Single-molecule Förster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I.
Single-molecule Förster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I.
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DOI：
10.1021/ja3038273
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发表时间：
2012-07-11
影响因子：
15
通讯作者：
Millar, David P.
中科院分区：
文献类型：
--
作者：
Berezhna, Svitlana Y.;Gill, Joshua P.;Lamichhane, Rajan;Millar, David P.
酶促反应通常涉及底物结合、构象重排、化学反应和产物释放过程中的复杂动力学。非共价步骤提供动力学检查点，有助于酶促反应的总体特异性。DNA聚合酶通过在反应途径的早期主动排斥非同源核苷酸底物，以出色的保真度进行DNA复制。当酶从开放构象转化为封闭构象时，底物通过酶的柔性手指亚结构域传递到活性位点。手指子结构域的构象动力学也可能在核苷酸选择中起作用，尽管确切的作用目前尚不清楚。利用单分子Förster共振能量转移，我们观察到单个大肠杆菌DNA聚合酶I （Klenow片段）分子进行底物选择。我们发现手指子域实际上通过三种不同的构象进行采样-开放，封闭和以前未被识别的中间构象。我们测量了聚合酶- dna复合物的总体解离率，以及在核苷酸底物存在和不存在的情况下各种构象状态之间的分布，这些分布要么正确，要么不正确。正确的底物促进聚合酶快速发展到具有催化能力的封闭构象，而不正确的核苷酸阻断中间构象中的酶并诱导与DNA的快速解离。值得注意的是，不正确的核苷酸底物也会促进DNA分裂到空间分离的3 ‘ -5 ’外切酶结构域，这为防止聚合酶活性位点的错误结合提供了额外的机制。这些结果揭示了早期先天保真度检查点的存在，在酶包围新生碱基对之前拒绝不正确的核苷酸底物。
Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry and product release. The non-covalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting non-cognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule Förster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformations - open, closed and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase-DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3′-5′ exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair.
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DOI：
10.1073/pnas.0403575101
发表时间：
2004-07-06
影响因子：
11.1
作者：
Rueda, D;Bokinsky, G;Walter, NG
通讯作者：
Walter, NG
影响因子：
2.9
作者：
Joyce, Catherine M.;Potapova, Olga;Grindley, Nigel D. F.
通讯作者：
Grindley, Nigel D. F.
影响因子：
4.8
作者：
Wu, Eugene Y.;Beese, Lorena S.
通讯作者：
Beese, Lorena S.
影响因子：
2.9
作者：
EGER, BT;BENKOVIC, SJ
通讯作者：
BENKOVIC, SJ
DOI：
10.1073/pnas.1012277107
发表时间：
2010-10-19
影响因子：
11.1
作者：
Datta, Kausiki;Johnson, Neil P.;von Hippel, Peter H.
通讯作者：
von Hippel, Peter H.