Department of Physics, University of Washington, Seattle, WA 98195.
Department of Physics, Johns Hopkins University, Baltimore, MD 21218.
Proc Natl Acad Sci U S A. 2022 Sep 6;119(36):e2202489119. doi: 10.1073/pnas.2202489119. Epub 2022 Aug 29.
We used single-molecule picometer-resolution nanopore tweezers (SPRNT) to resolve the millisecond single-nucleotide steps of superfamily 1 helicase PcrA as it translocates on, or unwinds, several kilobase-long DNA molecules. We recorded more than two million enzyme steps under various assisting and opposing forces in diverse adenosine tri- and diphosphate conditions to comprehensively explore the mechanochemistry of PcrA motion. Forces applied in SPRNT mimic forces and physical barriers PcrA experiences , such as when the helicase encounters bound proteins or duplex DNA. We show how PcrA's kinetics change with such stimuli. SPRNT allows for direct association of the underlying DNA sequence with observed enzyme kinetics. Our data reveal that the underlying DNA sequence passing through the helicase strongly influences the kinetics during translocation and unwinding. Surprisingly, unwinding kinetics are not solely dominated by the base pairs being unwound. Instead, the sequence of the single-stranded DNA on which the PcrA walks determines much of the kinetics of unwinding.
我们使用单分子皮米分辨率纳米孔镊子（SPRNT）来解析超家族 1 解旋酶 PcrA 在移动或解旋数千碱基长的 DNA 分子时的毫秒级单核苷酸步骤。我们在不同的三磷酸腺苷和二磷酸腺苷条件下记录了超过两百万个酶步骤，以全面探索 PcrA 运动的机械化学。SPRNT 中施加的力模拟了 PcrA 所经历的力和物理障碍，例如当解旋酶遇到结合蛋白或双链 DNA 时。我们展示了 PcrA 的动力学如何随这些刺激而变化。SPRNT 允许将潜在的 DNA 序列与观察到的酶动力学直接关联起来。我们的数据表明，穿过解旋酶的潜在 DNA 序列强烈影响了在迁移和解旋过程中的动力学。令人惊讶的是，解旋动力学不仅仅由要解开的碱基对主导。相反，PcrA 行走的单链 DNA 的序列决定了解旋动力学的大部分。
