Laboratory of Molecular Genetics
- Hisao MASUKATA
- Associate Professor
- Takuro NAKAGAWA
- Specially Appointed Researcher
- Shiho OGAWA
- Specially Appointed Researcher
- Yoshitaka Kawasoe
Molecular Cell Biology
Graduate School of Science, Department of Biological Science
DNA replication is an essential reaction to maintain genetic information. For complete duplication of large and complex genome in eukaryotic cells, DNA replication initiates at large number of sites, called replication origins, on the chromosomes and the initiation process is tightly regulated by progression of cell cycle. We have been interested in how individual replication origin is activated and how origins are coordinated to ensure replication of all the genome in limited time period. We are also interested in the mechanisms to recover replication forks, which are stalled by shortage of deoxynucleotides or by DNA damage. Failure in the recovery results in cell death or genome instability that may cause a cancer in multicellular organisms. To understand these mechanisms at a molecular level, we use fission yeast, Schizosaccharomyces pombe, which has similarities with higher eukaryotes and thus is a suitable model organism to study the fundamental and general biological functions.
Genome-wide identification of replication origins
Chromosomes are confined into different chromatin structures relating with chromosome functions, such as centromere, mating type locus and telomeres. We are studying distribution of replication origins on fission yeast chromosomes by means of ChIP-chip analysis of replication factors and mapping of nascent DNA synthesized in early S phase. We identified 460 intergenic regions where Orc1 and Mcm6 are colocalized, as pre-replicative complex (pre-RC) sites, and 276 active origins that fire in early S phase in the presence of hydroxyurea.
Molecular mechanism of activation of replication origins
Replication origins are recognized by origin recognition complex (ORC) and minichromosome maintenance (MCM) complex is loaded depending on loading factors Cdc18/Cdc6 and Cdt1 in G1 phase. Assembly of several initiation factors is necessary for initiation of replication in S phase and this process is regulated by two conserved proteins kinases, cyclin-dependent kinase (CDK) and Dbf4-depedent kinase (DDK). By means of thermosensitive mutants of initiation factors, we identified the activation process is composed of three reactions, DDK-dependent Sld3-loading, DDK-CDK-dependent GINS-loading and Cdc45 loading.
Maintenance of Chromosome Integrity
Correct and precise duplication of genetic materials and faithful transmission of chromosomes to daughter cells are important for maintaining genome integrity. Defects in chromosome metabolisms can cause gross chromosomal rearrangements (GCRs) resulting in cell death as well as cancer and premature aging in multicellular organisms. To gain insights into the molecular mechanisms by which chromosome integrity is maintained, we have developed an assay system to detect GCRs using fission yeast and are searching for the genes essential for chromosome maintenance.
Two-dimensional gel electrophoresis of replication intermediates at replication origin ars2004 on chromosome II in haploid fission yeast cells arrested in early S phase (A). DNA microarray results showing locations of newly synthesized DNA (green) and initiator protein Orc1 (brown) at the ars2004 locus (B).
Assemby of replication initiation factors on fission yeast replication origins.
Chromosome instability causes cell death and genetic diseases.
Mechanism of coupling of chromosome cohesion to the initiation of DNA replication by Cdc7
Onaka, AT., Toyofuku, N., Inoue, T., Okita, AK., Sagawa, M., Su, J., Shitanda, T., Matsuyama, R., Zafar, F., Takahashi, TS., Masukata, H., and Nakagawa, T. Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation Nucl. Acids Res. doi:10.1093/nar/gkw874 , (2016)
Kawasoe, Y., Tsurimoto, T., Nakagawa, T., Masukata, H., Takahashi, TS. MutSα maintains the mismatch repair capability by inhibiting PCNA unloading eLIFE 12;5 , e15155 - (2016)
Tashiro, T., Handa, T., Matsuda, A., Ban, T., Takigawa, T., Miyasato, K., Ishii, K., Kugou, K., Ohta, K., Hiraoka, Y., Masukata, H., Kanoh, J. Shugoshin forms a specialized chromatin domain at subtelomeres that regulates transcription and replication timing. Nature Communications 7 , 10393 - (2016)
Tazumi, A., Fukuura, M., Nakato, R., Kishimoto, A., Takenaka, T., Ogawa, S., Song, J., Takahashi, TS., Nakagawa, T., Masukata, H. Telomere-binding protein Taz1 controls global replication timing through its localization near late replication origins in fission yeast. Genes Dev. 26 , 2050 - 2062 (2012)
Handa, T., Kanke, M., Takahashi, TS, Nakagawa, T., Masukata, H. DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast. Mol. Biol. Cell 23 , 3240 - 3253 (2012)
Higashi, TL., Ikeda, M., Tanaka, H., Nakagawa, T., Bando, M., Shirahige, K., Kubota, Y., Takisawa, Masukata, H., Takahashi, TS. The Prereplication Complex Recruits XEco2 to Chromatin to Promote Cohesin Acetylation in Xenopus Egg Extracts. Current Biology, 22 , 977 - 988 (2012)
Kanke, M., Kodama, Y., Takahashi, TS., Nakagawa, T., Masukata, H. Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components. EMBO Journal, 31 , 2182 - 2194 (2012)
Maki K, Inoue T, Onaka A, Hashizume H, Somete N, Kobayashi Y, Murakami S, Shigaki C, Takahashi TS, Masukata H, Nakagawa T. Abundance of prereplicative complexes (Pre-RCs) facilitates recombinational repair under replication stress in fission yeast. J Biol Chem, 286 , 41701 - 41710 (2011)
Fukuura, M., Nagao, K., Obuse, C., Takahashi, TS., Nakagawa, T., and Masukata, H. CDK promotes interactions of Sld3 and Drc1 with Cut5 for initiation of DNA replication in fission yeast. Mol. Biol. Cell. 22(14) , 2620 - 2633 (2011)
Hayashi, T.M., Takahashi, S.T., Nakagawa, T., Nakayama, J., Masukata, H. SThe heterochromatin protein Swi6/HP1 activates replication origins at the pericentromeric region and silent mating-type locus. Nature Cell Biology 11 (doi:10.1038/ncb1845) , 357 - 362 (2009)
Dr. Hisao Masukata
Osaka University, Graduate School of Science
1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043