Volume 2, Issue 4, Pages 477-484 (October 1998) DNA Ligase IV Is Essential for V(D)J Recombination and DNA Double-Strand Break Repair in Human Precursor Lymphocytes  Ulf Grawunder, David Zimmer, Sebastian Fugmann, Klaus Schwarz, Michael R. Lieber  Molecular Cell  Volume 2, Issue 4, Pages 477-484 (October 1998) DOI: 10.1016/S1097-2765(00)80147-1

Figure 1 Organization and Targeting of the Human DNA Ligase IV Locus (a) Genomic organization of the germline and targeted human LIG4 loci. A combined PCR- and Southern-blot-mapping approach revealed that the entire open reading frame of human DNA ligase IV is encoded by a single exon (left). The nucleotide positions of some relevant restriction sites, relative to the first nucleotide of the published cDNA sequence (Wei et al. 1995) mapped by Southern blotting, is shown. The right panel shows the configuration of the targeted loci resulting from a homologous recombination event of the targeting vectors (see below). (b) Organization of the targeting vectors. Of the 5′ and 3′ regions of the LIG4 cDNA (indicated as LIG4-up and LIG4-do), 1.3 kb was disrupted by a either a 1.6 kb SV40 promoter/enhancer (pSV)-driven neomycin resistance gene (top) or a 1.4 kb SV40 promoter/enhancer-driven puromycin gene. Nucleotide positions of the LIG4 regions based on the published cDNA (Wei et al. 1995), size and position of probes used for Southern blotting (black bars), as well as the relative location of the diphtheria-toxin expression cassette are indicated. Molecular Cell 1998 2, 477-484DOI: (10.1016/S1097-2765(00)80147-1)

Figure 2 Analysis of Cells with Targeted Disruption of the LIG4 Locus (a) Southern blot analysis of wild-type Nalm-6 human precursor B lymphocytes (+/+), heterozygous LIG4 neo-disrupted Nalm-6 cells (+/−), and LIG4 neo/puro double-knockout Nalm-6 cells (−/−). Restriction enzymes used for the digestion of genomic DNA are indicated on top; probes used for the detection of specific signals are indicated below the blots. The germline bands for the LIG4 locus, when digested with AseI, NdeI, and StuI and probed with a LIG4− 5′ probe (left), migrate at 2.8 kb, 3.2 kb, and >20 kb (not visible on this blot), respectively. Bands in the neo-targeted locus migrate at 4.4 kb, 4.8 kb, and at 2.2 kb, respectively, and in the puro-targeted locus at 4.2 kb, 4.6 kb, and 2.5 kb. AseI-digested DNA was also probed with a LIG4− 3′ probe that results in two germline bands of 2.8 kb and 5.0 kb, since the probe hybridized to sequences 5′ and 3′ of the AseI site. The 2.8 kb band is changed to 4.4 kb upon neo insertion (see middle lane in AseI/LIG4− 3′ blot) and to 4.2 kb upon puro insertion, leading to 5.0, 4.4, 4.2 kb triple bands. Germline bands are not detectable in any of the LIG4−/− blots, hybridized with either of the LIG4− 5′ or LIG4- 3′ probes. The targeted alleles also hybridized to neo and puro probes of the correct sizes. The faint signal in the puro blot of the LIG4+/− line is the result of hybridization between the SV40 enhancer/promoter region that is also part of the neo-targeting construct. (b) Adenylation of DNA ligase IV coimmunoprecipitated with XRCC4. DNA ligase IV was coimmunoprecipitated with polyclonal antibodies against human XRCC4 and adenylated with [α32P-ATP] in vitro. Proteins were then fractionated by SDS-PAGE and analyzed by autoradiography. Adenylated DNA ligase IV was only detectable in the wild-type and LIG4+/− heterozygous cells, but not in the LIG4 double knockout. Molecular Cell 1998 2, 477-484DOI: (10.1016/S1097-2765(00)80147-1)

Figure 3 X-Ray Sensitivity Assay The percentage of clonogenic survival of cells with the three different LIG4 genetic backgrounds (as indicated) was determined by limiting dilution analysis. The average survival including standard deviation from two independent experiments is displayed for each data point. A percent survival value could not be calculated for 400 rad irradiated LIG4−/− cells, since no viable cells were observed under the assay conditions. Molecular Cell 1998 2, 477-484DOI: (10.1016/S1097-2765(00)80147-1)

Figure 4 Relative Levels of Expression and DNA Ligase Activity of DNA Ligase I, III, and IV (Upper panel) Expression vectors for myc epitope-tagged versions of DNA ligase I, III, and IV were transiently transfected into cells and harvested at 24 hr. Epitope-tagged proteins were immunopurified, and analyzed on PAGE and immunoblotted. C represents the empty pcDNA3 expression vector; L1, L3, and L4 represent the expression vectors for DNA ligase I, III, and IV, respectively. The band at 50 kDa in all lanes is the heavy chain of the c-myc monoclonal antibody used in the immunoprecipitation (see Experimental Procedures). (Lower panel) The ligase activity was measured by ligation of a nick in a double-stranded oligonucleotide for 30 min at 37°C in 60 mM Tris–Cl pH 7.9/10 mM MgCl2/2 mM DTT. The ligation products were analyzed on a denaturing polyacrylamide gel (Grawunder et al. 1997). Molecular Cell 1998 2, 477-484DOI: (10.1016/S1097-2765(00)80147-1)