::::::: My Cyber Lab(s) : ::::::
eIMBL Unit Labs
Faculty Members
DNA Replication
Systems Biology
Genomic Medicine
Molecular Oriental Medicine
Infectious Disease
DNA Replication
Hiroyuki ARAKI, Ph.D.
- A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2-Dpb11
Phosphorylation often regulates protein-protein interactions to control biological reactions. The Sld2 and Dpb11 proteins of budding yeast form a phosphorylation-dependent complex that is essential for chromosomal DNA replication. The Sld2 protein has a cluster of 11 cyclin-dependent kinase (CDK) phosphorylation-motifs (Ser/Thr-Pro), six of which match the canonical sequences Ser/Thr-Pro-X-Lys/Arg, Lys/Arg-Ser/Thr-Pro and Ser/Thr-Pro-Lys/Arg. Simultaneous alanine-substitution for serine or threonine in all the canonical CDK-phosphorylation motifs severely reduces complex formation between Sld2 and Dpb11, and inhibits DNA replication. Here we show that phosphorylation of these canonical motifs does not play a direct role in complex formation but rather regulates phosphorylation of another residue, Thr84. This constitutes a non-canonical CDK-phosphorylation motif within a 28-amino acid sequence that is responsible, after phosphorylation, for binding of Sld2 to Dpb11. We further suggest that CDK-catalysed phosphorylation of sites other than Thr84 renders Thr84 accessible to CDK. Finally, we argue that this novel mechanism sets a threshold of CDK activity for formation of the essential Sld2-Dpb11 complex and therefore prevents premature DNA replication.
Arturo FALASCHI, M.D. Ph.D.
- Functional interactions of DNA topoisomerases with the lamin B2 replication origin.
The human DNA replication origin located in the lamin B2 gene interacts with the DNA topoisomerases I and II in a cell cycle-modulated fashion. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in late M, early G1 and G1/S border and topoisomerase II in early M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it directly interacts with topoisomerase II during the assembly of the pre-replicative complex and with topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are members of the replicative complexes and DNA topology plays an essential functional role for origin activation.
Masatoshi FUJITA, M.D. Ph.D.
- Cell cycle regulation of DNA replication in mammalian cells and its implication in carcinogenesis
Genomic DNA has to be replicated completely and only once during the cell cycle. The MCM complex, a candidate replicative helicase, is loaded onto chromatin by ORC, CDC6 and Cdt1 proteins. After S phase, the actions of these initiation factors should be suppressed to prevent re-replication. We found that the activity of human Cdt1 is negatively regulated through Cdk phosphorylation and subsequent ubiquitination by SCFSkp2 as well as by an inhibitory protein geminin. Such strict regulation suggests that Cdt1 deregulation may have a deleterious effect. In fact, we found that Cdt1 is overexpressed in cancer cells. Interestingly, Cdt1 overexpression at pathophysiological levels induces chromosomal damage without rereplication and activates an ATM/Chk2 damage checkpoint pathway. More importantly, deregulated Cdt1 induces chromosomal instability in normal human cells. These data highlight a new and important link between deregulation of DNA replication initiation and genetic instability and, presumably, eventual carcinogenesis. We have identified several novel Cdt1-binding proteins by a proteomics approach. Among them, involvement of the APC/CCdh1 ubiquitin ligase in proteolytic regulation of Cdt1 was clarified. In addition, it turned out that Cullin 4-based ubiquitin ligase also controls Cdt1 during the S phase and that PCNA mediates this ubiquitination.
Deog Su HWANG, Ph.D.
- TopBP1 is required for the cell cycle progression in mammalian cells
TopBP1 (Topoisomerase II binding protein 1) contains eight BRCT domains and is mainly reported to be involved in DNA damage response pathway as many other DNA repair proteins which have BRCTs. Other roles of TopBP1 in human cells have yet to be found, but several studies on TopBP1 homologues such as Dpb11, Cut5, Mus101, and Xmus101/Xcut5 have shown that they are also required for progression of the cell cycle such as DNA replication or cell division. Here we used RNA interference (RNAi) method for the further study on the role of TopBP1. TopBP1 depletion do not activate the DNA checkpoint pathway, but it caused more than one defect in the cell cycle progression at different points, which suggests that TopBP1 protein plays a direct role in the normal cell cycle progression as well as under the DNA damaged condition.
- Knockdown of human MCM10 causes slowed and incomplete chromosome replication followed by G2 phase arrest
MCM10 protein is required for Cdc45 chromatin loading for chromosome replication initiation in S. pombe and X. laevis. However, the function of human MCM10 has not been addressed. To investigate the role of MCM10 in human chromosome replication, we performed MCM10 knockdown experiments using siRNA. The knockdown accumulated S and G2 phase cells. Under our experimental conditions, chromosome replication during early- and mid-S phase was retarded and late-S replication was defective and incomplete, which appeared to be caused by less efficient and defective initiations at replication origins. The defective replication, which was followed by DNA breaks and eventually cell death, activated a checkpoint pathway composed of Chk1 and Cdc25 to inhibit Cdk1 necessary for G2 to mitotic phase transition. Thus, MCM10-depleted cells were arrested in G2 phase. Our results indicate that human MCM10 is essential for human chromosome replication and cell growth.
Tsutomu KATAYAMA, Ph.D.
- Protein associations in DnaA-ATP hydrolysis mediated by the replicase clamp-Hda complex
In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the ?-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified Hda mutant proteins were used in a staged in vitro RIDA system followed by pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arginine-168 in the AAA+ Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis but not in clamp-binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain about 50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA+ proteins, including DnaA, suggest the following model: DnaA-ATP is hydrolyzed at a binding interface between the AAA+ domains of DnaA and Hda, the DnaA N-terminal domain supports this interaction, and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.
- Formation of an ATP-DnaA-specific initiation complex requires DnaA arginine-285, a conserved motif in the AAA+ protein family
Escherichia coli DnaA protein, a member of the AAA+ superfamily, initiates replication from the chromosomal origin oriC in an ATP-dependent manner. Nucleoprotein complex formed on oriC with the ATP-DnaA multimer but not the ADP-DnaA multimer is competent to unwind the oriC duplex. The oriC region contains ATP-DnaA-specific binding sites termed I2 and I3, which stimulate ATP-DnaA-dependent oriC unwinding. In this study, we show that the DnaA R285A mutant is inactive for oriC replication in vivo and in vitro and that the mutation is associated with specific defects in oriC unwinding. In contrast, activities of DnaA R285A are sustained in binding to the typical DnaA boxes and to ATP and ADP, formation of multimeric complexes on oriC, and loading of the DnaB helicase onto single-stranded DNA. Footprint analysis of the DnaA-oriC complex reveals that the ATP form of DnaA R285A does not interact with ATP-DnaA-specific binding sites such as the I sites. A subgroup of DnaA molecules in the oriC complex must contain the R285 residue for initiation. Sequence and structural analyses suggest that the DnaA R285 residue is an arginine finger, an AAA+ family-specific motif that recognizes ATP bound to an adjacent subunit in a multimeric complex. In the context of these and previous results, the DnaA R285 residue is proposed to play a unique role in the ATP-dependent conformational activation of an initial complex by recognizing ATP bound to DnaA and by modulating the structure of the DnaA multimer to allow interaction with ATP-DnaA-specific binding sites in the complex.
Joon Kyu LEE, Ph.D.
Chun LIANG, Ph.D.
Hisao MASAI, Ph. D.
- Biochemical characterization of MCM complexes
MCM is composed of six subunits, MCM2-7, which are members of AAA family proteins and share the conserved ATP binding domain. Previous biochemical characterization of mammalian MCM proteins indicated that MCM4-6-7 forms a complex, which shows DNA helicase activity. We have shown essential role of ATP binding domain of MCM6 and MCM7 for ATPase and DNA helicase activities of MCM4-6-7, while the ATP binding domain of MCM4 is required for its DNA binding activity. We then examined the effect of the substrate structures on DNA helicase activity of MCM4-6-7. We discovered that ATPase and DNA helicase activities of MCM4-6-7 are specifically activated by thymine-rich single-stranded DNA present on the substrate DNAs. Using a ¡°bubble¡± DNA substrate, which mimics the activated replication origin, we have shown that the T-rich sequence from the known human replication origin sequences, including lamin-B2 and c-myc origins, can efficiently activate the MCM helicase activity and that replacement of the thymine residues with guanine completely abrogated this activation. Thus, MCM helicase may be specifically activated at the replication origins through interaction with the melted thymine-rich strands frequently discovered at the initiation sites. On the basis of these results, we proposed a model in which MCM may play a critical role in the process of origin selection through site-specific activation of its helicase activity by the exposed thymine-rich single-stranded DNA.
