Journal: Science (New York, N.Y.)

Article Title: PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair **

doi: 10.1126/science.1261971

Figure Lengend Snippet: PAXX is required for DSB repair in human cells. (A) GFP-tagged PAXX accumulates at sites of laser micro-irradiation in U2OS cells. White arrowheads indicate the path of the laser used to induce DSBs. (B) Clonogenic survival assay showing that PAXX depletion in U2OS cells causes radiosensitivity and that this is rescued by exogenous expression of PAXXWT but not PAXXV199A/F201A. In this experiment and those below, error bars represent the standard error of the mean (SEM) from 3 independent experiments. (C) Clonogenic survival assay showing that PAXX−/− cells are radiosensitive and that PAXX loss is epistatic with XRCC4 depletion. (D) PAXX−/− cells display persistent γ-H2AX foci after IR. Cells with >5 foci were scored as positive, and were co-stained with Cyclin A to eliminate S and G2 cells from analysis. At least 100 cells were scored per condition. “cl.” indicates clone number. (E) PAXX is required for cellular DSB repair as measured by neutral comet assay. R/D ratios represent mean tail length of cells treated with 40 μg/ml phleomycin for 2 hours and allowed to recover (R) for 2 hours over mean tail length of cells damaged (D) for 2 hours without recovery.

Article Snippet: A peptide-competition analysis was carried out in a similar way using 20 nM PAXX 1-204 with and without 20 nM-2 μM of PAXX 177-204 or 2 μM PAXX 177-V199A/F201A-204 (Biomatik).

Techniques: Irradiation, Clonogenic Cell Survival Assay, Expressing, Staining, Neutral Comet Assay

Journal: Science (New York, N.Y.)

Article Title: PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair **

doi: 10.1126/science.1261971

Figure Lengend Snippet: PAXX promotes NHEJ in vitro and stabilizes NHEJ proteins on damaged chromatin. (A) EMSA of Ku and PAXX derivatives with 50-bp 6-FAM(6-carboxyfluorescein)-labelled DNA. 200 nM PAXX and 20 nM Ku were added where indicated. (B) Stimulation of DNA-end ligation by PAXX. 50 ng of pcDNA3.1(-) digested by EcoRV was incubated with XRCC4/LIG4 (25 nM), Ku (25 nM), PAXX (250 nM) and PAXXV199A/F201A (250 nM) as indicated (left). Ligation efficiency (right) was calculated as a percentage of ligated plasmid from four independent assays. (C) Chromatin fractionation of PAXX+/+ and PAXX−/− cells treated with phleomycin as indicated. Note that DNA-damage-dependent chromatin recruitment of proteins such as RPA, that function in DNA repair pathways other than NHEJ, were unaffected by PAXX loss. (D) Model of PAXX in NHEJ. Two Ku-bound DNA-ends are bound by a PAXX dimer via its C-termini, which stabilizes the NHEJ machinery to promote DNA-end ligation.

Article Snippet: A peptide-competition analysis was carried out in a similar way using 20 nM PAXX 1-204 with and without 20 nM-2 μM of PAXX 177-204 or 2 μM PAXX 177-V199A/F201A-204 (Biomatik).

Techniques: In Vitro, Ligation, Incubation, Plasmid Preparation, Fractionation