B. DEPARTMENT OF CELL GENETICS
B-b. Division of Microbial Genetics - Hiroyuki Araki Group

RESEARCH ACTIVITIES

--We have been studying on eukaryotic chromosomal DNA replication and its regulation by the cell cycle. For this purpose, we have employed budding yeast, Saccharomyces cerevisiae, as a model system of eukaryotic cells. Using strong genetics of budding yeast, we have identified novel factors involving in chromosomal DNA replication and revealed their functions in chromosomal DNA replication. We have been also interested in chromosome instability caused by replication defect.

(1) The interaction between replication proteins for the initiation of DNA replication

Sachiko Sakamoto, Kazuyuki Hirai, Yoichiro Kamimura and Hiroyuki Araki

--At replication origins in eukaryotes, the pre-RC (pre-Replicative Complex) forms from late M to G1 phases and other replication proteins including DNA polymerases assemble when CDK activity increases from G1/S boundary. We have reported three yeast complexes, Sld2-Dpb11, Sld3-Cdc45 and GINS, all of which associate with origins in a mutually dependent and the pre-RC dependent manner. Dpb11 has four BRCT repeats and form a complex with the Sld2 protein phosphorylated by CDK. The Sld3-Cdc45 complex is observed throughout the cell cycle. The GINS complex consisting of Sld5, Psf1, Psf2 and Psf3 subunits first associates with origins and then moves with replication forks.--To elucidate how the proteins assemble, we investigated the complex formation between GINS and other proteins by co-immunoprecipitation assay. GINS coprecipitated with Pol ε throughout the cell cycle and with Mcm, a component of the pre-RC, in the S phase. Moreover, when we treated the cells with the cross-linking agent before precipitation, Sld3, Sld2 and Dpb11 also coprecipitated with GINS. Even when S-CDK activity increased without the pre-RC in Cdc6-depleted cells, Sld2-Dpb11 and Pol ε coprecipitated with GINS, suggesting that they form a complex without origin-association. We therefore propose the pre-Landing Complex (pre-LC) containing Sld2, Dpb11, Pol ε and GINS, which is formed before associating with origins. The pre-LC seems to associate with the pre-RC via Sld3 since the C-terminal portion of Sld3 interacts with GINS whereas the N-terminal portion interacts with Cdc45. The pre-LC complex is observed only when CDK activity increases. Since the complex formation between Sld2 and Dpb11 depends on CDK-phosphorylation of Sld2 (see below), CDK regulates the pre-LC formation through the Sld2-Dpb11 complex formation.
--In parallel with the in vivo analysis, we have tried to purify the proteins related to the pre-LC to know their function. First, we expressed all the subunits of GINS in insect cells and purified GINS to be a near homogeneity. We also expressed Dpb11 and Sld3 in Escherichia coli and partially purified them. The purified GINS complex binds to the purified Dpb11 and Sld3, consistent with two hybrid assay and in vivo co-immunoprecipitation. We will further extend this analysis to all the components of the pre-LC.

(2) Regulatory mechanism of the complex formation between Sld2 and Dpb11 by cyclin-dependent kinase (CDK)

Yon-Soo Tak, Yoshimi Tanaka, Yoichiro Kamimura and Hiroyuki Araki

--The initiation of chromosomal DNA replication in eukaryotes requires the CDK activity. However, how CDK regulates the initiation of DNA replication is not well elucidated since the substrates of CDK in DNA replication have not been identified except the Sld2 protein. Sld2 has a cluster of eleven CDK-dependent phosphorylation sites (S/T-P), six of which are preferred CDK phosphorylation sites. Phosphorylation of Sld2 enhanced its binding to Dpb11, which appears to be necessary for onset of DNA replication. We have studied how the complex formation between Dpb11 and Sld2 is regulated by CDK activity. Dpb11 has four copies of the BRCT domain, which is recently reported as a phospho-peptide binding module. A C-terminal pair of BRCT domains of Dpb11 binds to a short stretch of Sld2.
--We purified the truncated Sld2 containing the Dpb11-binding stretch and 11-CDK phosphorylation sites and the C-terminal pair of BRCT domains of Dpb11. Using these purified proteins, we demonstrated that CDK-phosphorylation enhances the complex formation between Sld2 and Dpb11. To this in vitro binding reaction, we challenged various synthesized peptides with or without phosphorylation. It revealed that the 20-aa stretch with phosphorylation at threonine followed by proline (CDK-catalyzed phosphorylation site) competes with the complex formation between phosphorylated Sld2 and Dpb11. Thus, the 20-aa stretch with the phosphorylation functions as a binding stretch to Dpb11. Consistent with these observations, an alanine substitution of this threonine reduced the binding activity to Dpb11 in in vitro binding assay as well as two-hybrid assay. Furthermore, this alanine-substitution confers defect of cell growth.
--We previously reported that the simultaneous alanine substitutions of all the canonical (preferred) CDK-phosphorylation sites of Sld2 confers defect of cell growth as well as reduced affinity to Dpb11 (Masumoto et al., Nature 415, 651, 2002). The 20-aa stretch does not contain these canonical phosphorylation sites. Moreover, the phosphomimetic replacement of this threonine by asparagine overrides the defect of the simultaneous substitutions of canonical phosphorylation-sites in a two-hybrid assay. These observations suggest that phosphorylation of canonical CDK-sites affects the phosphorylation of the threonine in the 20-aa stretch.
--CDK is consisted of a catalytic subunit and a cyclin. Budding yeast has one catalytic subunit, Cdc28 and nine different cyclins, and thus 9 species of CDK. We expressed these CDK subunits in E. coli and purified typical CDK species. Using these purified CDKs, we found that Cdc28-Clb5, S-phase CDK most efficiently phosphorylates the Sld2 protein. We also identified efficient phosphorylation sites by CDK in both in vivo and in vitro using various Sld2 protein with mutations in CDK-phosphorylation sites. According to this assay, the threonine in the 20-aa stretch is an inefficient phosphorylation site. Moreover, the simultaneous mutations in canonical phosphorylation sites, some of which are actually efficient phosphorylation sites, reduced severely the phosphorylation of the threonine in the 20-aa stretch. We therefore propose that the threonine in the 20-aa stretch of Sld2 is phosphorylated cooperatively with other efficient phosphorylation sites and then Sld2 binds to Dpb11.

(3) CDK targets in the initiation of DNA replication

Seiji Tanaka and Hiroyuki Araki

--The initiation of eukaryotic DNA replication is triggered by two essential kinases, Cdc7 and CDK. Requirement of Cdc7 in initiation can be bypassed with a bob1-1 mutation, which is allelic to the one of the subunit of proposed replicative helicase, MCM5. Mutations that can bypass requirement of CDK, however, are not known so far. We have recently identified Sld2 as an essential substrate of CDK in the initiation of DNA replication. Phosphorylation of Sld2 by CDK enhances the interaction between Sld2 and its binding partner, Dpb11, which is likely to be important for association of the Sld2-Dpb11 complex to replication origins (Masumoto et al. Nature 415, 651-655, 2002).
--To understand the individual processes in the initiation of DNA replication, we have tested whether Sld2 phosphorylation by CDK is sufficient for initiation. We have constructed a Sld2 mutant which might mimic a phosphorylated status of wild type Sld2. Although this mutant could support cell growth, CDK activity was still required for initiation. This suggests that CDK regulates multiple targets in the initiation of DNA replication. In order to identify these CDK target(s) other than Sld2, we have set a screening of mutants that show synthetic lethality with phosphomimetic sld2. The rationale of the screening is that cells might to be a lethal because of untimely DNA replication when all of the CDK targets in the initiation process including Sld2 are freed from strict regulation of CDK. Although we have not been successful to isolate that kind of mutants so far, we will continue the screening.

(4) Mechanisms for generating genomic instability

Seiji Tanaka and Hiroyuki Araki

--Although genomic instability is a hallmark of human cancer cells, the mechanisms by which genomic instability is generated and selected for during oncogenesis remain obscure. In most human cancers, the pathway leading to the activation of the G1 cyclins is deregulated. Previously we hypothesized that G1 cyclin deregulation could cause genomic instability because the lack of a proper ‘low Cdk' period in G1 may reduce numbers of functional pre-RCs that confer replication competence to cells. Using budding yeast as a model, we have shown that overexpression of the G1 cyclin, Cln2, inhibits the assembly of pre-RCs and induces gross chromosome rearrangements (GCRs) such as translocation, deletion of a chromosome arm, interstitial deletions and inversions. These results suggest that deregulation of G1 cyclins, selected for in oncogenesis because it confers clonal growth advantage, may also provide an important mechanism for generating genomic instability by inhibiting replication licensing. However, we still do not know how reducing origin licensing/firing contributes to genomic instability. We have focused on this question.
--Reduced numbers of replication forks and slow DNA replication as a result may cause broken chromosomes in M phase or stalled replication forks that persist for longer periods. We have asked if these are good substrates for recombination and contribute to generating genomic instability. To test this, we have introduced a genomic sequence that is known to block the progression of replication fork and found that this induce higher rate of GCR. We are now proceeding the detailed analysis on this.

PUBLICATIONS

Papers
1. Iida, T. and Araki, H. (2004). Non-competitive counteractions of DNA polymerase ε and ISW2/yCHRAC for epigenetic inheritance of telomere-position effect in Saccharomyces cerevisiae. Mol. Cell. Biol. 24, 217-227.
2. Mimura, S., Seki, T., Tanaka, S. and Diffley, J.F.X. (2004). Phophorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control. Nature 431, 1118-1123.

Books
3. 荒木弘之(2004)真核生物の複製開始反応,DNA複製･修復がわかる(花岡文雄編集),羊土社.
4. 荒木弘之(2004)DNAの複製,バイオテクノロジーのための基礎分子生物学(大嶋泰治･北本勝ひこ･原島俊･宮川都吉編),化学同人.

ORAL PRESENTATIONS

1. Araki, H. The protein interactions to replicate chromosomal DNA in budding yeast. Chromatin/DNA replication workshop, Marie Curie Research Institute, Oxted, UK, April, 2004.
2. Tak, Y. -S., Muramatsu, S., Kamimura, Y. and Araki, H. Association of replication proteins with origins in budding yeast. The 5th UK-Japan Cell Cycle Workshop, Nara, April, 2004.
3. Tak, Y. -S., Muramatsu, S., Kamimura, Y. and Araki, H. Protein-protein interactions to promote the initiation of DNA replication. Yeast Chromosome Structure, Replication and Segregation, Pine Mountain, Georgia, USA, July, 2004.
4. Kamimura, Y. , Tak, Y. -S., Muramatsu, S. and Araki, H. Protein-assembly at replication origins in budding yeast. DNA Replication and Genome Integrity 2004, Salk Institute, La Jolla, California, USA, August, 2004.
5. Tak, Y. -S., Kamimura, Y. and Araki, H. Regulatory mechanism for the interaction between Sld2 and Dpb11 in S. cerevisiae. The 17th Workshop on DNA Replication and Segregation, Sendai, July, 2004.
6. Kamimura, Y., Tak, Y. -S., Hirai, K., Sakamoto, S. and Araki, H. Formation of the pre-Landing Complex (pre-LC) regulated by CDK activity in budding yeast. The 27th Annual Meeting of the Molecular Biology, Kobe, December, 2004.
7. 上村陽一郎,荒木弘之「真核生物のレプリソーム形成におけるダイナミクス」第17回複製・分配ワークショップ,仙台,2004年7月
8. 坂本佐知子,上村陽一郎,荒木弘之「染色体DNA複製開始領域への複製タンパク質の集合機構」第37回酵母遺伝学フォーラム研究報告会,松江市,2004年9月
9. 本間良美,上野勝,瓜谷真裕,荒木弘之,丑丸敬史「DNA複製に関与する核小体タンパク質Nog1複合体の解析」第37回酵母遺伝学フォーラム研究報告会,松江市、2004年9月

