Figure 4.

RecBC enzyme loads RecA protein only onto the DNA strand that terminates 3′ at the entry site. The starting dsDNA substrate consisted of two fragments, 3.7 and 0.6 kb, described in Materials and Methods. Each fragment has one end that was resected by Exo III and is inaccessible to RecBC enzyme and another AlwNI-restricted end at which the enzyme can enter. Exo I protection assays were performed. Control reactions were also performed in which either RecA protein or Exo I was omitted. After electrophoresis and Southern transfer, the DNA was probed with one of two strand-specific probes. (A) Top strand (probed with PB19); (B) bottom strand (probed with PB27). (The 0.6-kb fragment hybridizes to neither probe and is not shown.) The positions of 3.7-kb dsDNA starting material and ssDNA unwinding product are indicated. (Specificity marker) Lanes demonstrate the specificity of the two probes. These lanes contain a mixture of NdeI-restricted pBR322 χ⁰ dsDNA (the 4.3-kb fragment, which hybridizes to both probes) and pBR322 χ⁰ DNA restricted with EcoRI and AvaI (which produces fragments of 1.4 and 2.9 kb; probe PB19 hybridizes only to the smaller fragment, whereas PB27 hybridizes only to the larger fragment).