Identification of topoisomerase II cleavage sites in MLL and FRYL that form translocation breakpoint junctions. Substrates contained MLL bcr positions 5382-5570 (A) or positions 1309-1479 in FRYL intron 50 (B). Autoradiographs show cleavage products after 10-minute incubation at 37°C of singly 5′ end-labeled DNA (30 000 cpm) and unlabeled substrate (25 ng, labeled and unlabeled total) with 147 nmol/L human topoisomerase IIα, 1 mmol/L ATP and, where indicated, 20 μmol/L etoposide, etoposide catechol, etoposide quinone, or doxorubicin. Specified reactions were incubated for an additional 10 minutes at 75°C before trapping the covalent complexes (panels A,B). Jagged arrows designating translocation breakpoints (panels A,B far right) bracket the normal homologue of the deleted region in each substrate. Topoisomerase II creates staggered nicks in duplex DNA with 4-base 5′ overhangs⁴⁰ ; bases at the 5′ side of cleavage (−1 position) on the sense strand were used to designate cleavage site positions (dashes at right of gels). Amounts of cleavage at sites within the deleted regions (circled) were quantified by filmless autoradiographic analysis in (+) Topo reactions with specified compounds without subsequent 75°C incubation relative to cleavage at same sites without any drug (panels A,B bottom). The lanes shown in (A) are from a single large gel that was scanned in multiple sections because of its size. After scanning, the lanes were aligned together in their original positions as they appear in the complete gel. (C) Model for creation of t(4;11) breakpoint junctions by processing of the etoposide metabolite enhanced cleavage sites at MLL bcr position 5485 in intron 8(11) (top, red) and position 1409 in FRYL intron 50 (top, blue). The processing includes exonucleolytic digestion (italic) to form homologous overhangs and create both breakpoint junctions by classic error-prone NHEJ (boxes). In formation of the der(11), positions 5476-5485 on the sense strand and positions 5478-5489 on the antisense strand of MLL bcr and positions 1410-1421 on the sense strand and positions 1414-1423 on the antisense strand of FRYL intron 50 are deleted by exonucleolytic digestion (middle, italic) before NHEJ (middle, box) joins the indicated bases. Exonucleolytic digestion removes positions 1388-1409 on the sense strand and positions 1391-1413 on the antisense strand of FRYL intron 50 (bottom, italic) but all bases in the sense strand of the 4-base 5′ overhang in MLL are retained, and NHEJ (bottom, box) and gap fill-in create the der(4) breakpoint junction.