XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

Abstract

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…(40)(41)(42)(43)(44)(45)(46) Cdt1 physically associates with Cdc6 (41) suggesting that it also forms part of the pre-RC. In Xenopus egg extracts, Cdt1 is required for licensing chromatin in vitro.…”

Geminin Deficiency Causes a Chk1-dependent G2 Arrest inXenopus

“…MCM complex is thought to be the helicase that separates the two strands of the double helix. Geminin seems to affect replication by inhibiting Cdt1, an essential replication factor that must be incorporated into preRC before the MCM complex can be added (Maiorano et al, 2000). Geminin binds tightly to Cdt1, and overexpression of Cdt1 overcomes the inhibition of replication caused by geminin (Wohlschlegel et al, 2000;Tada et al, 2001).…”

An ATR- and BRCA1-Mediated Fanconi Anemia Pathway Is Required for Activating the G2/M Checkpoint and DNA Damage Repair upon Rereplication

“…The balance between Cdt1 and geminin is one such mechanism (23,31). Cdt1 is a replication initiation factor that promotes the assembly of the prereplicative complex (20,28). Overexpression of Cdt1 in human cells results in rereplication (43), and increased expression of Drosophila melanogaster Cdt1 (Dup) in diploid cells is sufficient to induce polyploidization (40).…”