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Summary of Research Projects (Supports in 2004
Fiscal Year)
| Subjects |
Dynamics of DNA binding of replication initiation proteins |
| Representative researcher |
Graduate School of Frontier Biosciences, Osaka University
Shou WAGA |
| I investigated the dynamics
of DNA binding of replication initiation proteins during formation
of the pre-replicative complex (pre-RC) on plasmids in Xenopus
egg extracts. The pre-RC was efficiently formed on plasmids
at 23°C, with one or a few ORC
molecules and about 10-20 MCM2 molecules loaded onto each plasmid.
MCM interacted weakly but
stoichiometrically with the plasmid in an ORC-dependent manner,
even in the presence of geminin (with about 10 MCM2 molecules
per plasmid). Interestingly, DNA binding of ORC, CDC6 and CDT1
was significantly stabilized in the presence of geminin, under
which conditions about 10-20 molecules each of ORC and CDC6
were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex
rapidly formed on DNA at lower temperature (0°C)
without geminin, with about 10-20 molecules each of ORC and
CDC6 bound to the
plasmid, but almost no binding of MCM. However, upon shifting
the temperature to 23°C, most ORC, CDC6 and CDT1 molecules
were displaced from the DNA, leaving about one ORC molecule
on the plasmid, while about 10 MCM2 molecules were loaded onto
each plasmid. These results imply that each DNA-bound ORC molecule
loads only one or a few MCM2-7 complexes during metazoan pre-RC
formation. |
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