Kimura Seisuke, Furukawa Tomoyuki, Kasai Nobuyuki, Mori Yoko, Kitamoto Hiroko K, Sugawara Fumio, Hashimoto Junji, Sakaguchi Kengo
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Chiba, 278-8510, Noda, Japan.
Gene. 2003 Sep 18;314:63-71. doi: 10.1016/s0378-1119(03)00694-2.
Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. Previously, we isolated and characterized a complementary DNA (cDNA) from rice (Oryza sativa) encoding a protein which shows homology with the eukaryotic flap endonuclease-1 (FEN-1). In this report, we found that rice (O. sativa L. cv. Nipponbare) possessed two FEN-1 homologues designated as OsFEN-1a and OsFEN-1b. The OsFEN-1a and OsFEN-1b genes were mapped to chromosome 5 and 3, respectively. Both genes contained 17 exons and 16 introns. Alignment of OsFEN-1a protein with OsFEN-1b protein showed a high degree of sequence similarity, particularly around the N and I domains. Northern hybridization and in situ hybridization analysis demonstrated preferential expression of OsFEN-1a and OsFEN-1b in proliferating tissues such as the shoot apical meristem or young leaves. The levels of OsFEN-1a and OsFEN-1b expression were significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to regrow following the addition of sucrose to the medium, both OsFEN-1a and OsFEN-1b were again expressed at high level. These results suggested that OsFEN-1a and OsFEN-1b are required for cell proliferation. Functional complementation assay suggested that OsFEN-1a cDNA had the ability to complement Saccharomyces cerevisiae rad27 null mutant. On the other hand, OsFEN-1b cDNA could not complement the rad27 mutant. The roles of OsFEN-1a and OsFEN-1b in plant DNA replication and repair are discussed.
瓣状核酸内切酶-1（FEN-1）是一种参与DNA复制和修复的重要酶。此前，我们从水稻（Oryza sativa）中分离并鉴定了一个互补DNA（cDNA），其编码的蛋白质与真核生物瓣状核酸内切酶-1（FEN-1）具有同源性。在本报告中，我们发现水稻（O. sativa L. cv. Nipponbare）拥有两个FEN-1同源物，分别命名为OsFEN-1a和OsFEN-1b。OsFEN-1a和OsFEN-1b基因分别定位在第5号和第3号染色体上。这两个基因都包含17个外显子和16个内含子。OsFEN-1a蛋白与OsFEN-1b蛋白的比对显示出高度的序列相似性，尤其是在N和I结构域周围。Northern杂交和原位杂交分析表明，OsFEN-1a和OsFEN-1b在增殖组织如茎尖分生组织或幼叶中优先表达。当从生长培养基中去除蔗糖导致细胞增殖暂时停止时，OsFEN-1a和OsFEN-1b的表达水平显著降低。当向培养基中添加蔗糖后生长停止的细胞开始重新生长时，OsFEN-1a和OsFEN-1b再次高水平表达。这些结果表明，OsFEN-1a和OsFEN-1b是细胞增殖所必需的。功能互补试验表明，OsFEN-1a cDNA具有互补酿酒酵母rad27缺失突变体的能力。另一方面，OsFEN-1b cDNA不能互补rad27突变体。本文讨论了OsFEN-1a和OsFEN-1b在植物DNA复制和修复中的作用。
