Binding of Drosophila Orc Proteins to Anaphase Chromosomes Requires Cessation of Mitotic Cyclin-Dependent Kinase Activity

Abstract

The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. We describe a transgenic Drosophila model which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes.Initiation of DNA replication on the chromosomes of eukaryotic cells requires the prior assembly of the pre-replicative complex (pre-RC), establishing the licensed chromatin state. Of the multiple proteins participating in the stepwise formation of the pre-RC, the initiator origin recognition complex (ORC) makes the first chromatin contacts. In Saccharomyces cerevisiae, the heterohexameric ORC binds to distinct sequence elements in the context of small, modular origins of DNA replication. These are occupied by ORC throughout the cell cycle. Thus, while the initial ORC-DNA interaction can define the position of origins on the chromosomes, it is an unlikely candidate to determine the timing of pre-RC formation, which is confined to the M and G 1 phases of the cell cycle. Instead, this process is mainly controlled by changes in cyclindependent kinase (CDK) activity during these cell cycle phases and is part of the safeguard mechanisms against unscheduled DNA synthesis (4,6,15,16,28).In contrast to yeast, origins in metazoans are less well defined and their distribution along chromosomes is subject to tissue-specific and developmental control. No conserved DNA sequence motifs that would indicate a sequence-specific DNA binding by initiator proteins have been identified within replication initiation regions. For these reasons, alternative modes of origin selection are discussed (13,18,30).It is nonetheless assumed that the position of metazoan origins is determined by nonrandom ORC-chromatin interactions. To what extent these interactions persist after origin firing has not been extensively studied. The same holds true for the issue of reoccupation of the additional potential ORC binding site emerging immediately after DNA replication initiation. It is clear, though, that ORC has to be chromatin bound at the latest toward the end of mitosis for the subsequent pre-RC assembly steps to occur. When exactly ORC associates with chromatin and, especially, whether ORC is bound to metaphase chromosomes have not been firmly establishe...

Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells

“…In Drosophila, binding of Orc2 to mitotic chromosomes post-anaphase is dependent on cessation of mitotic CDK activity (87). These results are consistent with Orc1 being the first determinant for chromatin binding and origin recognition.…”

Functional analysis of an Orc6 mutant in Drosophila

“…1). The observed pattern of Orc6 staining in this experiment is remarkably similar to those of both Drosophila Orc2 and Xenopus Orc1, which were also weakly associated with DNA at metaphase but present at the later stages of mitosis (4,24,25). Most likely, at these stages ORC is deposited onto the replication origins in preparation for the next cell cycle.…”

“…We have found that during mitotic stages, Orc6 localization is remarkably similar to that of other ORC subunits, such as Drosophila Orc2 and Xenopus Orc1. All of these proteins were weakly associated with DNA at metaphase but present at the later stages of mitosis (4,24,25). This pattern of binding of Drosophila ORC to the chromatin depends on the cessation of mitotic cyclin activity (25).…”

“…All of these proteins were weakly associated with DNA at metaphase but present at the later stages of mitosis (4,24,25). This pattern of binding of Drosophila ORC to the chromatin depends on the cessation of mitotic cyclin activity (25). Most likely, at these stages functional 6-subunit ORC is deposited onto the replication origins in preparation for the next cell cycle.…”

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

“…Since it is an established view that the mobility of nuclear chromatin-binding proteins is determined by their retention time on the relatively immobile chromosomal DNA, 32 we interpret the distinct decrease in mobility of Cdc6-YFP in telophase, as compared to the other cell cycle phases, as evidence that Cdc6 interacts with chromatin more often and/or longer during this phase. It is likely that the immobilization of Cdc6-YFP in telophase reflects the time frame at which most replication origins are licensed, since the second loading factor Cdt1, 33 the origin recognition complex ORC, 34 and human MCM proteins 13,24 are also shown to associate with chromatin mainly at the M/G1 transition. Of interest, it was recently shown that loading of the first MCM2-7 hexamer onto DNA occurrs within seconds, whereas the subsequent formation of a MCM2-7 double hexamer is slow and takes several minutes.…”