Figure 4: Cleavage of the cruciform structure by GEN1.
(a) Effect of candidate gene knockdown on cleavage. siRNAs for GEN1, SLX1 or MUS81, as well as control siRNA, were transfected into HEK 293 cells before transfection of a substrate plasmid. After a 3-h transfection of the plasmid, DNAs were collected and subjected to Southern blotting (upper panel), and the cleaved bands were quanrtified relative to the control siRNA (lower panel, n=6). Samples of untransfected plasmid DNA (plasmid) and DNA from untransfected HEK 293 cells (–) were loaded for control. (b) Double knockdown of the combination of the candidate genes. Cleavage was monitored by Southern blot (upper panel) and then quantified (lower panel, n=3). (c) Overexpression of recombinant GEN1 in HEK 293 cells. Only the expression of wild-type GEN1 (GEN1wt) induced the cleavage of the cruciform-extruding plasmid, whereas mutant GEN1 (GEN1mut) or transfection of a control empty vector (control) did not (n=3). (d) Recombinant GEN1 cleaved the PATRR-forming cruciform in vitro. Overexpressed GEN11–527-HIS in E. coli was purified and used for in vitro cleavage of the PATRR cruciform. Cleavage of the cruciform-extruding plasmid was observed in a way similar to the sample treated with T7 endonuclease I (T7 endo I) (n=3). (e) Translocation-like rearrangement products monitored by semi-quantitative PCR. Decrease of the translocation-like rearrangement molecules (arrow) was observed only in the GEN1 knockdown, which diminished cleavage of the cruciform structure (n=3). Data are presented as means±s.d. Statistical significance against control siRNA was assessed using a Student’s t-test: *P<0.01.
