PURIFICATION OF THE LARGE MOBILIZATION PROTEIN OF PLASMID RSF1010 ANDCHARACTERIZATION OF ITS SITE-SPECIFIC DNA-CLEAVING DNA-JOINING ACTIVITY

A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transf er during bacterial conjugation. We have previously reproduced this ni cking at the duplex oriT in vitro using purified preparations of the t hree known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase), MobB and MobC [Scherzinger, E., Lurz, R., Otto, S. & Dobrin ski, B. (1992) Nucleic Acids Res. 20, 41-48]. In this study we report the purification of MobA to apparent homogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying t he cleavage of sets of oligodeoxyribonucleotides varying successively by single nucleotides at the 5' or 3' end, the minimal substrate for c leavage has been defined. The results identify the MobA recognition se quence within the 11-residue oligonucleotide AAGTGCGC-CCT which is cle aved at the 3' side of the G at position 7. During the cleavage reacti on, MobA becomes covalently linked to the 5'-phosphate end of each bro ken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provid ed that the DNA molecule contains at its 3' end at least the seven nuc leotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a tyrosyl phosphate. Extensive digestion of the P-32-labeled MobA-oligon ucleotide complex with lysine carboxypeptidase yielded a single DNA-bo und peptide which was purified and sequenced. The resulting peptide se quence consists of amino acid residues at positions 22-30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the cova lent complex.