Paul K , Wang M , Mladenov E , Bencsik-Theilen A , Bednar T , Wu W , Arakawa H , Iliakis G .

	Figure 2. LIG3 supports processing of DSBs also after low doses of radiation.(A) Representative images of γ-H2AX foci formation in wt, LIG1−/−, LIG4−/− and LIG1−/−LIG4−/− DT40 cells after exposure to 1 Gy X-rays at the indicated times after IR. (B) Representative kinetics of γ-H2A.X foci formation and decay of wt DT40 cells as measured by immunostaining after exposure to 0.5 and 1 Gy X-rays. The results shown represent the analysis of 4000 nuclei in one representative experiment. (C) γ-H2A.X foci scored in wt, LIG1−/−, LIG4−/− and LIG1−/−LIG4−/− cells 8 h after exposure to 1 Gy X-rays. Foci measured in non-irradiated cells have been subtracted. Results of two independent experiments, in which 8000 nuclei were scored, were used to calculate the indicated means and standard errors.
	Figure 3. Conditional knockout of LIG3 reveals the function of LIG1 in the processing of IR-induced DSBs.(A) Kinetics of DSB processing in the indicated mutants after treatment with 4HT for the indicated periods of time. Other details are as in Fig. 1B. Results from three independent experiments with 3 samples each were used to calculate the indicated means and standard errors. (B) LIG3 mRNA levels measured by real-time PCR in wt and LIG3−/2loxP cells after different incubation times with 4HT. The mRNA level measured in wt cells was set to 100%. (C) Western blot analysis of LIG3 protein in LIG3−/2loxP cells after treatment with 4HT for the indicated periods of time. A mouse monoclonal antibody against human LIG3 (clone 1F3) that recognizes the chicken LIG3 was used. GAPDH is a loading control. (D) Apoptotic index measured by microscopically scoring nuclear fragmentation and pycnosis in wt and LIG3−/2loxP cells at various times after treatment with 4HT. Results from two independent experiments in each of which 1000 cells were scored were used to calculate the indicated means and standard errors. (E) As in A for the indicated mutants. (F) Representative cell-cycle distribution histograms obtained by flow cytometry in wt, LIG4−/− and LIG3−/2loxPLIG4−/− cells treated with 4HT for 3.5 days before and after enrichment by centrifugal elutriation in G2 phase of the cell cycle. (G) Kinetics of DSB processing in cells enriched by centrifugal elutriation in the G2 phase of the cell cycle as shown in F.
	Figure 4. Deletion of nuclear LIG3 reveals a function for LIG1 in the processing of IR-induced DSBs.(A) Western blot analysis of LIG3 protein in the cytosolic fraction (CP), the mitochondria fraction (MP) and the nuclear fraction (NP) of wt and LIG3−/M2I cells. GAPDH, α-Tubulin, and histone H1 are fractionation- and loading-controls. (B) Subcellular localization studies by live cell imaging using mtLIG3-GFP fusion protein to follow the intracellular distribution mtLIG3 and MitoTracker Deep Red to visualize the mitochondria. Cell nuclei were counterstained with Hoechst 33342. Note the colocalization between GFP and Deep Red that indicates the localization of mtLIG3 in the mitochondria. (C) Kinetics of DSB repair in wt, LIG3−/2loxP, LIG3−/M2I, LIG4−/− and LIG3−/M2ILIG4−/− after exposure to 40 Gy X-rays. Results from at least two independent experiments with 4 samples each were used to calculate the indicated means and standard errors. Other details are as in Fig. 1B. (D) Kinetics of DSB repair in wt and LIG3−/−Cdc9 cells measured in the presence or absence of 10 µM NU7441, a DNA-PKcs specific inhibitor. This LIG3−/− mutant is viable as a result of the expression of the yeast homolog of LIG1, Cdc9 [18]. Other details are as in C. (E) Kinetics of DSB repair in asynchronous LIG3−/2loxPLIG4−/− and clones 1, 3 and 7 of LIG3−/2loxPLIG4−/−mts-hLig1 cells. Other details are as in C. (F) Kinetics of DSB repair in asynchronous LIG3−/2loxPLIG4−/−, clones 1, 3 and 7 of LIG3−/−Lig4−/−mts-hLig1 cells and of LIG3−/2loxPLIG4−/−3.5 days after 4HT treatment, respectively. Other details are as in C.
	Figure 5. Dominant contribution of LIG3 in-vitro end joining.(A) Representative gels of in vitro DNA end joining of Sal I linearized pSP65 plasmid using whole cell extracts of asynchronous wt, LIG1−/−, LIG4−/−, and LIG1−/−LIG4−/− cells. The linearized input substrate (linear) and the rejoined products (circles, dimers and multimers) generated by end joining are indicated (B) As in A. for LIG3−/2loxP cells after treatment with 4HT for the indicated periods of time. (C) As in A. for LIG3−/2loxPLIG4−/− cells after treatment with 4HT for the indicated periods of time.
	Figure 6. LIG1 and LIG3 contribute to the survival of cells exposed to IR.(A) Cell survival measured by colony formation in wt, LIG1−/−, LIG4−/− and LIG1−/−LIG4−/− cells after exposure to increasing doses of X-rays. Results from three independent experiments with 3 replicates each were used to calculate the indicated means and standard errors. (B) As in A. for LIG3−/2loxP, and LIG3−/−Cdc9 cells after treatment for 1 h before and 4 h after IR with 10 µM NU7441. The dashed lines trace for comparison the results of wt and LIG1−/−LIG4−/− cells. (C) As in B. for LIG3−/2loxP, LIG3−/M2I, LIG3−/2loxPLIG4−/−, and LIG3−/M2ILIG4−/− cells. Results from at least two independent experiments with 3 replicates each were used to calculate the indicated means and standard errors. (D) As in B. for clone 3 of LIG3−/2loxPLIG4−/−mts-hLIG1 and LIG3−/−LIG4−/−mts-hLIG1. Results from at least two independent experiments with 3 replicates each were used to calculate the indicated means and standard errors.
	Figure 1. LIG3 processes IR-induced DSBs in LIG1 and LIG4 deficient cells.(A) Representative dose response curves for the induction of DSBs, as measured by PFGE, in cells exposed to increasing doses of X-rays. Images of ethidium bromide stained gels (upper panel) were analyzed to estimate the fraction of DNA released (FDR) from the well into the lane (regions defined as indicated) that is plotted as a function of IR dose for the indicated mutants (lower panel). Results from three independent experiments with 3 samples each were used to calculate the indicated means and standard errors. The dotted lines indicate the approach used to deduce Deq from FDR in DSB repair experiments (see text for details). (B) Repair kinetics of IR-induced DSBs in asynchronous wt, LIG1−/−, LIG4−/−, and LIG1−/−LIG4−/− cells after exposure to 40 Gy X-rays. The upper panel shows typical gels used to calculate the FDR at each repair time point, which was subsequently converted to Deq with the help of dose response curves such as those shown in A but generated with the same cell population used in the repair experiment (see text for details). Results of three determinations from at least two independent experiments were used to calculate the indicated means and standard errors (lower panel).

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