Figure 2.

RecD inhibits RecA loading during DNA unwinding by RecB^D1080ACD enzyme. RecBCD, RecBC, RecB^D1080ACD, and RecB^D1080AC enzymes were assayed by using 5′-³²P-labeled (*) pBR322 χ⁺F225 DNA (see diagram) as described in Materials and Methods. The DNA substrate (4.7 nM) and indicated amount of mutant or wild-type RecBCD enzyme were incubated at 37^o for 2 min in a reaction mix with RecA protein (lanes 3–6, 11–14, 19–22, and 27–30) or without RecA protein (lanes 7–10, 15–18, 23–26, and 31–34). An aliquot was removed for analysis (0 min Exo I). Exonuclease I was added to the remaining sample, and incubation was continued for the times indicated. The products of reaction were analyzed by electrophoresis in a 1% agarose gel (Materials and Methods). The position of ds substrate DNA (DS; lane 1), unwound ssDNA (SS; boiled, in lane 2), and the major product of Chi-dependent nuclease attenuation (Chi) are shown.