Okoniewski Stephen R, Uyetake Lyle, Perkins Thomas T
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309-0440, USA.
Department of Physics, University of Colorado, Boulder, CO 80309-0440, USA.
Nucleic Acids Res. 2017 Oct 13;45(18):10775-10782. doi: 10.1093/nar/gkx761.
Single-molecule force spectroscopy provides insight into how proteins bind to and move along DNA. Such studies often embed a single-stranded (ss) DNA region within a longer double-stranded (ds) DNA molecule. Yet, producing these substrates remains laborious and inefficient, particularly when using the traditional three-way hybridization. Here, we developed a force-activated substrate that yields an internal 1000 nucleotide (nt) ssDNA region when pulled partially into the overstretching transition (∼65 pN) by engineering a 50%-GC segment to have no adjacent GC base pairs. Once the template was made, these substrates were efficiently prepared by polymerase chain reaction amplification followed by site-specific nicking. We also generated a more complex structure used in high-resolution helicase studies, a DNA hairpin adjacent to 33 nt of ssDNA. The temporally defined generation of individual hairpin substrates in the presence of RecQ helicase and saturating adenine triphosphate let us deduce that RecQ binds to ssDNA via a near diffusion-limited reaction. More broadly, these substrates enable the precise initiation of an important class of protein-DNA interactions.
单分子力谱技术有助于深入了解蛋白质如何与DNA结合并沿其移动。此类研究通常会在较长的双链（ds）DNA分子中嵌入一个单链（ss）DNA区域。然而，制备这些底物仍然费力且低效，尤其是使用传统的三链杂交时。在此，我们开发了一种力激活底物，通过设计一个50%的GC片段使其没有相邻的GC碱基对，当部分拉入微拉伸转变（约65 pN）时，该底物会产生一个内部1000个核苷酸（nt）的ssDNA区域。一旦制备好模板，通过聚合酶链反应扩增，然后进行位点特异性切口，就能高效地制备这些底物。我们还构建了一种用于高分辨率解旋酶研究的更复杂结构，即与33 nt的ssDNA相邻的DNA发夹结构。在RecQ解旋酶和饱和三磷酸腺苷存在的情况下，单个发夹底物的时间定义生成使我们推断出RecQ通过近乎扩散限制的反应与ssDNA结合。更广泛地说，这些底物能够精确启动一类重要的蛋白质 - DNA相互作用。
