Kolpashchikov D M, Hughes P, Favre A, Baldacci G, Lavrik O I
Institut Jacques Monod (CNRS, Universite Paris 6, Universite Paris 7), 75351 Paris Cedex 05, France.
J Mol Recognit. 2001 Jul-Aug;14(4):239-44. doi: 10.1002/jmr.538.
Replication factor C (RFC) is a heteropentameric sliding clamp loader protein essential for processive synthesis of DNA by eukaryotic DNA polymerases delta and epsilon. To study the interaction of RFC with 3' and 5' ends of the DNA primer, we have developed chemical photocrosslinking assay using a synthetic DNA gap and DNA primer-template structures. We have found that the radioactively labeled primers containing a photoreactive group at their 5' end could crosslink with the largest RFC subunit (RFC140) on primer-templates and DNA gap structures, but that 3' end photoreactive primers could only crosslink with RFC140 within the DNA gap structure. Addition of replication protein A (RPA) to the reaction mixture resulted in the crosslinking of RPA subunits and inhibited crosslinking of RFC140 using 3' but not 5' photoreactive primers present at the gap. The results suggest specific contacts between RFC140 and the 5' end of the DNA primer. Together with previous data, these experiments allow us to propose a model for the DNA polymerase switch during eukaryotic DNA replication.
复制因子C（RFC）是一种异源五聚体滑动夹加载蛋白，对于真核生物DNA聚合酶δ和ε进行连续的DNA合成至关重要。为了研究RFC与DNA引物3'端和5'端的相互作用，我们利用合成的DNA缺口和DNA引物-模板结构开发了化学光交联测定法。我们发现，在其5'端含有光反应基团的放射性标记引物可以与引物-模板和DNA缺口结构上最大的RFC亚基（RFC140）交联，但3'端光反应性引物仅能在DNA缺口结构内与RFC140交联。向反应混合物中添加复制蛋白A（RPA）导致RPA亚基交联，并抑制了使用缺口处存在的3'而非5'光反应性引物时RFC140的交联。结果表明RFC140与DNA引物的5'端之间存在特异性接触。结合先前的数据，这些实验使我们能够提出真核生物DNA复制过程中DNA聚合酶转换的模型。
