Figure 3. Kat1 catalyzes strand-transfer.

(a) Schematic diagram of Kat1 cleavage. A DSB is formed between the flanking DNA and the transposon end. The products are a transposon end containing a free 3′-OH and hairpin-capped flanking DNA. Joining of a single 3´-OH end to target DNA leads to formation of a single end join (SEJ) product, whereas joining of two transposon ends to opposite strands of the target results in a double end join (DEJ) product. (b) Strand-transfer assay using WT Kat1 and DDE mutants as indicated, a ³²P-labeled PCR-product corresponding to TIR-R (197-bp, ~6 nM) and a circular target plasmid (pUC19). (c) Kat1 strand-transfer using pre-cleaved substrate. Schematic representation of 122-bp PCR amplified (using primers KKC55 and KKC52) DNA substrate with free 3´-OH transposon ends. The pre-cleaved transposon end (~10 nM) was incubated with pUC19, WT Kat1 and a panel of Kat1 mutant proteins. In all cases, samples were incubated with transposase for 3 hrs at 30 °C, deproteinized and separated on 1.3% native agarose gel.