Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced

Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs exhibited that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly mutant forms of XLF bearing point mutations at amino acid positions L115 and L179 also completely complemented the null phenotype suggesting in contrast to predictions to the contrary that these mutations do not abrogate XLF function. Finally we demonstrate that this absence of XLF causes a HKI-272 small but significant increase in homologous recombination implicating XLF in DSB pathway choice regulation. We conclude that human XLF is usually a non-essential but crucial C-NHEJ-repair factor. 1 Introduction DNA double-strand-breaks (DSBs) are the most cytotoxic form of DNA damage. They can occur following exposure of cells to exogenous brokers Mouse monoclonal to eNOS such as ionizing radiation (IR) topoisomerase inhibitors and radiomimetic drugs ([13]. This observation however is consistent with recent work showing that in XRCC4:XLF filaments the conversation with DNA is usually mediated almost exclusively via XLF’s C-terminus [22]. Like XRCC4 XLF is usually phosphorylated at C-terminal sites by the DNA-PK complex and this appears to regulate the ability of the XRCC4:XLF filaments to bridge DNA molecules and possibly regulate V(D)J recombination [23]. XLF is also phosphorylated by both ATM HKI-272 and DNAPK restriction enzyme fragment made up of the neomycin drug selection marker. The fusion PCR product was gel purified and ligated to the pAAV backbone using restriction enzyme sites to construct the final targeting vector. 2.3 Packaging and isolating computer virus The targeting vector (8.0 μg) was mixed with pAAV-RC and pHelper plasmids (8.0 μg of each) from the AAV Helper-Free System and was transfected into AAV 293 cells using Lipofectamine 2000. Computer virus was isolated from the AAV 293 cells 48 h after transfection using a freeze-thaw method [53]. 2.4 Infections HCT116 cells were produced to ~70-80% confluence in 6-well tissue culture plates. Fresh media (1.5 ml) was HKI-272 added to the cells 3 h prior to addition of the computer virus. The required volume of the computer virus was added drop-wise to the plates. After a 2 h incubation at 37°C another 1.5 ml of media was added to the plates. After a further 48 h incubation the cells were transferred to 96-well plates and placed under selection (1 mg/ml G418) to obtain single colonies. 2.5 Isolation of genomic DNA and Southern hybridizations Chromosomal DNA was prepared digested subjected to electrophoresis and then transferred to a nitrocellulose membrane as described [56]. The membrane was hybridized with probe (Fig. 1C) to detect correct targeting of the XLF targeting vector. The probe corresponds to ~550 bp and was made by PCR with the primers XLF5’ProbeF1 5 and XLF5’ProbeR1 5 The PCR product was electrophoresed on a 1% agarose gel and gel purified prior to use. Probe ‘and end-joining reporter plasmid pEGFP-Pem1-Ad2 has been described [52 59 The plasmid was digested to completion (8 to 12 h) with expression plasmid and 1.0 μg DR-GFP SA-GFP or EJ2-GFP+ assay substrates. HKI-272 GFP and mCherry expression was then analyzed 48 hr post transfection using flow cytometry as described above. The repair efficiency was calculated as the percentage of GFP and mCherry doubly positive cells divided by the mCherry-positive cells. 2.15 Microhomology assay The microhomology assay HKI-272 (which is an independent measure of A-NHEJ) was performed as described [52 63 In brief 2.5 μg HKI-272 of (to remove un-replicated plasmids) transfected into chemically competent Top10 cells and then plated on ampicillin (100 μg/ml) or ampicillin (100 μg/ml) and chloramphenicol (22 μg/ml) plates. DAC colonies (DAC = DpnI-treated-AmpR-CamR) represent V(D)J recombination events whereas DA colonies (DA = DpnI-treated-AmpR) are a measure of total plasmids recovered from each transfection. The percentage of signal joint or coding joint formation was calculated by dividing DAC by DA counts. 2.17 Telomere FISH Cells were treated with colcemid at 100 μg/ml for 3 h to obtain metaphases. The cells were then trypsinized and harvested by centrifugation. Metaphase spreads were prepared according to the manufacturer’s.