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

	Figure 1. Eukaryotic DNA ligases and targeting strategies. (A) Domain structure of chicken DNA ligases. Nc, nuclear; mt, mitochondrial. Red bars indicate regions deleted in the mutants generated. The β form of LigIII is inferred, as the corresponding exon could not be identified. (B) Vectors and approach taken to generate conditional and constitutive knockouts of DT40 LIG3 alleles. Gels show PCR products confirming genome editing steps. The steps followed to generate the indicated mutants are outlined in the lower right. (C) Approach to generate an allele expressing the mitochondrial version of LigIII.
	Figure 2. LIG3, but not LIG1 or LIG4, is essential for DT40 survival. (A) Determination of LIG3 mRNA level in LIG32loxP/− cells treated with 4HT. The mRNA levels of LIG1, LIG4 and LIG3 were measured by real-time PCR at different times after incubation with 4HT. (B) Western blot analysis of LigIII protein level in LIG32loxP/− cells treated with 4HT. A mouse monoclonal antibody, raised against human LigIII (clone 1F3), which efficiently recognizes the chicken LigIII was used. Protein loading was monitored with an anti-GAPDH antibody. (C and E) Growth kinetics of the indicated cell lines maintained in the exponential phase of growth by routine dilution in fresh growth medium. (D) Colony forming ability of wt and LIG32loxP/−DT40 cells as a function of time after incubation with 4HT. (F) Accumulation of Okazaki fragments in LigI-deficient human and mouse cells, as well as the indicated DT40 mutants. The graph shows the percentage of total radioactivity present in single-stranded DNA fragments <2.0 kb for each cell line.
	Figure 3. The role of mitochondria in DT40 cell viability. (A, B, D and E) Growth kinetics of indicated mutants in the presence or absence of 4HT. (C) Colony forming ability of indicated mutants incubated in the presence or absence of 4HT for the indicated periods of time.
	Figure 4. Analysis of LigI and LigIII functions in DNA replication (A) The upper panel on the left shows a representative flow-cytometry histogram of G1-enriched cells obtained by centrifugal elutriation; the lower panel shows the same population 6 h after incubation at 41°C to allow progression through the cell cycle. The horizontal bar shows the subpopulation used to estimate progression through the cycle of the population median. Middle panel: progression through S-phase calculated by following the relative increase in DNA content for the median of the population described in A. Right panel: progression through S-phase of LIG32loxP/− cells treated with 4HT for 3.5 and 4 days. (B) Representative flow-cytometry histograms of the indicated mutants with or without 4HT incubation. (C) Left panel: representative dot plot of BrdU-labeled wt cells. The gates applied to calculate the active S-phase fraction are shown. Middle panel: fraction of actively replicating cells. Right panel: fraction of actively replicating cells at various times after treatment with 4HT.

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