A. DEPARTMENT OF MOLECULAR GENETICS
A-b. Division of Mutagenesis - Fumiaki Yamao Group

RESEARCH ACTIVITIES

(1) Multifunctional role of Mcl1/Slr3 in DNA replication, repair and chromatin structure

Yasuhiro Tsutsui, Toyoaki Natsume and Fumiaki Yamao

--Slr3 in fission yeast is a member of eukaryotic family of WD repeat proteins and important for genome stability. slr3 gene was originally identified as mutants showing synthetic lethality with Rad2 defect. Fission yeast Rad2 is a FEN-1 family nuclease processing Okazaki fragment during lagging strand DNA synthesis. Since recombination activity is essential for survival of cells defective in Rad2 function, genes that show, when mutated, synthetic lethality with Rad2-defect was expected to be involved in recombination or recombination repair of damaged DNA. Actually slr3 mutants are sensitive to DNA damage reagents such as MMS, UV, bleomycin and hydroxyurea. Null mutant cell is temperature sensitive for growth, and shows severe growth retardation even at the permissive temperature. The mutant cells arrest at G2 phase with 2C DNA content at the restricted temperature, showing that the bulk DNA synthesis occurred. However, DNA double-strand breaks were found to accumulate in the arrested cells, which was a similar phenotype caused by defect of Okazaki fragment processing in such as dna2 or cdc17 mutants. It was further shown that Slr3 interacts not only genetically but also physically with DNA polymerase α. These results strongly suggest that Slr3 functions in lagging strand synthesis and Okazaki fragment processing in addition to DNA repair. On the other hand, slr3 gene was found allelic to mcl1 that is required for proper chromosomal segregation. Concomitantly slr3 mutant cells were sensitive to TBZ. Consequently, highly frequent chromosome loss observed in mcl1 mutant cells was attributed to defective cohesion of sister chromatids in centromere region. Recently, we also found that Slr3 affects heterochromatin structure, which is now under an extensive investigation (Natsume et. al). Thus, Slr3/Mcl1 protein plays multifunctional role to maintain chromosomal stability by connecting DNA replication, recombination, damage repair, cohesion and heterochromatin structure. This work was collaborated with H. Shinagawa's and H. Iwasaki's group in Osaka University and Yokohama-City University, respectively.

ORAL PRESENTATIONS

1. F. Yamao. Ubiquitin Pathway Regulating Recombination and DNA Damage Repair in Fission Yeast. Symposium on "The Role of Ubiquitylation in the DNA Repair Pathway" in The 57th Annual Meeting of Japan Society for Cell Biology, Osaka, May.
2. 夏目豊彰、筒井康博、森下卓、品川日出夫、山尾文明「ヘテロクロマティンの維持に関与する分裂酵母mcl1/slr3遺伝子の解析」第76回日本遺伝学会大会、大阪、9月

POSTER PRESENTATIONS

1. 井内勝哉、山尾文明、矢倉達夫.「エトポシド処理したHL-60細胞におけるKuの機能解析」第77回生化学会大会、横浜、10月
2. 夏目豊彰、筒井康博、森下卓、岩崎博史、品川日出夫、山尾文明.「ヘテロクロマティンの維持に関与する分裂酵母mcl1/slr3遺伝子の解析」第27回日本分子生物学会年会、神戸、12月
3. 井内勝哉、水野めぐみ、山尾文明、矢倉達夫.「TPAまたはエトポシドによりアポトーシス誘導したHL-60細胞におけるKuタンパク質レベル及びDNA結合性に関する研究」第27回日本分子生物学会年会、神戸、12月

EDUCATION

1. Dr. Yamao was invited to give a seminar on a title of “Ubiquitin as a Multi-Player in Cell" at Keihanna Interaction Plaza, Kyoto, March (in Japanese).
2. Dr. Yamao gave a lecture at Department of Veterinary Medicine, Yamaguchi University, December (in Japanese).