Abstract
Reverse genetics by gene disruption is an informative and powerful tool for investigating protein functions in the cells. The chicken B lymphocyte line DT40 is widely used because of the relative ease of gene manipulation including targeted gene disruption.
Here we present our two resent research topics as follows:
1) Requirement of the non-homologous end-joining (NHEJ) DNA repair proteins DNA-PKcs and Ku70 required for inducing apoptosis following X-ray irradiation.
Using altogether 11 mutant DT40 cell lines lacking various DNA repair related genes, we have examined apoptosis induction, judged by DNA ladder formation, after X-ray irradiation. Most of the cell lines showed similar apoptotic pattern as wild type cells, however, two cell lines, deficient in either DNA-PKcs or Ku70, failed to die by apoptosis. Neither DNA ladder formation nor mitochondrial membrane permeabilization could be detected in these cells. Loss of Artemis or DNA Ligase IV, downstream factors of the NHEJ pathway, did not affect X-ray induced apoptosis. These results suggested that DNA-PKcs and Ku70 themselves rather than the NHEJ pathway have essential roles in mediating apoptotic signals.
2) FancI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway
A rare hereditary disorder Fanconi anemia (FA) is clinically characterized by progressive bone marrow failure, and developmental abnormalities. Altogether 13 genes have been implicated in FA, and their products constitute a common pathway in DNA damage signaling termed the "FA pathway". In response to DNA damage, the FA pathway is activated, leading to monoubiquitination and colocalizing foci formation of the key proteins FancD2 and FancI. In DT40 cell system, mutations in S/TQ motifs of FancI largely abrogate monoubiquitination as well as foci formation of both FancD2 and FancI, resulting in loss of DNA repair function. We propose that the multiple phosphorylation of FancI serves as a molecular switch in activation of the FA pathway.
