Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis.
Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions. Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008297:Male; D051738:Origin Recognition Complex; D010957:Plasmids; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate