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History of the laboratory Goals of the laboratory Ongoing projects and recent publications Lab introduction (PDF)

Genome Dynamics Project,
Tokyo Metropolitan Institute of Medical Science

Ongoing projects and recent publications

1 Genetic program for replication origins and timing

The genetic program for where and when replication origins are activated is still elusive in mammalian cells. We have chosen cytokine cluster regions on the human chromosome 5 and mouse chromosome 11 as a model to elucidate the replication program of mammalian cells. These regions contain multiple cytokine genes, whose expression is coordinatedly and inducibly regulated during T cell activation. A large body of knowledge on transcription factors and chromatin structures involved in cytokine gene regulation have been accumulated. Furthermore, various cis-regulatory sequence mutant cells are available and differentiation from common precursor cells into specific cell lineage can be manipulated in vitro. These make the cytokine cluster region an excellent model system to elucidate how transcription and chromatin structures regulate origin activation and its timing. It also gives an opportunity to investigate how DNA replication program is influenced by differentiation processes.

Todorovic, V., Giadrossi, S., Pelizon, C., Masai, H. and Giacca, M. (2005)
"Novel human origins of DNA replication selected from a library of nascent DNA."
Mol. Cell 19, 567-575.
Hayashida,T.^#, Oda , M^#., Ohsawa K.,Yamaguchi A., Giacca, M., Locksley, R.M., Masai H.*, and Miyatake, S.* (2006) "Replication initiation from a novel origin identified in the Th2 cytokine cluster locus requires a distant conserved non-coding sequence." (^#cofirst authors; *cocommunicating authors)
J. Immunol. 176, 5446-5454.


2 Functional and structural dissection of Cdc7 kinase

Cdc7 was originally identified as a budding yeast mutant defective in initiation of DNA replication. Cdc7 encodes a serine-threonine kinase and forms a complex with Dbf4 to become an active kinase. We and others showed that Cdc7 and Dbf4 are conserved through evolution, and are required for DNA replication. Dbf4 carries three conserved motifs, namely Dbf4-motif-N. –M and –C, and mutation analyses in fission yeast Him1/Dfp1 revealed that both Dbf4-motif-M and –C binds to Hsk1 (fission yeast Cdc7 homologue) on its own, and combination of the two motifs are required for the maximum kinase activation and cell viability. The results indicate that the segment between motif-M and –C is dispensable for kinase activation, suggesting that bipartite binding of the two motifs to Hsk1 is required for efficient kinase activation. Similar conclusion was reached by the study on mammalian ASK protein, an activation subunit for mammalian Cdc7 kihase.
In order to elucidate the roles of Cdc7 kinase in mammalian cell proliferation, we attempted to generate a mutant mice lacking the Cdc7 genes. The Cdc7-/- embryo died between E3.5 and 6.5, suggesting an essential role of Cdc7 kinase in cell proliferation and/or early embryonic development. We then generated a mutant ES cell line, in which Cdc7 genes are inactivated in the presence of a flox-transgene encoding the wild-type Cdc7 cDNA. The removal of the transgene by Cre recombinase resulted in arrest of cell cycle within S phase, and accumulation of DNA damages. Cells attempt to repair the damages through recombination and induceG2/M checkpoint signals, but eventually underwent p53-dependent cell death. Similarly, conditional knockout of ASK gene in ES cells resulted in S phase arrest and cell death. Thus, a high level of p53 protein in tautipotent stem cells ensures the preservation of correct genetic information by disposing of the cells suffering from genetic damages rather than repairing them.
The knockdown of Cdc7 protein levels in cells and mice resulted in reduced growth rate and reduced body size, respectively, suggesting that the sufficient expression level of Cdc7 is required for proper cell growth. Most notably, reduction of Cdc7 protein level in germ cells resulted in severe impairment of germ cell development (see the item #7).
In human, a second activation subunit for Cdc7 kinase, ASKL1, was identified. ASKL1 forms an active kinase complex with Cdc7 and is required for efficient progression of S phase. ASKL1 is expressed at late S through M phase, and is also required for progression of M phase.

