine (Pu·Py) tract ofPKD1intron 21 may play a role in promoting somatic mutations. To better characterize this tract and to evaluate its potential to participate in mutagenesis, we investigated the thermodynamics of intramolecular triplex formation by 15 Pu·Py mirror repeat tracts fromPKD1intron 21 by 2D gel electrophoresis. We demonstrate that intramolecular triplexes form with modest superhelical tensions for all the tracts examined. Primer extension studies demonstrated significant polymerase arrest within the Pu·Py tracts in one direction of replication only. We found correlation between polymerization arrest and both the potential length of the triplex and superhelical tension of intramolecular triplex formation. The presence of a Pu·Py tract also led to a replication blockade and double‐strand breakage using an SV40in vitroreplication assay with HeLa cell extracts. During DNA replication, the G‐rich template of the PKD1 Pu·Py tracts may form a triplex structure with the nascent strand, thereby blocking replication and potentially leading to recombination and mutation.