Investigation of t(15;17) translocation mechanism in UPN 2 by in vitro topoisomerase IIα DNA cleavage assay. Chromosomal breakpoint junctions were examined in an in vitro topoisomerase IIα cleavage assay using substrates containing PML (A) and RARA (B) translocation breakpoints in the APL case of UPN 2. Reactions in lane 1 were performed without DNA topoisomerase IIα and lanes 2 to 5 show dideoxy sequencing reactions. DNA cleavage reactions were performed in the presence of 147 nM of human DNA topoisomerase II alpha and in the absence (lanes 6 and 8) or presence of 20 μM mitoxantrone (lanes 7 and 9). Reactions in lanes 8 and 9 were incubated at 75°C to assess the heat stability of the cleavage products seen in lanes 6 and 7. In each case, the location of the relevant heat stable cleavage site is indicated by an arrow on the far right. (C) Native PML and RARA sequences are shown in red and blue, respectively. In the creation of the PML-RARA genomic fusion, processing includes exonucleolytic deletion to form a 2-base homologous overhang that facilitates repair via the error prone NHEJ pathway. In the creation of the reciprocal RARA-PML genomic fusion, 2-base homologies facilitate NHEJ repair, whereas in both instances polymerization of the relevant overhangs fills in any remaining gaps (shown black font).