PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step.
PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step.
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DOI：
10.1093/nar/gkn745
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发表时间：
2008-12
影响因子：
14.9
通讯作者：
White, Malcolm F.
中科院分区：
文献类型：
--
作者：
Hutton, Richard D.;Roberts, Jennifer A.;Penedo, J. Carlos;White, Malcolm F.
滑动钳增殖细胞核抗原（PCNA）作为各种DNA修饰酶的招募者和组织者，包括核酸酶，解旋酶，聚合酶和糖基化酶。5 ' -flap内切酶Fen-1在真核生物和古细菌中对冈崎片段加工至关重要，被PCNA靶向到复制叉上。Crenarchaeal XPF，一种3 ' -flap内切酶，也能在体外被PCNA刺激。使用一种新的连续荧光测定，我们证明了PCNA通过根本不同的机制激活这两种核酸酶。如前所述，PCNA通过增加酶对底物的结合亲和力来刺激Fen-1。然而，PCNA通过将催化速率常数提高4个数量级而不影响KM来激活XPF。PCNA可以作为XPF施加力扭曲DNA底物的平台，使底物不稳定和/或稳定过渡态结构。这表明，在某些情况下，PCNA可以作为一种必要的辅助因子直接支持催化作用，这是一种在分子生物学中通常被认为具有被动靶向和组织功能的蛋白质的新角色。这可能为针对特定情况（如复制分叉或预装载PCNA的受损DNA）精确控制核酸酶活性提供了一种机制。
The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5′-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3′-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the KM. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA.
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影响因子：
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通讯作者：
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