Nucleic Acids Research, 2008, Vol. 36, No. 9 2943 Figure 2. PHF1 is recruited to laser-irradiated sites via two indepen- dent domains of the N-terminus Tudor domain and the central region (aa 340–431). (A) Schematic presentation of PHF1 domains (top) and deletion mutants (left) and the results of recruitment experiments (right). (B) Live cell imaging of irradiated HeLa cells expressing GFP- tagged deletion mutants of PHF1. Arrows indicate the sites of irradiation. from the Chinese hamster ovary (CHO) cell lines of XR-C1 (DNA-PKcs-deﬁcient), XR-1 (XRCC4-deﬁcient) (Figure 3B and C). However, PHF1 was not recruited to laser-irradiated sites in XR-V15B (Ku80-deﬁcient), while PHF1 is recruited in XR-V79B (Ku80 proﬁcient) (Figure 3A). We generated a V15B cell line expressing His-ku80, in which expression of Ku80 as well as recruitment of Ku70 to laser-irradiated site is restored. In this cell line, recruitment of EGFP-PHF1 is also restored (Figure 3A). Thus, this indicates that PHF1 is recruited to the sites of DSBs in a Ku70/80-dependent manner. Like NHEJ factors, PHF1 is recruited to DSBs sites both in G1 and S/G2 phases (Figure 3E) without cell-cycle dependency. PHF1 is associated physically with ku70/ku80 Having known that the recruitment of PHF1 is Ku70/Ku80-dependent, we tested whether or not PHF1 is associated with Ku70/Ku80 proteins. In order to check the interaction, we generated two human 293 cell lines stably expressing either PHF1 or Ku80 fusion protein containing FLAG-HA tags. For prevention of any DNA-dependent association, coimmunoprecipitation was carried out in the presence of ethidium bromide (26,27). PHF1 was immunoprecipitated from 293 cells extracts by using anti-FLAG antibody and the immuno- precipitated products were analyzed by western blotting Figure 3. PHF1 is recruited to DSBs in dependence on Ku70/Ku80. (A) EGFP-PHF1 accumulates at laser-irradiated site in wild-type V79B. (B) EGFP-PHF1 does not accumulate at irradiation sites in V15B cell (Ku80-deﬁcient) but do in V15B cell (+His-Ku80). (C) EGFP-PHF1 accumulates at irradiation sites both in DNA-Pkcs-deﬁcient and proﬁcient CHO cells. (D) EGFP-PHF1 accumulates at irradiation sites in XR-1 cell (XRCC4-deﬁcient). (E) EGFP-PHF1 accumulates at irradiation sites in 1022QVA (NBS1-deﬁcient). (F) HeLa cells were synchronized to G1- and G2/S-phase by thymidine and hydroxyurea treatment. PHF1 accumulates at irradiation sites in HeLa cells both in G1 phase and S phase. Arrows indicate the sites of irradiation. with anti-Ku70 antibody. As shown in Figure 4A, Ku70 was coimmunoprecipitated with Flag-HA-PHF1, but was not present in control immunoprecipitants from the extracts of cells transfected with a blank vector. Moreover, PHF1 also coimmunoprecipitated with FLAG-HA-Ku80 from extracts of cells transfected with FLAG-HA-Ku80, but was not present in control immu- noprecipitants (Figure 4B). These results indicate that PHF1 is associated physically with Ku70/Ku80 in the cells. RNA interference of PHF1 causes cell sensitivity to X-Ray and increases HR frequency In light of the above data, we speculated that PHF1 might be involved in NHEJ. Either NHEJ or HR deﬁciency will lead cells to sensitivity to X-Ray or chemical reagent. In order to analyze this, we generated a stable PHF1 knockdown cell line using a vector-based siRNA approach. Characterization of the established cell line indicates that about 77% knockdown is achieved at mRNA level by quantitative RT–PCR (Figure 4C), but only 50% knockdown at the protein level (Figure 4D). Downregulation of PHF1 causes mild cell sensitivity to X-ray (Figure 4E). It is probably due to low eﬃciency of