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首页> 外文期刊>Molecular and Cellular Biology >Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA.
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Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA.

机译：哺乳动物DNA聚合酶辅助蛋白：分析复制因子C催化的增殖细胞核抗原负载到环状双链DNA上。

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To understand the mechanism of action of the two eukaryotic replication auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C), we constructed a plasmid for producing PCNA which could be 32P labelled in vitro. This allowed us to analyze the assembly of the auxiliary proteins directly on DNA and to examine this process in the absence of DNA synthesis. By using closed circular double-stranded DNA or gapped circular DNA for protein-DNA complex formation, the following results were obtained, (i) RF-C can load PCNA in an ATP-dependent manner directly on double-stranded DNA, and no 3'-OH ends are required for this reaction; (ii) the RF-C-PCNA complex assembled on closed circular DNA differs from those assembled on gapped or nicked circular DNA; (iii) the stable RF-C-PCNA complex can be assembled on circular but not on linear DNA; and (iv) only gapped DNA can partially retain the assembled RF-C-PCNA complex upon the linearization of the template. We propose that RF-C first binds unspecifically to double-stranded DNA in the presence of ATP and then loads PCNA onto DNA to yield a protein complex able to track along DNA. The RF-C-PCNA complex could slide along the template until it encounters a 3'-OH primer-template junction, where it is likely transformed into a competent clamp. The latter complex, finally, might still be able to slide along double-stranded DNA.
机译：为了了解两种真核复制辅助蛋白增殖细胞核抗原（PCNA）和复制因子C（RF-C）的作用机理，我们构建了一种可在体外制备32P标记的PCNA的质粒。这使我们能够直接在DNA上分析辅助蛋白的装配，并在没有DNA合成的情况下检查该过程。通过使用封闭的环状双链DNA或带缺口的环状DNA形成蛋白质-DNA复合物，获得了以下结果：（i）RF-C可以以ATP依赖的方式直接将PCNA加载到双链DNA上，而没有3该反应需要'-OH端; （ii）组装在封闭环状DNA上的RF-C-PCNA复合物不同于组装在缺口或刻痕环状DNA上的RF-C-PCNA复合物; （iii）稳定的RF-C-PCNA复合物可以组装成环状，但不能组装成线性DNA; （iv）只有空缺的DNA可以在模板线性化后部分保留组装的RF-C-PCNA复合物。我们建议，RF-C首先在ATP存在下非特异性结合至双链DNA，然后将PCNA加载到DNA上以产生能够沿DNA追踪的蛋白质复合物。 RF-C-PCNA复合物可以沿着模板滑动，直到遇到3'-OH引物-模板连接点为止，在此处可能会转变为有效的钳位。最后，后者可能仍然能够沿着双链DNA滑动。

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