The human pre-replication complex is an open complex

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Transient dsDNA breaks during pre-replication complex assembly

Initiation of DNA replication involves the ordered assembly of the multi-protein pre-replicative complex (pre-RC) during G₁ phase. Previously, DNA topoisomerase II (topo II) was shown to associate with the DNA replication origin located in the lamin B2 gene locus in a cell-cycle-modulated manner. Here we report that activation of both the early-firing lamin B2 and the late-firing hOrs8 human replication origins involves DNA topo II-dependent, transient, site-specific dsDNA-break formation. Topo IIβ in complex with the DNA repair protein Ku associates in vivo and in vitro with the pre-RC region, introducing dsDNA breaks in a biphasic manner, during early and mid-G₁ phase. Inhibition of topo II activity interferes with the pre-RC assembly resulting in prolonged G₁ phase. The data mechanistically link DNA topo IIβ-dependent dsDNA breaks and the components of the DNA repair machinery with the initiation of DNA replication and suggest an important role for DNA topology in origin activation.     

Distinct phosphoisoforms of the Xenopus Mcm4 protein regulate the function of the Mcm complex

Initiation of DNA replication in eukaryotes requires the assembly of prereplication complexes (pre-Rcs) at the origins of replication. The assembly and function of the pre-Rcs appear to be controlled by phosphorylation events. In this study we report the detailed characterization of the cell cycle phosphorylation of one component of the Xenopus pre-Rcs, the Mcm protein complex. We show that individual Mcm subunits are differentially phosphorylated during the cell cycle. During mitosis, the Mcm4 subunit is hyperphosphorylated, while the other subunits are not actively phosphorylated. The mitotic phosphorylation of Mcm4 requires Cdc2-cyclin B and other unknown kinases. Following exit from mitosis, the Mcm4 subunit of the cytosolic interphase complex undergoes dephosphorylation, and the Mcm2, Mcm3, or Mcm6 subunits are then actively phosphorylated by kinase(s) other than cyclin-dependent kinases (Cdks) or Cdc7. The association of the Mcm complex with the pre-Rcs correlates with the formation of a transient interphase complex. This complex contains an intermediately phosphorylated Mcm4 subunit and is produced by partial dephosphorylation of the mitotic hyperphosphorylated Mcm4 protein. Complete dephosphorylation of the Mcm4 subunit inactivates the Mcm complex and prevents its binding to the chromatin. Once the Mcm complex is assembled on the chromatin the Mcm4 and the Mcm2 proteins are the only subunits phosphorylated during the activation of the pre-Rcs. These chromatin-associated phosphorylations require nuclear transport and are independent of Cdk2-cyclin E. These results suggest that the changes in Mcm4 phosphorylation regulate pre-Rc assembly and the function of the pre-Rcs on the chromatin.     

Mcm10 proteolysis initiates before the onset of M-phase

Background  

Mcm10 protein is essential for initiation and elongation phases of replication. Human cells proteolyze Mcm10 during mitosis, presumably to ensure a single round of replication. It has been proposed that anaphase promoting complex ubiquitinates Mcm10 in late M and early G₁ phases.     

The basal body-root complex ofChlamydomonas reinhardtii during mitosis

Summary The results of this work clarify several structural and temporal aspects of biogenesis of the basal body-root complex inChlamydomonas reinhardtii. The two phases of basal body development (probasal body assembly and conversion of probasal body into mature basal body) occur at identical mitotic stages in successive mitoses during multiple fission, which indicates a tight coupling between basal body development and the mitotic cycle. The two steps of basal body development are separated from one another in time,i.e. immature probasal bodies originate during an interval lasting ca. 5 min between mid-metaphase and early telophase, but mature after a quasi-dormant period only during early prophase of the next mitotic round. The duration of the dormant period depends on the interval between two mitoses: during synchronized vegetative growth there is an interval of ca. 20 h (interphase growth) between two rounds of multiple fissions, but only a maximum interval of 1.5 h between the successive mitoses of one round of multiple fissions.The microtubular root system, which is bisected at the same time as the basal body apparatus in a plane perpendicular to the distal connecting fiber during prophase, and whose roots seem to be reduced in length, starts duplication at early metaphase with the successive origin of two short bud-like partner roots just opposite the remnants. These initial roots elongate during subsequent phases by unilateral and radial growth from the basal bodies and along the cell's periphery, but exactly where they terminate is not known. The two-stranded roots opposite each other appear to be again connected as early as anaphase.The striation pattern of the distal connecting fiber is lost during early prophase thus indicating a partial breakdown of the fiber.Dedicated to Prof. Dr. C.-G. Arnold (Erlangen) on the occasion of his 60th birthday.     

Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.     

Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle

We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase, approximately 30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins.     

Origin single-stranded DNA releases Sld3 protein from the Mcm2-7 complex, allowing the GINS tetramer to bind the Mcm2-7 complex

The replication fork helicase in eukaryotic cells is comprised of Cdc45, Mcm2-7, and GINS (CMG complex). In budding yeast, Sld3, Sld2, and Dpb11 are required for the initiation of DNA replication, but Sld3 and Dpb11 do not travel with the replication fork. Sld3 and Cdc45 bind to early replication origins during the G(1) phase of the cell cycle, whereas Sld2, GINS, polymerase ε, and Dpb11 form a transient preloading complex that associates with origins during S phase. We show here that Sld3 binds tightly to origin single-stranded DNA (ssDNA). CDK-phosphorylated Sld3 binds to origin ssDNA with similar high affinity. Origin ssDNA does not disrupt the interaction between Sld3 and Dpb11, and origin ssDNA does not disrupt the interaction between Sld3 and Cdc45. However, origin ssDNA substantially disrupts the interaction between Sld3 and Mcm2-7. GINS and Sld3 compete with one another for binding to Mcm2-7. However, in a mixture of Sld3, GINS, and Mcm2-7, origin ssDNA inhibits the interaction between Sld3 and Mcm2-7, whereas origin ssDNA promotes the association between GINS and Mcm2-7. We also show that origin single-stranded DNA promotes the formation of the CMG complex. We conclude that origin single-stranded DNA releases Sld3 from Mcm2-7, allowing GINS to bind Mcm2-7.     

Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus

The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing. Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea. The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue. The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro. The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA. Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA. The purified Orc/Cdc6 homologue was also able to bind DNA. Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates. Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap. The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer.     

Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex

Mcm, which is composed of six structurally related subunits (Mcm2-7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the Mcm4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the Mcm4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the Mcm4/6/7 complex containing a zinc finger mutant of Mcm4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The Mcm4/6/7 complex containing this Mcm4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of Mcm4 is involved in subunit interactions of trimers. The Mcm4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric Mcm4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003-8015), our data indicate distinct roles of Mcm4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the Mcm4/6/7 complex.     

The chromosomal passenger complex takes center stage during mitosis

The chromosomal passenger complex plays important roles in key mitotic events, including chromosome bi-orientation, the spindle assembly checkpoint, and cytokinesis. Two groups now report the identification of a novel component of the Incenp/survivin/auroraB complex (Gassmann et al., 2004; Sampath et al., 2004) and show that different subcomplexes may exist during mitosis. Exciting data support the involvement of the passenger complex in yet another key event, the assembly of the mitotic spindle.     

Localization of human Mcm10 is spatially and temporally regulated during the S phase

Mcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle. However, the precise roles that Mcm10 plays are still unknown. To study the localization dynamics of human Mcm10, we established HeLa cell lines expressing green fluorescent protein (GFP)-tagged Mcm10. From early to mid-S phase, GFP-Mcm10 appeared in discrete nuclear foci. In early S phase, several hundred foci appeared throughout the nucleus. In mid-S phase, the foci appeared at the nuclear periphery and nucleolar regions. In the late S and G phases, GFP-Mcm10 was localized to nucleoli. Although (2)the distributions of GFP-Mcm10 during the S phase resembled those of replication foci, GFP-Mcm10 foci did not colocalize with sites of DNA synthesis in most cases. Furthermore, the transition of GFP-Mcm10 distribution patterns preceded changes in replication foci patterns or proliferating cell nuclear antigen foci patterns by 30-60 min. These results suggest that human Mcm10 is temporarily recruited to the replication sites 30-60 min before they replicate and that it dissociates from chromatin after the activation of the prereplication complex.     

