The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication

Abstract

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat-containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin.

Figure 1.

Xmus101 is required for DNA replication in Xenopus. (A) The Mus101 protein family. Shown are schematic depictions of Mus101-related proteins from divergent organisms. The shaded gray boxes indicate the position of the BRCT domains, and the numbers to the right indicate the size of the protein, in amino acids. For comparison, the domain structure of budding yeast Dpb11 and fission yeast Cut5 is also shown. (B) Immunoblot analysis of Xenopus egg extract (XEE), or egg extract that had been immunodepleted of Xmus101 (α-Xmus101), probed with affinity-purified anti-Xmus101 antibodies. The asterisk denotes a background band recognized by the antibody that is not diminished in the depleted extract. The numbers to the left of the gel denote the migration position and molecular mass, in kD, of molecular mass markers. (C) Egg extracts were prepared, and then depleted of Xmus101 protein (α-Xmus101) or mock-depleted (mock). Additionally, Xmus101-depleted extracts were supplemented with in vitro transcription and translation (IVT) reactions programmed by the Xmus101 cDNA. In vitro transcription was performed in either the sense, or anti-sense orientation. Sperm chromatin was then added, along with ³²P-dATP, and DNA replication in the given extract was measured at the indicated times. The graph depicts the amount of DNA replication observed after quantification of the dried gels by PhosphoImager analysis. The data shown are from a single experiment, and are representative of four independent trials. The amount of replication observed in the mock-depleted sample at the 90-min time point was set to 100, and all other values adjusted accordingly.

Figure 2.

Xmus101 is required for the loading of Cdc45 onto chromatin during the initiation of DNA replication. (A) Immunoblot of either total extract samples from mock-depleted (mock, lane 1), or Xmus101-depleted (α-Xmus101, lane 2) extract, or chromatin isolated after a 45-min incubation in either mock- (mock, lane 3), or Xmus101-depleted (α-Xmus101, lane 4) extract. The blots were probed with antibodies against Xenopus Xmus101 (panel I), pol α (pol α p60 subunit, panel II), RPA (the 34-kD subunit, panel III), pol ɛ (pol α p70 subunit, panel IV), Cdc45 (panel V), MCM10 (panel VI), Cdc7 (panel VII), or the MCM7 component of the MCM2-7 complex (panel VIII). (B) Immunoblot of complete egg extract, both total extract (Tot.) and the isolated chromatin fraction (Chr.), and membrane-free egg extract, or egg cytosol, both total extract and the isolated chromatin fraction. (C) Egg extracts were prepared and supplemented with sperm chromatin (lane 2), or sperm chromatin plus recombinant geminin (500 nM, lane 3), or sperm chromatin plus recombinant p27Kip (500 nM, lane 4). After a 45-min incubation, the chromatin was isolated and probed, by immunoblotting, for the presence of Cdc45 and Xmus101. Lane 1 shows a reaction lacking sperm chromatin.

Figure 3.

Xmus101 requires ORC for association with sperm chromatin. In lanes 1–4, egg cytosol (EC) that either contained or lacked ORC, as indicated, was mixed with sperm chromatin and incubated for 30 min. The chromatin was then isolated and probed by immunoblotting for the presence of Xmus101, MCM10, MCM7, or ORC2. In lanes 5–8, egg cytosol that either contained or lacked ORC, as indicated, was mixed with sperm chromatin and incubated for 30 min. After incubation, NPE that either contained or lacked ORC, as indicated, was then added and incubation continued for an additional 30 min before isolation of the chromatin and immunoblotting for the indicated factor. The NPE contained the replication inhibitor aphidicolin, to trap assembled replication complexes and prevent disassembly following the completion of DNA replication.

Figure 4.

Xmus101 is dispensable for DNA replication after initiation is complete. (A) Experimental scheme, see text for details. (B) DNA replication was measured in the given extract at the given time. (C) Immunoblotting of either mock-depleted (mock), or Xmus101-depleted (α-Xmus101) extract after transfer of the chromatin, showing that no detectable Xmus101 is transferred along with the chromatin.

Figure 5.

A model for the replication initiation pathway. The data presented here suggest two parallel pathways, one MCM2-7 dependent, the other Mus101 dependent, which cooperate to load Cdc45 at the G1/S transition. See Discussion for details.