KENBIKYO Vol.49▶No.2 2014
â– Reviews

Single Particle Analysis of Replication Fork Complex

Kouta Mayanagi

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 (PolB). PolB interacts with the DNA clamp, PCNA, which forms a trimeric ring structure that encircles the DNA, and increases the processivity of the bound PolB by tethering it to the DNA. It is known now, that PCNA also interacts with various protein factors, to control DNA replication, DNA repair, and cell cycle progression.
We have investigated the structures of PolB-PCNA-DNA and DNA ligase-PCNA-DNA, which play very important roles in replisome. The obtained structures, both revealed unexpected configurations of the complexes. Intriguingly, besides the authentic interaction through a PCNA-interacting protein box, a novel contact was found between both polymerase-PCNA and ligase-PCNA. The obtained molecular architecture of the complexes, including the newly found contacts, provides clearer insights into the mechanism of the regulation of the fork complex.

Key words: Single particle analysis, DNA clamp, DNA replication, replication fork, macromolecular complex