Suppression of targeting of Dbf4-dependent kinase to pre-replicative complex in G0 nuclei.

Intact G0 nuclei isolated from quiescent cells are not capable of DNA replication in interphase Xenopus egg extracts, which allow efficient replication of permeabilized G0 nuclei. Previous studies have shown multiple control mechanisms for maintaining the quiescent state, but DNA replication inhibition of intact G0 nuclei in the extracts remains poorly understood. Here, we showed that pre-RC is assembled on chromatin, but its activation is inhibited after incubating G0 nuclei isolated from quiescent NIH3T3 cells in the extracts. Concomitant with the inhibition of replication, Mcm4 phosphorylation mediated by Dbf4-dependent kinase (DDK) as well as chromatin binding of DDK is suppressed in G0 nuclei without affecting the nuclear transport of DDK. We further found that the nuclear extracts of G0 but not proliferating cells inhibit the binding of recombinant DDK to pre-RC assembled plasmids. In addition, we observed rapid activation of checkpoint kinases after incubating G0 nuclei in the egg extracts. However, specific inhibitors of ATR/ATM are unable to promote DNA replication in G0 nuclei in the egg extracts. We suggest that a novel inhibitory mechanism is functional to prevent the targeting of DDK to pre-RC in G0 nuclei, thereby suppressing DNA replication in Xenopus egg extracts.

MCM interference during licensing of DNA replication in Xenopus egg extracts-Possible Role of a C-terminal region of MCM3.

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.

RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in Xenopus egg extracts.

Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase α and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.

Evolutionary diversification of MCM3 genes in Xenopus laevis and Danio rerio.

Embryonic cell cycles of amphibians are rapid and lack zygotic transcription and checkpoint control. At the mid-blastula transition, zygotic transcription is initiated and cell divisions become asynchronous. Several cell cycle-related amphibian genes retain 2 distinct forms, maternal and zygotic, but little is known about the functional differences between these 2 forms of proteins. The minichromosome maintenance (MCM) 2-7 complex, consisting of 6 MCM proteins, plays a central role in the regulation of eukaryotic DNA replication. Almost all eukaryotes retain just a single MCM gene for each subunit. Here we report that Xenopus and zebrafish have 2 copies of MCM3 genes, one of which shows a maternal and the other a zygotic expression pattern. Phylogenetic analysis shows that the Xenopus and zebrafish zygotic MCM3 genes are more similar to their mammalian MCM3 ortholog, suggesting that maternal MCM3 was lost during evolution in most vertebrate lineages. Maternal MCM3 proteins in these 2 species are functionally different from zygotic MCM3 proteins because zygotic, but not maternal, MCM3 possesses an active nuclear localization signal in its C-terminal region, such as mammalian MCM3 orthologs do. mRNA injection experiments in zebrafish embryos show that overexpression of maternal MCM3 impairs proliferation and causes developmental defects, whereas zygotic MCM3 has a much weaker effect. This difference is brought about by the difference in their C-terminal regions, which contain putative nuclear localization signals; swapping the C-terminal region between maternal and zygotic genes diminishes the developmental defects. This study suggests that evolutionary diversification has occurred in MCM3 genes, leading to distinct functions, possibly as an adaption to the rapid DNA replication required for early development of Xenopus and zebrafish.

Non-SCF-type F-box protein Roy1/Ymr258c interacts with a Rab5-like GTPase Ypt52 and inhibits Ypt52 function.

Skp1/Cul1/F-box (SCF)-type F-box proteins are a component of the Cullin-RING SCF ubiquitin E3 ligase, which is involved in numerous cellular processes. However, the function of non-SCF-type F-box proteins remains largely unknown. The Rab5-like small guanosine 5'-triphosphatase Vps21/Ypt51 is a key regulator of intracellular transportation; however, deletion of its isoforms, Ypt52 and Ypt53, results in only a modest inhibition of intracellular trafficking. The function of these proteins therefore remains largely elusive. Here we analyze the role of a previously uncharacterized non-SCF-type F-box protein, Roy1/Ymr258c, in cell growth and intracellular transport in Saccharomyces cerevisiae. Roy1 binds to Ypt52 under physiological conditions, and Skp1 is indispensable for the association of Roy1 with Ypt52. The vps21Δ yeast cells exhibit severe deficiencies in cell growth and intracellular trafficking, whereas simultaneous deletion of roy1 alleviates the defects caused by deletion of vps21. However, additional disruption of ypt52 in roy1Δvps21Δ cells largely suppresses the cell growth and trafficking observed in roy1Δvps21Δ cells. We demonstrate that Roy1 interacts with guanosine 5'-diphosphate-bound and nucleotide-free Ypt52 and thereby inhibits the formation of guanosine 5'-triphosphate-bound, active Ypt52. These results thus indicate that Roy1 negatively modulates cell viability and intracellular transport by suppressing Ypt52.

Cul8/Rtt101 forms a variety of protein complexes that regulate DNA damage response and transcriptional silencing.

