Relaxosomes are presumably composed of supercoiled plasmid DNA with transfer factors complexed at the oriT site (Lanka and Wilkins, 1995) . To confirm whether the MobA89K and MobC89K proteins can induce the site-and strand-specific DNA cleavage reaction, the relaxation activities of these purified proteins on supercoiled plasmid DNA were tested. The supercoiled oriT-containing plasmid pMD-oriT was used as substrate DNA in an in vitro cleavage reaction. As shown in Fig. 5B , no relaxation was observed in the absence of either protein component. However, MobA89K showed an obvious relaxation activity in the presence of the accessory protein MobC89K, yielding 56% nicked, open circular (OC) plasmid DNA compared with 10% in the untreated control. These findings suggested that both MobA89K and MobC89K are required for the nicking reaction. Meanwhile, MobA89KN258 also increased the conversion of the covalently closed circular (CCC) plasmid DNA into the relaxed form (OC) by the addition of MobC89K, yielding 45% nicked products (i.e. slightly less than that observed for the full-length MobA89K protein).