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Figure 7
D-loop formation by DNA with 5'-single-stranded ends mediated by RecT. pUC18 DNA was linearized by ScaI and partially degraded with exonuclease III, as described in Materials and methods, to generate a resected duplex carrying 5'-single-stranded tails about 100 bases long, designated 5'-ssDNA. 5'-ssDNA (20  M) was incubated with the indicated amounts of RecT for 20 min at 25°C. Supercoiled pHV2900OB DNA (40 M) was added to initiate the reactions (lanes d–k) or was omitted in controls (lanes a–c). The reaction mixtures were incubated for 50 min at 37°C, deproteinized and the products resolved by electrophoresis in a 0.9% agarose gel revealed by SYBR green staining. Unreacted supercoiled pHV2900OB DNA is in lane l. jm, joint molecules; nc, nicked circular duplex; ccc, supercoiled pHV2900OB.
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