Analysis of the role of PCNA-DNA contacts during clamp loading
Analysis of the role of PCNA-DNA contacts during clamp loading
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DOI：
10.1186/1472-6807-10-3
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发表时间：
2010-01-30
影响因子：
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通讯作者：
Kuriyan, John
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文献类型：
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作者：
McNally, Randall;Bowman, Gregory D.;Kuriyan, John
背景资料：滑动夹，例如真核生物中的增殖细胞核抗原（PCNA），是环状蛋白质复合物，其包围DNA并通过充当DNA聚合酶的对接位点而实现高度进行性DNA复制。在一个ATP依赖性的反应中，钳装载复合物，如复制因子-C（RFC）在真核生物中的复合物，打开钳和负载它周围的引物模板DNA.Results：我们建立了一个模型的RFC绑定到PCNA和DNA的基础上现有的晶体结构的钳装载。该模型表明，DNA将在夹加载期间以一定角度进入夹，从而与PCNA中心的带正电荷的残基相互作用。我们发现，同时突变的赖氨酸20，赖氨酸77，精氨酸80，和精氨酸149，这与DNA的RFC-PCNA-DNA模型中的相互作用，妥协的能力，酵母PCNA刺激DNA依赖的ATP酶活性的RFC时，DNA是足够长的延伸通过钳。荧光各向异性结合实验表明，突变的钳蛋白刺激RFC ATP酶活性的能力可能是由RFC-PCNA复合物对DNA的亲和力降低引起的。我们获得了几种晶体形式的酵母PCNA-DNA复合物，测量X-射线衍射数据到3.0埃分辨率的一个这样的复合物。由此产生的电子密度图显示，DNA是结合在一个倾斜的方向相对于PCNA，但使不同的接触比那些牵连在钳加载。由于DNA中明显的部分无序，我们将DNA的细化限制在刚体模型中。这一结果与以前的分析细菌钳绑定到DNA，其中的DNA是很好地resolved.Conclusion：PCNA突变分析表明，带正电荷的残基在钳的中心创建一个结合表面，使接触DNA。破坏这种阳性表面，这在以前没有涉及到钳加载功能，降低RFC ATP酶活性的DNA存在下，最有可能通过降低RFC和PCNA的DNA的亲和力。然而，DNA的相互作用并不限于一个方向，如PCNA-DNA共晶体的分析所示。
Background: Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.Results: We built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 angstrom resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved.Conclusion: Mutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.