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

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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).