- Processing of arrested replication forks and cellular responses for maintenance of genetic integrity
The moving replication forks are interfered by a number of internal and external causes. It is becoming apparent that the progression of replication forks pauses or stalls even during normal course of DNA replication, and intricate networks of cellular responses to stalled replication forks have evolved. If something goes wrong with these systems, cells cannot maintain the integrity of the fork, leading to incomplete S phase, and eventually to cell death due to extensive DNA damages. The replication machinery as well as other factors at the replication forks play a major role in cellular responses to stalled replication forks. We have recently shown that Cdc7 kinase plays a major role in transmission of arrested replication fork signal to downstream mediator kinases. We also have evidence that Cdc7 interacts with replication fork protection factors to stabilize arrested replication forks (see below) and would like to elucidate its molecular mechanisms.
We are also studying E. coli protein PriA, a DEXH-type helicase which recognizes the arrested replication forks. PriA recognizes and binds to the arrested forks, and can form a stable complex through its 3¡¯-end recognition pocket when the fork carries a 3¡¯-end of the arrested leading strand at the junction. We have demonstrated that PriA, in collaboration with another helicase RecG, binds and stabilize the arrested replication forks. We wish to identify eukaryotic proteins with similar structure and functions.
- Functional dissection of Hsk1 kinase, a fission yeast homologue of Cdc7 kinase
Hsk1, a fission homologue of Cdc7 kinase, is required for S phase initiation and progression. We have isolated a hsk1ts mutant (hsk1-89) and identified a number of genes which genetically interact with hsk1-89. These include rad3, cdc19 (Mcm2), rad26, swi1 and mrc1 (Claspin). We have shown that Hsk1 is required for activation of Cds1 kinase in response to replication fork block. When Hsk1 kinase is blocked, cells accumulate DNA damages and eventually die. This phenotype is augmented in combination with swi1 mutation. Hsk1 and Swi1 physically interact and contribute to the stabilization of arrested replication forks. We hope to elucidate the molecular mechanism of how Cdc7 protects replication forks in conjunction with Swi1/ Mrc1 fork protection factors.
- Role of Cdc7 kinase in meiotic recombination
We previously generated a mutant mice in which Cdc7 kinase activity is decreased. The mutant mice were born but reduced in body size mainly due to decreased cell numbers. Most notable feature of the mutant mice is infertility and almost complete loss of germ cell development. Generation of both testes and ovaries is impaired. The examination of the defective testes in the mutant mice indicated arrest of testes development at early premiotic stage.
We therefore examined the roles of Cdc7 kinase during meiosis in fission yeast. We have used hsk1-89, a temperature-sensitive mutant of Hsk1, for this purpose. In hsk1-89 diploid cells, meiosis was arrested at a premeiotic stage, indicating an essential role of Hsk1 kinase in fission yeast meiosis. We then analyzed the effect of hsk1-89 mutation in pat-1 induced meiosis in haploid cells. Unexpectedly, premeiotic DNA replication completed in hsk1-89 cells. We then examined the frequency of meiotic recombination in hsk1-89 cells, which may occur in a manner coupled to premeiotic DNA replication, and found out the meiotic recombination frequency is reduced by one order of magnitude in hsk1-89 cells. The expression of various rec+ genes, which are essential for meiotic recombination, was induced in a timing identical to the wild-type cells in the mutant. However, we found out that the double-stranded DNA breaks which are essential for initiation of meiotic recombination does not occur in hsk1-89 cells. Meiosis I and II are also inhibited in hsk1-89 cells, and Cdc2 was in an inactive state carrying the phosphorylated tyrosine 15. All theses defects were not restored by inactivation of known checkpoint kinases, namely Chk1, Cds1 and Mek1, suggesting that Hsk1 may be directly involved in DSB formation and meiosis. We further observed a loss of chromatin remodeling at the recombination hot spot, ade6-M26, suggesting a role of Hsk1 kinase in chromatin regulation essential for initiation of meiotic recombination.
