Figure 5.

In vitro HJ formation by XerCD on a supercoiled plasmid carrying two dif sites. (A) restriction map of plasmid pSDC124 and of the HJ created by action of XerCD. (B) One-hour reactions in 40% glycerol. (C) Time course experiment in 40% glycerol and effect of the glycerol concentration in 1-h reactions. Molecular weights are in kilobase pairs. (D) Determination of the Xer recombinase whose catalytic activity is necessary for HJ formation. One-hour reactions in 40% glycerol used catalytically inactive mutants of XerC (C^YF) and XerD (D^YF) mixed with their functional partner. pSCD124 was left untreated (uncut, linear) or treated with the Xer recombinases (Xer). EtBr was added at the end of reactions as indicated. For the 20′ time point, EtBr was added at the beginning of the reaction. sc 1, sc 2, lin, and oc, supercoiled monomer, supercoiled dimer, linear, open circle forms of pSDC124, respectively. s, restriction fragments from pSCD124; χ, χ-molecule containing a HJ; α and α′, supercoiled and nicked α-molecules containing a HJ.