Abstract
Pre-RC formation on chromosomal replication origins is an essential process for eukaryotic DNA replication in order for the origins to form pre-IC complexes. Though purification of each pre-RC component is of great importance to conduct quantitative analyses in vitro, recombinant MCM in budding yeast was a challenge to purify as a form active in pre-RC formation. We purified recombinant MCM complexed with Cdt1 and then reconstituted pre-RC formation using only purified, recombinant proteins. The established reaction could occur on representative early and late origins and was repressed by certain origin mutations. Reconstituted pre-RC had affinity for recombinant pre-IC components, indicating that a recombinant pre-IC could be assembled in vitro.
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