Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg S41296, Sweden.
Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):15179-84. doi: 10.1073/pnas.1213172109. Epub 2012 Sep 4.
Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60-70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271:792-794; Smith SB, Cui Y, Bustamante C (1996) Science 271:795-799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106:18231-18236]. Here, we resolve this controversy by using optical tweezers to extend small 60-64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.
混合序列 DNA 分子在 60-70 pN 下经历约 70%的机械拉伸。自 15 年前首次发现以来，关于该分子是否采用新形式[Cluzel P，等。（1996）科学 271:792-794; Smith SB，Cui Y，Bustamante C（1996）科学 271:795-799]，还是在张力下简单变性[van Mameren J，等。（2009）美国国家科学院院刊 106:18231-18236]，一直存在争议。在这里，我们使用光学镊子来扩展小的 60-64 bp 单 DNA 双链分子，其碱基含量可以随意设计，从而解决了这一争议。我们表明，当 AT 含量高（70%）时，在 62 pN 下会发生 DNA 螺旋的力诱导变性，伴随着分子的大约 70%的延伸。相比之下，富含 GC 的序列（60%GC）被发现经历可逆的过度拉伸转变为一种独特的形式，其特征是 51%的延伸，并且仍然碱基配对。首次可以呈现证明存在拉伸碱基配对形式的 DNA 的结果。在可逆转变中观察到的延伸与细菌 RecA 和人 Rad51 结合在 DNA 上产生的延伸相吻合，表明其在同源重组中可能具有相关性。
