Kobayashi Masahiko, Hirano Atsushi, Kumano Tomoyasu, Xiang Shuang-Lin, Mihara Keiko, Haseda Yasunari, Matsui Osamu, Shimizu Hiroko, Yamamoto Ken-ichi
Department of Molecular Pathology, Cancer Research Institute, Kanazawa University, Ishikawa 920-0934, Japan.
Genes Cells. 2004 Apr;9(4):291-303. doi: 10.1111/j.1356-9597.2004.00728.x.
The Rad17-replication factor C (Rad17-RFC) and Rad9-Rad1-Hus1 complexes are thought to function in the early phase of cell-cycle checkpoint control as sensors for genome damage and genome replication errors. However, genetic analysis of the functions of these complexes in vertebrates is complicated by the lethality of these gene disruptions in embryonic mouse cells. We disrupted the Rad17 and Rad9 loci by gene targeting in the chicken B lymphocyte line DT40. Rad17-/- and Rad9-/- DT40 cells are viable, and are highly sensitive to UV irradiation, alkylating agents, and DNA replication inhibitors, such as hydroxyurea. We further found that Rad17-/- and Rad9-/- but not ATM-/- cells are defective in S-phase DNA damage checkpoint controls and in the cellular response to stalled DNA replication. These results indicate a critical role for chicken Rad17 and Rad9 in the cellular response to stalled DNA replication and DNA damage.
Rad17复制因子C（Rad17-RFC）和Rad9-Rad1-Hus1复合物被认为在细胞周期检查点控制的早期阶段发挥作用，作为基因组损伤和基因组复制错误的传感器。然而，这些复合物在脊椎动物中的功能的遗传分析因这些基因在胚胎小鼠细胞中的破坏导致的致死性而变得复杂。我们通过基因靶向在鸡B淋巴细胞系DT40中破坏了Rad17和Rad9基因座。Rad17-/-和Rad9-/- DT40细胞是有活力的，并且对紫外线照射、烷化剂和DNA复制抑制剂（如羟基脲）高度敏感。我们进一步发现，Rad17-/-和Rad9-/-细胞而非ATM-/-细胞在S期DNA损伤检查点控制和对停滞的DNA复制的细胞反应中存在缺陷。这些结果表明鸡Rad17和Rad9在细胞对停滞的DNA复制和DNA损伤的反应中起关键作用。
