Pandey Shristi, Ogloblina Anna M, Belotserkovskii Boris P, Dolinnaya Nina G, Yakubovskaya Marianna G, Mirkin Sergei M, Hanawalt Philip C
Department of Biology, Stanford University, Stanford, CA 94305, USA.
Blokhin Cancer Research Center RAMS, Moscow, Russia.
Nucleic Acids Res. 2015 Aug 18;43(14):6994-7004. doi: 10.1093/nar/gkv622. Epub 2015 Jun 22.
DNA sequences that can form unusual secondary structures are implicated in regulating gene expression and causing genomic instability. H-palindromes are an important class of such DNA sequences that can form an intramolecular triplex structure, H-DNA. Within an H-palindrome, the H-DNA and canonical B-DNA are in a dynamic equilibrium that shifts toward H-DNA with increased negative supercoiling. The interplay between H- and B-DNA and the fact that the process of transcription affects supercoiling makes it difficult to elucidate the effects of H-DNA upon transcription. We constructed a stable structural analog of H-DNA that cannot flip into B-DNA, and studied the effects of this structure on transcription by T7 RNA polymerase in vitro. We found multiple transcription blockage sites adjacent to and within sequences engaged in this triplex structure. Triplex-mediated transcription blockage varied significantly with changes in ambient conditions: it was exacerbated in the presence of Mn(2+) or by increased concentrations of K(+) and Li(+). Analysis of the detailed pattern of the blockage suggests that RNA polymerase is sterically hindered by H-DNA and has difficulties in unwinding triplex DNA. The implications of these findings for the biological roles of triple-stranded DNA structures are discussed.
能够形成异常二级结构的DNA序列与基因表达调控及基因组不稳定性的产生有关。H-回文序列是这类DNA序列中的一个重要类别，它能够形成一种分子内三链结构，即H-DNA。在一个H-回文序列中，H-DNA与标准的B-DNA处于动态平衡，随着负超螺旋增加，平衡会向H-DNA方向移动。H-DNA与B-DNA之间的相互作用，以及转录过程会影响超螺旋这一事实，使得阐明H-DNA对转录的影响变得困难。我们构建了一种不能转变为B-DNA的H-DNA稳定结构类似物，并在体外研究了这种结构对T7 RNA聚合酶转录的影响。我们发现多个转录阻断位点位于参与这种三链结构的序列附近及内部。三链介导的转录阻断会随环境条件的变化而显著不同：在存在Mn(2+)时或K(+)和Li(+)浓度增加时会加剧。对阻断详细模式的分析表明，RNA聚合酶在空间上受到H-DNA的阻碍，难以解开三链DNA。本文讨论了这些发现对三链DNA结构生物学作用的意义。
