Figure 5: Artemis cleaves newly formed hairpin ends.

(a) Rearrangement-specific PCR of the samples obtained after knockdown of candidate genes. Several siRNAs for Artemis, CtIP, MRE11 or ligase IV, as well as a control siRNA, were transfected into HEK 293 cells before the transfection of substrate plasmids. At 24 h after transfection of the plasmids, the DNAs were collected and translocation-specific PCR was carried out (left). The top band at ~650 bp (an arrow) corresponds to a product derived from fusion at the tips of the hairpins of PATRR11 and 22. Knockdown of Artemis and ligase IV specifically reduced the top band, indicating that these enzymes were involved in generation of the translocation. Some minor bands below the top band (bracket) were by-products of the model system, because these shorter products cannot be observed when the same translocation-specific PCR was performed to detect de novo translocations in sperm samples from human males. (b) Quantification of the ~650 bp PCR products (n=3). (c) Schematic representation of the central inversion produced by Artemis cleavage at 2 bp distal from the tip of the hairpin. Data are presented as means±s.d. Statistical significance against control siRNA was assessed using a Student’s t-test: *P<0.01.