Abstract
BRCT domain is a highly conserved domain found in many proteins involved in DNA damage checkpoint, DNA repair, and cell cycle control. PTIP protein carries 6 BRCT domains and forms nuclear foci after the irradiation with ionizing radiation (IR). To know whether PTIP acts in these pathways, we constructed chicken DT40 PTIP disruptants. Expression of PTIP was detected in DT40 cells. Chicken PTIP is located on chromosome 2, which is trisomy in DT40 cells. We disrupted three alleles of the gene and obtained two PTIP disruptants. Doubling time of these disruptants was 50% longer than that of wild type cells. This prolonged doubling time of disruptants was largely ascribed to the appearance of many disintegrated cells. Colony forming ability of these disruptants was less than 1 % of that of wild type cells. Expression of human PTIP complemented these phenotypes of the disruptants. Phosphorylation of CHK1 after the irradiation with IR, continued longer in the disruptant cells than in wild type cells. We also constructed conditional DT40 PTIP disruptant with Tet-off human PTIP expression system. At 24 hours after the addition of doxycycline (Dox), the conditional disruptant expressed human PTIP at slightly detectable level. These conditional disruptants with or without Dox treatment were exposed to IR and colony forming survivals were compared. LD10 of Dox treated cells was 0.8 of that of non-treated cells. These results indicate that PTIP protein is involved in DNA repair and signal transduction pathway of DNA damage.