The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical analysis of Cul8 complexes. Cul8 was found to form a Cul8-Mms1-Mms22-Esc4 complex under physiological conditions, with Mms1 bridging Cul8 and Mms22 and Mms22 bridging Mms1 and Esc4. Domain analysis demonstrated that the N-terminal region of Mms1 and the C-terminal region of Mms22 are required for the Mms1-Mms22 interaction, whereas the N-terminal region of Mms22 is required for the Mms22-Esc4 interaction. We also found other Cul8-Mms1-binding proteins Ctf4, Esc2, and Orc5 using yeast two-hybrid screening. Esc4 and Ctf4 bound to Mms22 directly and bound to Cul8-Mms1 in the presence of Mms22, whereas Esc2 and Orc5 interacted with both Cul8 and Mms1, independently. We found that Cul8, Mms1, and Mms22 participated in the regulation of transcriptional silencing of yeast telomeres. These results suggest that Cul8-Mms1, as part of various protein complexes, is involved in the regulation of chromatin metabolism.

SCF(Dia2) regulates DNA replication forks during S-phase in budding yeast.

Dia2 is an F-box protein, which is involved in the regulation of DNA replication in the budding yeast Saccharomyces cerevisiae. The function of Dia2, however, remains largely unknown. In this study, we report that Dia2 is associated with the replication fork and regulates replication fork progression. Using modified yeast two-hybrid screening, we have identified components of the replisome (Mrc1, Ctf4 and Mcm2), as Dia2-binding proteins. Mrc1 and Ctf4 were ubiquitinated by SCF(Dia2) both in vivo and in vitro. Domain analysis of Dia2 revealed that the leucine-rich repeat motif was indispensable for the regulation of replisome progression, whereas the tetratricopeptide repeat (TPR) motif was involved in the interaction with replisome components. In addition, the TPR motif was shown to be involved in Dia2 stability; deleting the TPR stabilized Dia2, mimicking the effect of DNA damage. ChIP-on-chip analysis illustrated that Dia2 localizes to the replication fork and regulates fork progression on hydroxyurea treatment. These results demonstrate that Dia2 is involved in the regulation of replisome activity through a direct interaction with replisome components.

A longevity protein, Lag2, interacts with SCF complex and regulates SCF function.

SCF-type E3-ubiquitin ligases control numerous cellular processes through the ubiquitin-proteasome pathway. However, the regulation of SCF function remains largely uncharacterized. Here, we report a novel SCF complex-interacting protein, Lag2, in Saccharomyces cerevisiae. Lag2 interacts with the SCF complex under physiological conditions. Lag2 negatively controls the ubiquitylation activities of SCF E3 ligase by interrupting the association of Cdc34 to SCF complex. Overexpression of Lag2 increases unrubylated Cdc53, whereas deletion of lag2, together with the deletions of dcn1 and jab1, results in the accumulation of Rub1-modified Cdc53. In vitro rubylation assays show that Lag2 inhibits the conjugation of Rub1 to Cdc53 in competition with Dcn1, which suggest that Lag2 down-regulates the rubylation of Cdc53 rather than promoting derubylation. Furthermore, Dcn1 hinders the association of Lag2 to Cdc53 in vivo. Finally, the deletion of lag2 combined with the deletion of either dcn1 or rub1 suppresses the growth of yeast cells. These observations thus indicate that Lag2 has a significant function in regulating the SCF complex by controlling its ubiquitin ligase activities and its rubylation cycle.

Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control.

Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae. CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms. We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs. We present evidence indicating that this Clb2-dependent mechanism contributes to preventing re-replication in vivo. CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides. Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites. Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides. We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.

CDK- and Cdc45-dependent priming of the MCM complex on chromatin during S-phase in Xenopus egg extracts: possible activation of MCM helicase by association with Cdc45.

BACKGROUND: MCM and Cdc45 are required for the initiation and elongation stages of eukaryotic DNA replication. Recent studies show that a purified Mcm4/6/7 complex has DNA helicase activity. However, the biochemical function of the MCM complex and Cdc45 bound to chromatin has not been elucidated. RESULTS: We have examined the biochemical properties of MCM proteins bound to chromatin fractions using Xenopus egg extracts. Immunoprecipitation of MCM proteins extracted under denaturing conditions reveals that all six subunits of MCM and Cdc45 form a tight complex following the initiation of DNA replication, and that both CDK activity and Cdc45 are essential for the complex formation. Chromatin immunoprecipitation of MCM proteins and Cdc45 shows that a complex containing MCM and Cdc45 has a DNA helicase activity which is dependent on CDK activity and Cdc45 in the extracts. Furthermore, both the complex and the helicase activity are resistant to treatment with phosphatase and high salt. CONCLUSIONS: Following the initiation of DNA replication, a tight MCM-Cdc45 complex is formed on chromatin and its formation is closely correlated with the DNA helicase activity of chromatin immunoprecipitates containing MCM and Cdc45. We propose that the tight MCM-Cdc45 complex functions as a replicative DNA helicase in vivo.