Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol epsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.

The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G 1 /S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was ϳ80 bp, and the MCM2-7: ORC ratio on this fragment was ϳ1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.In eukaryotic organisms, DNA replication initiates at many sites (1). In Saccharomyces cerevisiae, DNA replication initiates every ϳ40 kb at autonomously replicating sequences that recruit the origin recognition complex (ORC), 1 the sixsubunit initiator protein. In metazoans, initiation sites are less rigidly defined. In embryonic cells of Xenopus laevis, DNA replication initiates once every ϳ10 kb without sequence specificity (2). In somatic cells, initiation events are less frequent, occurring once every ϳ150 kb, and recent evidence indicates that initiations are controlled by genetic elements (1). At some loci, replication initiates at a precise location, whereas at other loci, initiation events are distributed throughout zones spanning up to 50 kb.The mechanism of DNA replication initiation is highly conserved among eukaryotic organisms (3, 4). A representative model system is Xenopus egg extracts (2, 4) where two factors, Cdc6 and Cdt1, bind to sites on chromatin that are marked by ORC. Subsequently, the hexameric MCM2-7 complex binds to the ORC-Cdc6-Cdt1 complex to establish the pre-replication complex (pre-RC). At the onset of DNA replication, the pre-RC is activated by the sequential action of the protein kinases Cdc7/Dbf4 and Cdk2/cyclin E (Cdk) (5, 6). Genetic and biochemical experiments suggest that the function of Cdc7/Dbf4 is the phosphorylation of the MCM2-7 complex (7). After MCM phosphorylation by Cdc7/Dbf4, Cdk2/cyclin E stimulates the association of Cdc45 with the pre-RC, likely via a direct interaction with the MCM2-7 complex (8, 9). The binding of Cdc45 coincides with activation of a chromatin-bound helicase that unwinds the DNA, allowing binding of the single-stranded...

Embryonic stem (ES) cells require a coordinated network of transcription factors to maintain pluripotency or trigger lineage specific differentiation. Central to these processes are the proteins Oct4, Nanog, and Sox2. Although the transcriptional targets of these factors have been extensively studied, very little is known about how the proteins themselves are regulated, especially at the post-translational level. Post-translational modifications are well documented to have broad effects on protein stability, activity, and cellular distribution. Here, we identify a key lysine residue in the nuclear export signal of Sox2 that is acetylated, and demonstrate that blocking acetylation at this site retains Sox2 in the nucleus and sustains expression of its target genes under hyperacetylation or differentiation conditions. Mimicking acetylation at this site promotes association of Sox2 with the nuclear export machinery. In addition, increased cellular acetylation leads to reduction in Sox2 levels by ubiquitination and proteasomal degradation, thus abrogating its ability to drive transcription of its target genes. Acetylation-mediated nuclear export may be a commonly used regulatory mechanism for many Sox family members, as this lysine is conserved across species and in orthologous proteins. STEM CELLS

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.

Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single-and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase ␣-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase ␣-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.Eukaryotic DNA replication is carried out by large multiprotein machines that coordinate DNA unwinding and synthesis at the replication fork. Initiation of replication involves ordered assembly of the replisome and local denaturation of duplex DNA at the origin followed by replisome activation. Screens for mutants defective in minichromosome maintenance (Mcm) 4 and DNA replication in yeast identified a number of factors essential for replication (1-4). Pre-replicative complexes composed of the origin recognition complex, Cdc6, Cdt1, and the hexameric Mcm2-7 helicase are assembled in G 1 (for review, see Ref. 5) and converted into active replication forks at the onset of S phase. Mcm10 loads onto chromatin after pre-replicative complex assembly (6, 7) and stimulates phosphorylation of Mcm2-7 by Dbf4-Cdc7 kinase (8). Once Mcm10 is present, Cdc45 and GINS are loaded onto chromatin (6, 9, 10) and form a Cdc45/Mcm2-7/GINS helicase complex (11-14). Cyclinand Dbf4-dependent kinases together with Sld2, Sld3, and Dpb11 in budding yeast (15, 16) stimulate origin unwinding, which is signified by recruitment of replication protein A to singlestranded DNA (17, 18). Mcm10, Cdc45, and replication protein A facilitate subsequent loading of DNA polymerase ␣-primase (pol ␣) onto chromatin (7,9,19,20). The association of proliferating cell nuclear antigen, RFC, and replicative DNA polymerases ␦ and ⑀ with the origin completes the replisome (for review, see Ref. 21).A number of interactions have been observed between Mcm10 and proteins found in the pre-replicative complexes and at the replication fork. Mcm10 is a component of active replication complexes in Xenopus and budding yeast (12,14) and is associated with chromatin throughout S-phase (7). Mcm10 interacts genetically w...