CtIP and Exo1 promote DNA resection during the joining of intrachromosomal I-SceI–induced DSBs. (A) Schematic representation of the IgH^I-96k allele before (top) and after (bottom) recombination by I-SceI. Blue circles indicate the I-SceI sites. Black arrows represent the position of the PCR primers for amplifying the recombination products. The perfect (nonresected) join is 336 nt. Sμ and Sγ1 are Switch μ and Switch γ1 regions, respectively. (B, left) Identification of sh-RNAs against CtIP. Western Blot analysis of protein lysates from activated B cells infected with candidate shRNAs and analyzed after 96 h in culture. The two best shRNAs were selected for the experiments (sh-CtIP1 and sh-CtIP2). (right) Time course of knock down by sh-CtIP1 (d = days since beginning of the culture). Two and one experiment, respectively. (C, left) Representative ethidium bromide–stained agarose gels with PCR products obtained after I-SceI recombination in IgH^I-96k/+53BP1^−/−AID^−/− B cells infected with I-SceI and sh-RNAs against CtIP (sh-CtIP1) or lacZ (sh-control), followed by sorting of the double-infected population. Arrows point to the 300 nt size, under which bands are scored as extensively resected. Vertical dotted line indicates merge of two adjacent pictures of the same gel. (middle) Histogram with the frequency of I-SceI–induced recombination products with extensive resection (>35 nt). (right) Resection dot plot, with each dot representing one sequence. On the Y axis is the number of resected nucleotides. (D) Same as in Fig. 1 C (middle), but for IgH^I-96k/+AID^−/− B cells (53BP1 proficient). (E) As in Fig. 1 C for IgH^I-96k/+53BP1^−/−AID^−/−Exo1^−/− B cells and Exo1-proficient control. (F) Same as in Fig. 1 C (middle), but for IgH^I-96k/+AID^−/−Exo1^−/− and IgH^I-96k/+AID^−/−Exo^+/+ B cells (53BP1 proficient). (G) Dot plots showing the number of nucleotides microhomology at the junctions from recombination products of I-SceI–infected IgH^I-96k/+53BP1^−/−AID^−/− B cells from the indicated experimental conditions. Sequences were grouped according to the extent of resection (more or less than 30 nt). Error bars in histograms indicate standard deviations. Horizontal lines in dot plots indicate the means. P-values were calculated using the unpaired two-tailed Student’s t test. All graphs represent data from at least two separate PCR reactions for each of two independent experiments.