SUNY at Albany
June 19-23, 2001
Eletron microscopic image analysis of macromolecules involved in DNA replication, repair, and recombination
Several proteins involved in DNA replication, repair, and recombination, assemble to form a huge macromolecular superstructure. Electro microscopy is one of the most powerfull methods to observe such comples, and by means of image precessing a high resolution 2-D and 3-D map can be obtained. Using this method, called "Single-particle analysis", we studied the structure of Thermus thermophilus RuvB oligomer, a motor protein for branch migration of the Holliday junction.
Through RuvB has been regarded as a meber of hexameric helicase, we found a clear seven-fold symmetry in the averaged projection map of RuvB oligomer in the absence of DNA, suggesting that it exists as a heptamer itself. The averaged image of RuvB, oligomerized in the presence of DNA, exihibited a six-fold symmetry, which suggested a transition from heptamer to hexamer upon binding to DNA (1). At the late stage of homologous recombination two RuvB hexamers and two RuvA-Holliday junction crystal structure (2). The diffrence suggests a large movement of RuvA domain III, induced by the interaction with RuvB.
We also studied the structure of Pyrcoccus furiosus replication factor C small subunits (RFCS). RFC is a clamp-loading protein which stimulate DNA synthesis activity of DNA ploymerase I by loading a ring shaped PCNA trimer on DNA. RFCD mostly forms ring-shaped hexamers at pH 9.0, although they tend to form C-shaped tetramers or pentamers at a lower pH (pH 5.5). The 3-D map revealed that the six subunits are arranged in a heat-to-tail configuration. Now we are analysing he structure of RFC-complex (including large subunit) and RFC-PCNA-DNA complex.References and footnotes.
Mayanagi, K. (1), Miyata, T. (1), Yamada, K.(1,2), Ariyoshi, M.(1), Cann, I.(1) Yuasa, M.(1), Ishino, Y.(1), Ohnishi, T.(2), Iwasaki, H.(2,3), Shinagawa, H.(2) and Morikawa, K.(1)
Biomolec. Engineering Research Inst.(1), Dept. of Molec. Microbiol.(2), Research Inst. For Microbial Diseases(3), Osaka Univ. JST