Analysis of mammalian origin specification in ORC-depleted Xenopus egg extracts

Abstract

Xenopus embryonic ORC is clearly not required for random origin site selection in Xenopus egg extracts. We conclude that a modification of Chinese Hamster chromatin takes place shortly after metaphase that complements a lack of XORC activity. This modification most likely represents an interaction of mammalian ORC with chromatin that is required for replication but, that is not sufficient for origin specification.

Mammalian Orc1 Protein Is Selectively Released from Chromatin and Ubiquitinated during the S-to-M Transition in the Cell Division Cycle

“…Three differences were detected between nocodazole-arrested metaphase chromatin and G 1 -phase chromatin that were relevant to the cell cycle-dependent affinity of Orc1 for chromatin. First, chromatin from M-phase cells that had been arrested in nocodazole for up to 4 h did not initiate DNA replication when incubated in a Xenopus egg extract depleted of Xenopus Orc proteins (25,34,59). Therefore, M-phase chromatin contains few, if any, active ORCs.…”

“…9). In mammals, Orc1 and Orc2 are both tightly bound to chromatin during late G 1 phase (33,34,43), and late G 1 -phase nuclei contain active ORCchromatin sites by virtue of the fact that they can initiate DNA replication at specific genomic sites when incubated in an Orcdepleted Xenopus egg extract (25,34,59). In contrast to yeast in which all six ORC subunits are stably bound to chromatin throughout the cell cycle (11,17,22,27,29), the affinity of mammalian Orc1 for chromatin is selectively reduced during S phase, such that lysis of cells in 0.1% Triton X-100, 0.15 M NaCl, and 1 mM Mg 2ϩ -ATP releases Orc1, but not Orc2, into the non-chromatin-bound fraction (Fig.…”

“…In mammals, the total amounts of Orc1 and Orc2 present throughout the cell cycle are constant (33,34,43,47), but Orc1 is selectively released from chromatin during M phase (34) and S phase (23). This accounts for the fact that M-phase chromatin does not contain functional ORCs (34,59) and for the disappearance of an ORC-like footprint at the human lamin B 2 origin during mitosis (1). ORC activity is restored during the transition from M to G 1 phase, concomitant with the appearance of Orc1 tightly bound to chromatin and the assembly of pre-RCs at specific genomic sites (25,34).…”

Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis

“…Interaction of Orc1 with the ORC core complex in the presence of ATP is essential for binding ORC to DNA and for assembly of pre-RCs in vitro (GiordanoColtart et al, 2005). Selective inhibition of Orc1 synthesis in cultured cells confirms that it is also required for assembly of pre-RCs in vivo , and the selective loss of Orc1 from chromatin during mitosis accounts for the fact that metaphase mammalian chromatin will not replicate in ORCdepleted Xenopus egg extracts Natale et al, 2000;Yu et al, 1998).…”

“…Since inhibition of CDK activity in metaphase cells causes rapid binding of Orc1 (Li et al, 2004) and minichromosome maintenance (MCM) proteins (Ballabeni et al, 2004) to chromatin, and premature initiation of DNA replication (Itzhaki et al, 1997), hyperphosphorylation of Orc1 during the G2-M phase presumably prevents its stable association with ORC(2-5)-chromatin sites. This would account for the absence of functional ORC-chromatin sites on metaphase chromatin Natale et al, 2000;Yu et al, 1998). Orc1 reassociates tightly with chromatin during the transition from M to G1 phase (Li and DePamphilis, 2002;Mendez et al, 2002;Natale et al, 2000) followed closely by the appearance of pre-RCs at specific genomic sites .…”