Ogino, K., Takeda, T., Matsui, E., Iiyama, H., Taniyama, C., Arai, K. and Masai, H. (2001)
"Bipartite binding of a kinase activator activates Cdc7-related kinase essential for S phase."
J. Biol. Chem. 276, 31376-31387.
Kim, J. M., Nakao, K., Nakamura, K., Saito, I., Katsuki, M., Arai, K. and Masai, H. (2002)
"Inactivation of Cdc7 kinase in mouse embryonic stem cells results in S phase arrest and p53-dependent cell death."
EMBO J. 21, 2168-2179.
Yamada, M., Sato, N., Taniyama, C., Ohtani, K., Arai, K. and Masai, H. (2002)
"A 63 base-pair DNA segment containing a Sp1 site but not a canonical E2F site can confer growth-dependent and E2F-mediated transcriptional stimulation of the human ASK gene encoding the regulatory subunit for human Cdc7-related kinase."
J. Biol. Chem. 277, 27668-27681.
Sato, N., Sato, M., Nakayama, M., Saitoh, R., Arai, K. and Masai, H. (2003)
"Cell cycle regulation of chromatin binding and nuclear localization of Cdc7-ASK kinase complex."
Genes to Cells, 8, 451-463.
Kim, J.M., Takemoto, N., Arai K. and Masai, H. (2003)
"Hypomorphic mutation in an essential cell-cycle kinase causes growth retardation and impaired spermatogenesis."
EMBO J. 22, 5260-5272.
Yoshizawa, N.,Ishii, A., Taniyama, C., Matsui, E., Arai, K., and Masai, H. (2005)
"A second human Dbf4/ASK-related protein, Drf1/ASKL1 is required for efficient completion of S and M phases."
J. Biol. Chem. 280, 13062-13070.
Yamashita, N., Kim, J-M., Koiwai, O., Arai, K. and Masai, H. (2005)
"Functional analyses of mouse ASK, an activator subunit for Cdc7 kinase, using conditional ASK knockout ES cells."
Genes to Cells 50, 551-563.


3 Architecture and regulation of replication forks during cell cycle

Replication fork is the center of DNA synthesis. A highly efficient machinery for efficient synthesis of both lagging and leading strands is assembled at the replication fork. This assembly is initiated at late M to early G1, when MCM (minichromosome maintenance) protein complexes are loaded onto chromatin through the actions of ORC, Cdc6 and Cdt1 proteins. This preRC (prereplicative complex) further matures at the G1-S boundary, by participation of additional factors required for replisome assembly. This step is regulated by phosphorylation by two kinases, namely cyclin-dependent kinase and Cdc7 kinase. MCM plays a major role at the replication forks by promoting DNA unwinding process through its DNA helicase activity. The replisome machinery at the replication forks is likely to be functionally and physically linked to other numerous proteins required for various chromosome dynamics including checkpoint regulation, sister chromatid cohesion, repair and bypass of DNA damages, chromatin assembly, heterochromatin formation and telomere maintenance. Thus, the replication forks serve as a hub for coordinated regulation of chromosome maintenance and functions. Our goal is to elucidate the architecture of this giant “replication fork complex” and elucidate how these various chromosome dynamics are coordinatedly regulated.

Masai, H., Zhiying, Y. & Arai, K. (2005)
"Control of DMA replication: regulation and activation of the eukaryotic replicative helicase , MCM."
IUBMB Life 57, 323-335.


4 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 by site-specific activation of its helicase activity by the exposed thymine-rich single-stranded DNA.

Ishimi, Y., Komamura-Kohno, Y., Arai, K. and Masai, H. (2001)
"Biochemical activities associated with mouse Mcm2 protein."
J. Biol. Chem. 276, 42744-42752.
You, Z., Ishimi, Y., Masai, H. and Hanaoka, F. (2002)
"Roles of Mcm7 and Mcm4 subunits in DNA helicase activity of mouse Mcm4/6/7 complex."
J. Biol. Chem. 277, 42471-42479.
You, Z., Ishimi, Y., Mizuno, T., Sugasawa, K., Hanaoka, F., and Masai, H. (2003)
"Thymine-rich single-stranded DNA sequences specifically activate mouse Mcm4/6/7 helicase on Y-fork and bubble-like substrates."
EMBO J. 22, 6148-6160.
You, Z. and Masai, H. (2005)
"DNA binding and helicase actions of mouse Mcm4/6/7 helicase."
Nucl. Acids Res. 33, 3033-3047.


