Makoto Ohtsuki

Publications (2)6.62 Total impact

• Article: BLM is an early responder to DNA double-strand breaks.

  Parimal Karmakar, Masayuki Seki, Makoto Kanamori, Kazunari Hashiguchi, Makoto Ohtsuki, Eriko Murata, Eri Inoue, Shusuke Tada, Li Lan, Akira Yasui, Takemi Enomoto
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  ABSTRACT: Bloom syndrome (BS) is an autosomal recessive disorder characterized by a marked predisposition to cancer and elevated genomic instability. The defective protein in BS, BLM, is a member of the RecQ helicase family and is believed to function in various DNA transactions, including in replication, repair, and recombination. Here, we show that both endogenous and overexpressed human BLM accumulates at sites of laser light-induced DNA double-strand breaks within 10s and colocalizes with gammaH2AX and ATM. Like its RecQ helicase family member, WRN, the defective protein in Werner syndrome, dissection of the BLM protein revealed that its HRDC domain is sufficient for its recruitment to the damaged sites. In addition, we confirmed that the C-terminal region spanning amino acids 1250-1292 within the HRDC domain is necessary for BLM recruitment. To identify additional proteins required for the recruitment of BLM, we examined the recruitment of BLM in various mutants generated from chicken DT40 cells and found that the early accumulation of BLM was not dependent on the presence of ATM, RAD17, DNA-PKcs, NBS1, XRCC3, RAD52, RAD54, or WRN. Thus, HRDC domain in DNA helicases is a common early responder to DNA double-strand breaks, enabling BLM and WRN to be involved in DNA repair.
  Biochemical and Biophysical Research Communications 10/2006; 348(1):62-9. · 2.48 Impact Factor
• Article: Analyses of the interaction of WRNIP1 with Werner syndrome protein (WRN) in vitro and in the cell.

  Yoh-ichi Kawabe, Masayuki Seki, Akari Yoshimura, Katsuaki Nishino, Tomoko Hayashi, Takashi Takeuchi, Sohta Iguchi, Yumiko Kusa, Makoto Ohtsuki, Takashi Tsuyama, Osamu Imamura, Takehisa Matsumoto, Yasuhiro Furuichi, Shusuke Tada, Takemi Enomoto
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  ABSTRACT: Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.
  DNA Repair 08/2006; 5(7):816-28. · 4.14 Impact Factor