Friday, August 28, 2009, 11:00 am — John Dunn Seminar Room, Bldg. 463
Initiation of DNA replication depends on loading of the MCM2-7 helicase at a replication origin, activation of the helicase and consequential processive DNA unwinding at the replication fork. We have reconstituted the first step of budding yeast replication initiation -- formation of the pre-Replication Complex (pre-RC) using purified proteins. During pre-RC formation ORC, Cdc6 and Cdt1 cooperate to load the MCM2-7 helicase onto DNA. During pre-RC formation ORC recruits Cdc6 to origin DNA. Cdc6 ATP-hydrolysis regulates ORC-Cdc6 complex stability in a DNA sequence dependent manner. The reconstituted pre-RC reaction depends on all 14 pre-RC proteins, DNA and Cdc6 ATP-hydrolysis. MCM2-7, in the absence of pre-RC proteins, adopts a hexameric structure, as analysed by gel-filtration and electron microscopy. During pre-RC formation the MCM2-7 structure changes and loaded MCM2-7 assume a configuration consistent with a double-hexamer. This structural change could be necessary for pre-Initiation Complex formation and activation of the MCM2-7 helicase.
Hosted by: Huilin Li
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