Abstract
A number of proteins are involved in DNA replication, which is essential for the inheritance of genetic information. These proteins assemble to form a huge complex, called replisome, and accomplish each function through highly regulated manner. Electron microscopic single particle analysis is one of the most powerful methods to study such complex system, which is difficult to study by X‐ray crystallography. DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). PCNA has a trimeric ring structure that encircles the DNA, and increases the processivity of the bound DNA polymerase by tethering it to the DNA. It is known now, that PCNA also interacts with multiple partners to control DNA replication, DNA repair, and cell cycle progression, and works not only as the platform, but also as the conductor for the recruitment and release of these factors. However, the molecular architectures as well as the mechanism of the regulation of these replication factors are not known in detail. We have been focusing our interest on the mechanism of synthesis and maturation of Okazaki fragments during lagging strand DNA replication in which three replication factors, i.e. DNA polymerase, Flap endonuclease, DNA ligase, are playing essential roles (Fig. 1A‐C). As all of these 3 enzymes are known to interact with PCNA trimer, a switching mechanism between these factors has been proposed, called PCNA tool belt model, which is considered to increase the efficiency of these sequential reactions (Fig 1D). Recent biochemical study, on the other hand, suggests a sequential switching mechanism of these factors. Little is known regarding the switching mechanism, due to the lack of the structural data of these complexes. We have investigated the three‐dimensional structure of the core components of the replisome, such as DNA polymerase B(PolB)‐PCNA‐DNA, and DNA ligase‐PCNA‐DNA ternary complexes, by single particle analysis (2‐ 3). Besides the authentic interaction through a PCNA‐interacting protein box (PIP‐box), we could find a novel contact between both polymerase‐PCNA and ligase‐PCNA. Mutant analysis showed that these contacts are involved in the regulation of the replication factors, such as the switching between the polymerizing and editing modes of the PolB. Our results, showing that both factors interacting with two subunits of the PCNA trimer ring, were inconsistent with the standard tool belt model. However, the third PCNA subunit was still free in both complexes, thus we analyzed the complex structures with two replication factors bound to a PCNA ring, in order to investigate the switching mechanism between them in more detail.
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