Analysis of Homologous DNA Recombination using the chicken DT40 B cell line

在高等真核细胞中，转基因DNA偶尔会以随机整合的方式整合到基因组中。这一规则的一个新的例外是鸡B淋巴细胞系，包括DT40细胞，其中随机整合和靶向整合的频率相似。该项目的目标是确定DT40细胞中有效的基因靶向的分子机制。虽然机制尚未阐明，但我们已经揭示了一些因素在同源DNA重组(HR)中的作用，如下所述：(1)酵母Rad18，E3泛素连接酶是复制后修复(PRR)所必需的，但不参与HR。我们发现Rad18抑制了非同源末端连接的毒性效应，从而促进了HR(Saberi等人，2007)。(2)在低等真核生物中，UBC13，E3泛素连接酶参与了一种类型的PRR，但不参与HR。我们发现UBC13在HR依赖的DSB修复的初始步骤中起着关键作用(赵等人，2007)。(3)FBH1似乎通过抑制RAD51在分裂酵母DNA损伤处的组装来防止HR异常。在DT40细胞中，我们发现FBH1不影响RAD51的积累，但在后期与Bloom解旋酶平行地抑制HR(Kohzaki等人，2007年)。

In higher eukaryotic cells, transfected DNA is occasionally integrated into the genome predominantly through random integration. A novel exception of this rule is chicken B lymphocyte lines, including DT40 cells, in which random integration and targeted one occur with similar frequencies. The goal of this project is to identify the molecular mechanisms underlying the efficient gene targeting in DT40 cells. While the mechanisms have not yet clarified, we have revealed a role of some factors in Homologous DNA Recombination (HR), as delineated below.(1) Yeast Rad18, E3 ubiquitin ligase is essential for postreplicational repair (PRR), but is not involved in HR. We found that Rad18 suppresses the toxic effect of nonhomolgous end-joining, and thereby facilitates HR (Saberi et al., 2007).(2) Ubc13, E3 ubiquitin ligase is involved in a type of PRR, but not in HR in lower eukaryotes. We found that Ubc13 plays a critical role in an initial step of HR dependent DSB repair (Zhao et al., 2007).(3) FBH1 seems to prevent aberrant HR by suppressing the assembly of Rad51 at DNA damage in the fission yeast. In DT40 cells, we found that FBH1 does not affect Rad51 accumulation, but acts in parallel with Bloom helicase to suppress HR at a late step (Kohzaki et al., 2007).