Waga S, Stillman B
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Mol Cell Biol. 1998 Jul;18(7):4177-87. doi: 10.1128/MCB.18.7.4177.
The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPgammaS-activated complex was no longer displaced from the DNA by p21. We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.
p21蛋白是一种细胞周期蛋白依赖性激酶（CDK）抑制剂，能够与细胞周期蛋白-CDK以及增殖细胞核抗原（PCNA）结合。通过与PCNA结合，p21可以在复制和修复过程中差异性地调节PCNA的功能。为了深入了解p21影响PCNA功能的精确机制，我们设计了一种新的检测方法，用于检测复制因子C（RFC）催化PCNA加载到DNA上的过程，该方法利用通过生物素-链霉亲和素附着在琼脂糖珠上的引物-模板DNA。使用该检测方法，我们发现加载反应后RFC在DNA上与PCNA短暂结合。添加p21并不抑制RFC依赖的PCNA加载；相反，p21在DNA上与PCNA形成了稳定的复合物。相比之下，DNA上p21-PCNA复合物的形成导致RFC从DNA上解离。ATP的不可水解类似物腺苷-5'-O-（3-硫代三磷酸）（ATPγS）和腺苷亚氨基二磷酸，在没有p21的情况下，各自稳定了包含RFC和PCNA的引物识别复合物。ATPγS激活复合物中的RFC不再被p21从DNA上置换。我们提出，p21在一个需要ATP水解的过程中刺激RFC从PCNA-DNA复合物中解离，然后抑制DNA复制中随后的PCNA依赖事件。数据表明，引物识别复合物中RFC的构象可能在ATP水解时发生变化。我们还提出，仍然附着在DNA上的p21-PCNA复合物可能起到将细胞周期蛋白-CDK复合物拴系到基因组特定区域的作用。
