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出芽酵母pre-RC形成の精製リコンビナント蛋白を用いた試験管内再構成と性状解析
講演者：川上広宣博士 所属：米国CSHL, Bruce Stillman Lab開催日：2010-12-14 16:00
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演題
出芽酵母pre-RC形成の精製リコンビナント蛋白を用いた試験管内再構成と性状解析
講演者
川上広宣博士（米国CSHL, Bruce Stillman Lab）
日時場所
１２月１４日 午後４時から 薬学部第３講義室（中央棟３階）
要旨
Chromosomal DNA replication is tightly regulated to occur only once per cell cycle. In eukaryotic cells, the pre-replicative complex (pre-RC) assembles at replication origins in G1 phase and is essential for replication licensing prior to S phase. Pre-RC assembly in budding yeast requires the origin recognition complex (ORC), Cdc6, and Cdt1 that recruit the minichromosome maintenance (MCM) heterohexamer onto replication origins, called autonomous replication sequences (ARS's). Although purification of each component is of great importance to conduct quantitative biochemical analyses on pre-RC assembly in vitro, recombinant MCM hexamer consisting of Mcm2/3/4/5/6/7 (Mcm2-7) has been difficult to purify as a form active in pre-RC assembly. In the present study, a recombinant, budding yeast Mcm2-7 was successfully purified to near homogeneity using a newly-developed method with recombinant Cdt1. Mcm2-7 and Cdt1 co-migrated during every fractionation step and eluted at a molecular mass of ~700 kDa upon gel-filtration, consistent with the theoretical mass of Mcm2-7/Cdt1 heptamer. The Mcm2-7 complex had affinity for DNA in a manner dependent on purified, recombinant ORC and Cdc6, wild-type ARS sequence, and ATP. The resultant bound Mcm2-7 was high salt wash-resistant, but that in the presence of ATP-γ- S was not. Analyses using mutant ARS's identified sequences important for Mcm2-7 loading. These results suggest that the Mcm2-7 complex co-purified with Cdt1 could not simply associate with ARS but was loaded onto the ARS via mechanisms mediated by ATP hydrolysis and the functional sequences within ARS. A model for MCM loading during pre-RC assembly will be discussed.
世話人
片山 勉（薬学研究院）
釣本敏樹（理学研究院）