Part of Xenopus translin is localized in the centrosomes during mitosis

During oogenesis, maternal mRNAs are synthesised and stored in a translationally dormant form due to the presence of regulatory elements at the 3' untranslated regions (3'UTR). In Xenopus oocytes, several studies have described the presence of RNA-binding proteins capable to repress maternal-mRNA translation. The testis-brain RNA-binding protein (TB-RBP/Translin) is a single-stranded DNA- and RNA-binding protein which can bind the 3' UTR regions (Y and H elements) of stored mRNAs and can suppress in vitro translation of the mRNAs that contain these sequences. Here we report the cloning of the Xenopus homologue of the TB-RBP/Translin protein (X-translin) as well as its expression, its localisation, and its biochemical association with the protein named Translin associated factor X (Trax) in Xenopus oocytes. The fact that this protein is highly present in the cytoplasm from stage VI oocytes until 48 h embryos and that it has been described as capable to inhibit paternal mRNA translation, indicates that it could play an important role in maternal mRNA translation control during Xenopus oogenesis and embryogenesis. Moreover, we investigated X-translin localisation during cell cycle in XTC cells. In interphase, although a weak and diffuse nuclear staining was observed, X-translin was mostly present in the cytoplasm where it exhibited a prominent granular staining. Interestingly, part of X-translin underwent a remarkable redistribution throughout mitosis and associated with centrosomes, which may suggest a new unknown role for this protein in cell cycle.     

The Mcm467 complex of Saccharomyces cerevisiae is preferentially activated by autonomously replicating DNA sequences

We have analyzed the role of single-stranded DNA (ssDNA) in the modulation of the ATPase activity of Mcm467 helicase of the yeast Saccharomyces cerevisiae. The ATPase activity of the Mcm467 complex is modulated in a sequence-specific manner and that the ssDNA sequences derived from the origin of DNA replication of S. cerevisiae autonomously replicating sequence 1 (ARS1) are the most effective stimulators. Synthetic oligonucleotides, such as oligo(dA) and oligo(dT), also stimulated the ATPase activity of the Mcm467 complex, where oligo(dT) was more effective than oligo(dA). However, the preference of a thymidine stretch appeared unimportant, because with yeast ARS1 derived sequences, the A-rich strand was as effective in stimulating the ATPase activity, as was the T-rich strand. Both of these strands were more effective stimulators than either oligo(dA)( )()or oligo(dT). The DNA helicase activity of Mcm467 complex is also significantly stimulated by the ARS1-derived sequences. These results indicate that the ssDNA sequences containing A and B1 motifs of ARS1, activate the Mcm467 complex and stimulate its ATPase and DNA helicase activities. Our results also indicate that the yeast replication protein A stimulated the ATPase activity of the Mcm467 complex.     

Deubiquitinating enzyme USP36 contains the PEST motif and is polyubiquitinated

The ubiquitin-mediated protein degradation pathway has been emphasized for the regulation of numerous cellular mechanisms and the significance of deubiquitination, mediated by deubiquitinating (DUB) enzymes, has been emerging as an essential regulatory step to control these cellular mechanisms. Previously, we demonstrated a human DUB enzyme, HeLa DUB-1, expressed in human ovarian cancer cells. Here, we report human USP36, which has the extension of the C-terminal region of HeLa DUB-1 and has conserved amino acid domains as previously shown in other DUBs. Human USP36, encoding a DUB enzyme, was isolated from ovarian cancer cells using RT-PCR and characterized. We identified DUB enzyme activity of USP36 by analyzing its capability to cleave the ubiquitin. Interestingly, structural and immunoprecipitation analyses revealed for the first time that USP36 contains the PEST motif and is polyubiquitinated.