Department of Physics and the Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Nat Chem Biol. 2011 Jun;7(6):367-74. doi: 10.1038/nchembio.561. Epub 2011 May 8.
λ exonuclease degrades one strand of duplex DNA in the 5'-to-3' direction to generate a 3' overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure, and most studies have focused on the processive phase. Here we have used single-molecule fluorescence resonance energy transfer (FRET) to reveal three phases of λ exonuclease reactions: the initiation, distributive and processive phases. The distributive phase comprises early reactions in which the 3' overhang is too short to stably engage with the enzyme. A mismatched base is digested one-fifth as quickly as a Watson-Crick-paired base, and multiple concatenated mismatches have a cooperatively negative effect, highlighting the crucial role of base pairing in aligning the 5' end toward the active site. The rate-limiting step during processive degradation seems to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.
λ 核酸外切酶从 5' 到 3' 方向降解双链 DNA 的一条链，产生重组所需的 3' 突出端。其能够进行几千个核苷酸的连续水解归因于其环状结构，大多数研究都集中在连续阶段。在这里，我们使用单分子荧光共振能量转移 (FRET) 来揭示 λ 核酸外切酶反应的三个阶段：起始阶段、分布阶段和连续阶段。分布阶段包括早期反应，其中 3' 突出端太短而无法与酶稳定结合。一个错配碱基的消化速度比 Watson-Crick 配对碱基快五分之一，而多个串联错配碱基具有协同的负效应，突出了碱基配对在将 5' 末端对齐到活性位点方面的关键作用。在连续降解过程中，限速步骤似乎是末端碱基对的酶切后解链。我们还发现，从已知的暂停序列中逃脱需要酶回溯。
