Graham James E, Marians Kenneth J, Kowalczykowski Stephen C
Department of Microbiology and Molecular Genetics and Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA.
Molecular Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
Cell. 2017 Jun 15;169(7):1201-1213.e17. doi: 10.1016/j.cell.2017.05.041.
It has been assumed that DNA synthesis by the leading- and lagging-strand polymerases in the replisome must be coordinated to avoid the formation of significant gaps in the nascent strands. Using real-time single-molecule analysis, we establish that leading- and lagging-strand DNA polymerases function independently within a single replisome. Although average rates of DNA synthesis on leading and lagging strands are similar, individual trajectories of both DNA polymerases display stochastically switchable rates of synthesis interspersed with distinct pauses. DNA unwinding by the replicative helicase may continue during such pauses, but a self-governing mechanism, where helicase speed is reduced by ∼80%, permits recoupling of polymerase to helicase. These features imply a more dynamic, kinetically discontinuous replication process, wherein contacts within the replisome are continually broken and reformed. We conclude that the stochastic behavior of replisome components ensures complete DNA duplication without requiring coordination of leading- and lagging-strand synthesis. PAPERCLIP.
人们一直认为，复制体中前导链和后随链聚合酶的DNA合成必须协调，以避免新生链中形成明显的缺口。通过实时单分子分析，我们确定前导链和后随链DNA聚合酶在单个复制体内独立发挥作用。尽管前导链和后随链上DNA合成的平均速率相似，但两种DNA聚合酶的个体轨迹都显示出合成速率可随机切换，并穿插着明显的停顿。在这种停顿期间，复制解旋酶的DNA解旋可能会继续，但一种自我调节机制（其中解旋酶速度降低约80%）允许聚合酶与解旋酶重新耦合。这些特征意味着一个更动态、动力学上不连续的复制过程，其中复制体内的接触不断被打破和重新形成。我们得出结论，复制体组件的随机行为可确保DNA完全复制，而无需前导链和后随链合成的协调。回形针。
