Cellular & Molecular Biology Unit

The major goal of our research is to understand the molecular mechanisms and principles governing homologous DNA (or genetic) recombination. Homologous recombination is the rearrangements of segments between a pair of homologous DNAs in living cells, and plays a role in DNA double-stranded break (DSB) repair for the genome stability. On the other hand, it has partly been proven by our "ADLib system", that in response to environmental changes, using preceding DNA sequences, homologous recombination creates new genes for adapting to pathogens and environmental changes, such as antibody production and sexual reproduction. A key reaction in homologous recombination is the formation of a heteroduplex joint with a D-loop (homologous pairing), in which a single-stranded DNA tail derived from an end of a DSB, is base-paired with a complementary sequence in homologous double-stranded DNA. I discovered RecA recombinase-catalyzed homologous pairing, which requires ATP (Shibata et al., PNAS 1979). We identified a new class of ATP-independent homologous pairing enzymes (Kagawa et al., JBC 2001; Ling & Shibata, EMBO J 2002). Using NMR, we found that a unique extended DNA structure was induced in a common homologous pairing intermediates by both the RecA-family recombinases (RecA, Rad51) and the ATP-independent homologous pairing enzymes from various origins (Masuda et al., JBC 2009). Thus, homologous pairing is an intrinsic molecular function of DNA, rather than a reaction catalyzed by a specific class of enzymes (Shibata et al., PNAS 2001 for review). We found a novel role of a homologous pairing enzyme in homoplasmy (a type of genome homogenization) in yeast mitochondrial DNA inheritance, in which ROS (reactive oxygen species) act as a regulatory mediator (Hori et al., NAR 2009; see Ling & Shibata 2011 for review). Using a novel bioassay, we identified a new rice SPO11 orthologue (OsSPO11D) as a meiosis-induced protein with a double-strand breaking activity (Shingu BMC Mol Biol 2012). We invented the ADLib System, for the rapid immunotolerance-free ex-vivo antibody production (Seo et al., Nature Biotech 2005).

Research Subjects

• Biochemistry and molecular & structural biology on the initiation of homologous DNA recombination
• Molecular regulation on the initiation of plant homologous recombination and its applications
• The functions of genetic recombination in mitochondrial genetic inheritance

Publications

1. Shingu, Y., Tokai, T., Agawa, Y., Toyota, K., Ahamed, S., Kawagishi-Kobayashi, M., Komatsu, A., Mikawa, T., Yamamoto, M. T., Wakasa, K., Shibata, T. and Kusano, K.:
   "The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay"
   BMC Mol. Biol., 13, 1 (2012)
2. Arai, N., Kagawa, W., Saito, K., Shingu, Y., Mikawa, T., Kurumizaka, H. and Shibata, T.:
   "Vital roles of the second DNA-binding site of Rad52 in yeast homologous recombination"
   J. Biol. Chem., 286, 17607-17617 (2011)
3. Inoue, J., Nagae, T., Mishima, M., Ito, Y., Shibata, T. and Mikawa, T.:
   "A mechanism for SSB displacement from single-stranded DNA upon SSB-RecO interaction"
   J. Biol. Chem., 286, 6720-6732 (2011)
4. Ling, F., Mikawa, T. and Shibata, T.:
   "Enlightenment of yeast mitochondrial homoplasmy: diversified roles of gene conversion"
   Genes, 2, 169-190 (2011)
5. Masuda, T., Ito, Y., Terada, T., Shibata, T. and Mikawa, T.:
   "A non-canonical DNA structure enables homologous recombination in various genetic systems"
   J. Biol. Chem., 284, 30230-30239 (2009)
6. Seo, H., Masuoka, M., Murofushi, H., Takeda, S., Shibata, T. and Ohta, K.:
   "Rapid generation of specific antibodies by enhanced homologous recombination"
   Nature Biotechnol., 23, 731-735 (2005)
7. Kagawa, W., Kurumizaka, H., Ikawa, S., Yokoyama, S. and Shibata, T.:
   "Homologous pairing promoted by the human Rad52 protein"
   J. Biol. Chem., 276, 35201-35208 (2001)
8. Shibata, T., Nishinaka, T., Mikawa, T., Aihara, H., Kurumizaka, H., Yokoyama, S. and Ito, Y.:
   "Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material"
   Proc. Natl. Acad. Sci. USA, 98, 8425-8432 (2001)
9. Nishinaka, T., Ito, Y., Yokoyama, S. and Shibata, T.:
   "An extended DNA structure through deoxyribose-base stacking induced by RecA protein"
   Proc. Natl. Acad. Sci. USA, 94, 6623-6628 (1997)
10. Shibata, T., DasGupta, C., Cunningham, R. P. and Radding, C. M.:
    "Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments"
    Proc. Natl. Acad. Sci. USA, 76, 1638-1642 (1979)