- Cell cycle regulation and differentiation
Mouse embryonic stem cells are tautipotent stem cells which can maintain the undifferentiated state, while capable of differentiating into various cell linages. In order to unravel molecular basis which underlie the characteristic cell cycle profiles of undifferentiated ES cells (namely the virtual absence of G1 and G2 phases), we have examined expression of various cell cycle/ replication regulators in undifferentiated ES cells as well as in the differentiated ES cells. We have identified four key cell cycle regulators which are highly overexpressed in undifferentiated ES cells and are quickly downreglated upon induction of differentiation. They are Cdc6, ASK (Cdc7 activation subunit), CyclinA and CyclinB. It is noteworthy that these factors regulate, respectively, preRC (prereplicative complex) formation, S phase activation, S phase progression and M phase initiation.
We have found that Cdc6 protein is stabilized in undifferentiated ES cells, while it undergoes proteasome-dependent degradation upon induction of differentiation, suggesting a possibility that differential proteasome activity in undifferentiated ES cells may be responsible for the characteristic cell cycle profile. We are now investigating detailed mechanisms of how the proteasome activity is regulated in response to differentiation.
Hisao MASUKATA, Ph.D.
- Genome-wide identification of replication origins in fission yeast: Uneven distribution of active replication origins
DNA replication of eukaryotic chromosomes initiates at numbers of discrete loci, called replication origins. For duplication of entire chromosomes consisting of different chromatin structures, activation of individual replication origins is regulated. Nonetheless, precise locations of replication origins have not been determined in genome-wide range in most eukaryotes except budding yeast Saccharomyces cerevisiae. Fission yeast Schizosaccharomyces pombe is a suitable model organism to study genome-wide regulation of chromosome replication, because the structures of replication origins have similarities with those in more complex organisms with respect to the lack of any short consensus sequence and the presence of AT-rich sequences. We mapped locations of Orc1 and Mcm6 in G1 phase, which are components of pre-replicative complexes (pre-RCs), using a high-resolution tiling array covering almost the entire genome. The pre-RCs were mapped at 460 intergenic regions, distributed rather evenly with an average distance of about 25 kb except enrichment at centromeres and subtelomeric regions. However, active replication origins that incorporate 5-bromo-2¡¯-deoxyuridine (BrdU) in the presence of hydroxyurea (HU) were co-localized at 276 pre-RC sites and distributed preferentially in mega-base regions of chromosome arms. Interestingly, replication of centromeric and subtelomeric heterochromatin is regulated distinctly.
- Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins
Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. We here investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with un-replicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, while recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.
Yeon-Soo SEO, Ph.D.
Katsuhiko SHIRAHIGE, Ph.D.
Haruhiko TAKISAWA, Ph.D.
Toshiki TSURIMOTO, Ph.D.
- Molecular links between DNA replication and maintenance of genomic stability by clamp and clamp loader proteins
DNA replication fork in eukaryotes functionally links with various reactions for maintenance of genomic stability. Characteristic proteins, clamps and clamp loader complexes, play central roles for the mechanism as indicated by their multiple activities not only for DNA replication but also for DNA repair, recombination, modification, chromatin assembly and chromosome cohesion. I have been working on structures and functions of replicative clamp PCNA, its loader protein RFC and their related complexes.
My group has constructed expression systems for human proteins involved in the replication fork, and studied functions of the purified proteins. Recent results we obtained are as follows.
1. We have identified a chromosome cohesion factor, Ctf18 as a novel PCNA binding protein. This protein forms a loader-type complex, Chl12-RFC with RFC small subunits, and functions as the second PCNA loader. We have searched a DNA polymerase specifically stimulated by Ctf18-RFC together with PCNA from a human cell extract, and identified one of translesion DNA polymerases, from the active fraction.
2. We purified one of RFC-related proteins, WRNIP1, which has been identified as a Werner DNA helicase binding protein. This protein forms a self-oligomerized complex and exhibits ATPase activity. It also interacts specifically with replicative DNA polymerase ? and stimulates the activity, suggesting its role in regulation of DNA synthesis by pol ?.
3. It has been reported that PCNA is mono-ubiqitinated as a DNA damage response and switches its functions. We have established a reaction system which efficiently mono-ubiqitinates PCNA loaded on DNA. Using the mono-ubiquitinated PCNA, we have studied biochemical effects exerted by this modification.
