Figure 6.

An uncleaved Mu DNA end inhibits strand transfer of the cleaved partner Mu DNA end. (A) Percent of the bio_c substrate that was joined. Reactions were performed as described in Fig. 4. The 5′ end of the cleaved strand of the bio substrate was ³²P-labeled. Reactions contained either the nonbio partner substrate (solid bars) or the precleaved partner substrate (open bars). (B) Types of complexes released from the avidin matrix in reactions containing the nonbio partner substrate. (white circles) DDE⁺ transposase; (black circles) DDE⁻ transposase; (striped circles) DDE⁺ or DDE⁻ transposase. DNA 3′ of cleavage site is dashed and light gray to signify that it has been cleaved. (C) Agarose gel electrophoresis of strand transfer products generated by complexes released from the matrix in reactions containing the nonbio partner. (Lanes 1–5) The products of complexes that have cleaved both Mu DNA ends (DECL complexes). The products of DECL complexes were observed by labeling the 5′ end of the cleaved strand of the nonbio substrate. (Lanes 6–10) The products of both DECL complexes and SECL complexes; these products were observed by similarly labeling the bio substrate. Products in which both Mu DNA ends (DEST) or a single Mu DNA end (SEST) are joined to the target DNA comigrate with linear and nicked target DNA, respectively. Numbers (average of three to five experiments, ± 1 s.d.) below lanes 1, 3–5 were calculated by dividing the percent of nonbio substrate joined by the percent of nonbio substrate cleaved (before addition of the target DNA); numbers >1.0 could reflect a stimulation of cleavage by the target DNA. The percent of observed strand transfer products that were single ended (%SEST) (average of three to four experiments) was calculated by comparing the amount of radioactivity in the SEST and DEST bands. The predicted %SEST was calculated based on the probability of obtaining DDE⁺ transposase at the R1 cis position.