POSTER PRESENTATIONS

1. Kamimura, Y. and Araki, H. The replicsome formation in the initiation of eukaryotic chromosomal DNA replication. The 5th UK-Japan Cell Cycle Workshop, Nara, April, 2004.
2. Tanaka, S and Araki, H. Sld2 is an essential but not a sufficient target of CDK in the initiation of DNA replication. Yeast Chromosome Structure, Replication and Segregation, Pine Mountain, Georgia, USA, July, 2004.
3. Tak, Y. ﾐS., kamimura, Y. and Araki, H. A novel mechanism in the interaction between the two yeast replication proteins, the BRCT-protein Dpb11 and the Sld2 protein phosphorylated by CDK activity. The 27th Annual Meeting of the Molecular Biology, Kobe, December, 2004.
4. Tanaka, S and Araki, H. Sld2 is an essential but not a sufficient target of CDK in the initiation of DNA replication. The 27th Annual Meeting of the Molecular Biology, Kobe, December, 2004.
5. Katou, Y, Araki, H. and Shirahige, K. Identification of peotins involved in arrest and recover of replication fork under replicative stress. The 27th Annual Meeting of the Molecular Biology, Kobe, December, 2004.
6. 岡さとみ,池西淳,園池公毅,荒木弘之,中谷洋一郎,瀬々潤,森下真一,湯川格史,佐野史,大矢禎一「出芽酵母の細胞形態情報に基づく遺伝子の機能予測」第27回日本分子生物学会年会,神戸市,2004年12月
7.本間良美,上野勝,瓜谷真裕,荒木弘之,丑丸敬史「出芽酵母新規DNA複製制御因子Nog1によるMCM量の維持」第27回日本分子生物学会年会,神戸市,2004年12月
8. 田中尚美,遠藤静子,荒木弘之「出芽酵母複製タンパク質Sld2を用いたサイクリン依存性キナーゼの基質特異性の解析」第27回日本分子生物学会年会,神戸市,2004年12月
9. 梅森稔子,平井和之,坂本佐知子,上村陽一郎,荒木弘之「出芽酵母複製タンパク質Sld3の機能解析」第27回日本分子生物学会年会,神戸市,2004年12月
10. 坂本佐知子,上村陽一郎,荒木弘之「染色体DNA複製開始領域への複製タンパク質の集合機構」第27回日本分子生物学会年会,神戸市,2004年12月
11. 関丘,橋本恵至,王成忠,坪田智明,真木智子,上村陽一郎,荒木弘之,杉野明雄「出芽酵母のGINSとDNAポリメラーゼε間の相互作用の解析」第27回日本分子生物学会年会,神戸市,2004年12月

EDUCATION

1. Dr. H. Araki gave a lecture at Medical School, University of Tokyo, February, 2004 (in Japanese).
2. Dr. H. Araki gave a seminar at Paterson Institute at Manchester, UK, April, 2004.
3. Dr. H. Araki gave a seminar at Clare Hall Laboratories, Cancer Research UK at South Mims, UK, April, 2004.
4. Dr. H. Araki gave a seminar at National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA, July, 2004.
5. Dr. H. Araki gave a lecture at Shizuoka University, October, 2004 (in Japanese).
6. Dr. H. Araki gave a lecture and a seminar at Tokyo Institute of Technology, November, 2004 (in Japanese).