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J Cell Biol
2002 Nov 25;1594:541-7. doi: 10.1083/jcb.200207090.
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The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication.
Van Hatten RA
,
Tutter AV
,
Holway AH
,
Khederian AM
,
Walter JC
,
Michael WM
.
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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.
Araki,
Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.
1995, Pubmed
Araki,
Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.
1995,
Pubmed
Bell,
DNA replication in eukaryotic cells.
2002,
Pubmed
Boyd,
Isolation and characterization of X-linked mutants of Drosophila melanogaster which are sensitive to mutagens.
1976,
Pubmed
Edwards,
MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts.
2002,
Pubmed
,
Xenbase
Jares,
Xenopus cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading.
2000,
Pubmed
,
Xenbase
Kelly,
Regulation of chromosome replication.
2000,
Pubmed
Labib,
Is the MCM2-7 complex the eukaryotic DNA replication fork helicase?
2001,
Pubmed
Masumoto,
Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast.
2000,
Pubmed
Michael,
Activation of the DNA replication checkpoint through RNA synthesis by primase.
2000,
Pubmed
,
Xenbase
Mimura,
Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk.
1998,
Pubmed
,
Xenbase
Mimura,
Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts.
2000,
Pubmed
,
Xenbase
Mäkiniemi,
BRCT domain-containing protein TopBP1 functions in DNA replication and damage response.
2001,
Pubmed
Orr,
Mutants suppressing in trans chorion gene amplification in Drosophila.
1984,
Pubmed
Saka,
Fission yeast cut5+, required for S phase onset and M phase restraint, is identical to the radiation-damage repair gene rad4+.
1993,
Pubmed
Saka,
Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1.
1997,
Pubmed
Tada,
Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin.
2001,
Pubmed
,
Xenbase
Tanaka,
Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases.
1998,
Pubmed
Toyoshima,
p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21.
1994,
Pubmed
Walter,
Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts.
2000,
Pubmed
,
Xenbase
Walter,
Regulated chromosomal DNA replication in the absence of a nucleus.
1998,
Pubmed
,
Xenbase
Walter,
Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha.
2000,
Pubmed
,
Xenbase
Wang,
Genetic and physical interactions between DPB11 and DDC1 in the yeast DNA damage response pathway.
2002,
Pubmed
Wang,
DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae.
1999,
Pubmed
Wohlschlegel,
Inhibition of eukaryotic DNA replication by geminin binding to Cdt1.
2000,
Pubmed
,
Xenbase
Wohlschlegel,
Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and stimulates origin binding of Cdc45.
2002,
Pubmed
,
Xenbase
Yamamoto,
The Drosophila mus101 gene, which links DNA repair, replication and condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1 C-terminus domains.
2000,
Pubmed
Yamane,
A DNA damage-regulated BRCT-containing protein, TopBP1, is required for cell survival.
2002,
Pubmed
Yamane,
A DNA-topoisomerase-II-binding protein with eight repeating regions similar to DNA-repair enzymes and to a cell-cycle regulator.
1997,
Pubmed
Zou,
Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin.
1998,
Pubmed
Zou,
Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase.
2000,
Pubmed
