Gening Leonid V, Klincheva Svetlana A, Reshetnjak Anastasia, Grollman Arthur P, Miller Holly
Laboratory of Chemical Biology, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651, USA.
Nucleic Acids Res. 2006 May 17;34(9):2579-86. doi: 10.1093/nar/gkl326. Print 2006.
DNA polymerase beta (polbeta), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polbeta from a variable pool of 8 x 10(12) individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polbeta activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polbeta active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polbeta also inhibited DNA polymerase kappa, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polbeta and kappa, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase activity in cells.
DNA聚合酶β（polβ）是DNA聚合酶X家族的成员，是碱基切除修复途径中的主要聚合酶。通过体外筛选，我们从一个包含30个随机核苷酸的8×10¹²个个体RNA序列的可变文库中获得了针对polβ的RNA适配体。对经过七轮筛选后选出的60个个体克隆进行了抑制polβ活性能力的筛选。所有分析的抑制性适配体都具有预测的三叶结构。凝胶迁移率变动分析表明，这些适配体可以从polβ活性位点取代DNA底物。适配体的抑制作用并非聚合酶特异性的；polβ的抑制剂也抑制DNA聚合酶κ，一种Y家族DNA聚合酶。然而，RNA适配体不抑制DNA聚合酶I的Klenow片段，并且对RB69 DNA聚合酶活性只有轻微影响。尽管polβ和κ的序列相似性很小且属于不同的DNA聚合酶家族，但它们具有相似的开放活性位点，并且与它们的DNA底物的相互作用相对较少。这可能使适配体能够结合并抑制聚合酶活性。具有抑制特性的RNA适配体可能有助于调节细胞中的DNA聚合酶活性。