5 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 response 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. The target of Cdc7 appears to be Claspin, a replication fork factor implicated in protection of replication forks. We would like to elucidate molecular mechanism for how Cdc7-Claspin contributes to the stabilization of stalled replication forks.
We are also studying E. coli protein PriA, which recognizes the arrested replication forks. PriA recognizes and binds to the arrested forks, and can stabilize it through its 3’-end recognition pocket when the fork carries a 3’-end of the arrested leading strand at the junction. We wish to identify eukaryotic proteins with similar structure and functions.

Tanaka, T., Mizukoshi, T., Taniyama, C., Kohda, D., Arai, K. and Masai H. (2002)
"DNA binding of PriA protein requires cooperation of the N-terminal D-loop/arrested-fork binding and C-terminal helicase domains."
J. Biol. Chem. 277, 38062-38071.
Tanaka, T., Taniyama, C., Arai, K., and Masai, H. (2003)
"ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination-dependent DNA replication."
Genes to Cells, 8, 251-261.
Mizukoshi, T., Tanaka, T., Arai, K., Kohda, D. and Masai, H. (2003)
"A critical role of the 3’-terminus of nascent DNA chains in recognition of stalled replication forks."
J. Biol. Chem. 278, 42234-42239.
Matsumoto, S., Ogino, K., Noguchi, E., Russell, P. and Masai, H. (2005)
"Hsk1-Dfp1/Him1, the Cdc7-Dbf4 Kinase in Schizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex."
J. Biol. Chem., 280, 42536-42542.
Tanaka, T. and Masai, H. (2006)
"Stabilization of a stalled replication fork by concerted actions of two helicases."
J. Biol. Chem., 281, 3484-3493.
Sasaki, K., Ose, T., Tanaka, T., Mizukoshi, T., Ishigaki, T., Maenaka,K., Masai, H. and Kohda, D. (2006)
"Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli."
Biochim. Biophys. Acta., 1764, 157-160.


6 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.

Takeda, T., Ogino, K., Tatebayashi, K. Ikeda, H., Arai, K. and Masai, H. (2001)
"Regulation of initiation of DNA replication and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast."
Mol. Biol. Cell 12, 1257-1274.
Matsumoto, S., Ogino, K., Noguchi, E., Russell, P. and Masai, H. (2005)
"Hsk1-Dfp1/Him1, the Cdc7-Dbf4 Kinase in Schizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex."
J. Biol. Chem., 280, 42536-42542.


7 Role of Cdc7 kinase in meiotic recombination

We have generated a mutant mice in which Cdc7 kinase activity is decreased. The mutamt 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 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.

Kim, J.M., Takemoto, N., Arai K. and Masai, H. (2003)
"Hypomorphic mutation in an essential cell-cycle kinase causes growth retardation and impaired spermatogenesis."
EMBO J. 22, 5260-5272.
Ogino, K., and Masai, H. (2006)
"Rad3-Cds1 mediates coupling of initiation of meiotic recombination with DNA replication: Mei4-dependent transcription as a potential target of meiotic checkpoint."
J. Biol. Chem., 281,1338-1344.
Ogino, K., Hirota, K., Matsumoto, S., Takeda, T., Ohta, K., Arai, K. and Masai, H. (2006)
"Hsk1 kinase is required for induction of meiotic double-stranded DNA breaks without involving checkpoint kinases in fission yeast."
Proc Natl Acad Sci U S A. 2006 May 23;103(21):8131-6. Epub 2006 May 12.


8 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 linage. In order to unravel molecular basis which underlie the characteristic cell cycle structures of undifferentiated ES cells (namely the virtual absence of G1 and G2 phase), 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 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.

Fujii-Yamamoto, H., Kim, J-M., Arai, K. and Masai, H. (2005)
"Cell cycle and developmental regulations of replication factors in mouse embryonic stem cells."
J. Biol. Chem. 280, 12976-12987.

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