Dynamics of pre-replicative complex assembly.
The pre-replicative complex (pre-RC) is formed at all potential origins of replication through the action of the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex. The end result of pre-RC formation is the loading of the Mcm2-7 replicative helicase onto origin DNA. We examined pre-RC formation in vitro and found that it proceeds through separable binding events. Origin-bound ORC recruits Cdc6, and this ternary complex then promotes helicase loading in the presence of a pre-formed Mcm2-7-Cdt1 complex. Using a stepwise pre-RC assembly assay, we investigated the fate of pre-RC components during later stages of the reaction. We determined that helicase loading is accompanied by dissociation of ORC, Cdc6, and Cdt1 from origin DNA. This dissociation requires ATP hydrolysis at a late stage of pre-RC assembly. Our results indicate that pre-RC formation is a dynamic process.
Tsakraklides
Vasiliki
V
Department of Biology, Cambridge, Massachusetts 02139.
Bell
Stephen P
SP
Department of Biology, Cambridge, Massachusetts 02139; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. Electronic address: spbell@mit.edu.
D000255:Adenosine Triphosphate / Q000737:chemistry; D018797:Cell Cycle Proteins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D016193:G1 Phase; D015966:Gene Expression Regulation, Fungal; D006868:Hydrolysis; D008954:Models, Biological; D051738:Origin Recognition Complex / Q000235:genetics*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C499592:TAH11 protein, S cerevisiae; D000255:Adenosine Triphosphate; D004247:DNA; C069199:MCM2 protein, S cerevisiae