酵母夹的逐步加载通过整体和单分子研究揭示。
Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies.
机构信息
Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
出版信息
Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):19736-41. doi: 10.1073/pnas.1014139107. Epub 2010 Nov 1.
Abstract
In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC·PCNA·DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC·PCNA·DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC·PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of polδ only one species converts to the RFC·PCNA·DNA·polδ holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.
摘要
在使用酵母增殖细胞核抗原 (PCNA，夹子) 和复制因子 C (RFC，夹子加载器) 的组合和单分子实验中，我们研究了 RFC·PCNA·DNA 复合物的组装及其在加入聚合酶 δ (polδ) 后向全酶的进展。我们获得的数据表明：(i) PCNA 在 DNA 上的加载通过多个构象中间体进行，并且在几次失败的尝试后成功；(ii) RFC 对引物 45-mer 模板叉没有催化作用；(iii) 在 ATP 存在下形成的 RFC·PCNA·DNA 复合物来自至少两种动力学上可区分的物种；(iv) 这些物种通过从 DNA 上卸下 RFC·PCNA 或 PCNA 解离成其组成亚基而解体；(v) 在 polδ 的存在下，只有一种物种转化为 RFC·PCNA·DNA·polδ 全酶。这些发现重新定义并加深了我们对夹子加载过程的理解，并揭示了它是一个令人惊讶的反复试验以达到启发式解决方案的过程。
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