INITIATION OF REPLICATION OF PLASMID PMV158 - MECHANISMS OF DNA STRAND-TRANSFER REACTIONS MEDIATED BY THE INITIATOR REPB PROTEIN

The initiator RepB protein of the rolling circle-replicating plasmid p MV158 has nicking-closing (topoisomerase I-like) activities on super-c oiled DNA. RepB is also able to perform a st-rand-transfer reaction on a single-stranded DNA substrate that contains its target. Several att empts at capturing covalent protein-DNA intermediates were made to ide ntify the mechanism of RepB-mediated activity. Whereas RepB did not ge nerate stable complexes with its target DNA, employment of single-stra nded oligonucleotides containing a chiral phosphorothioate in the targ et DNA allowed us to follow the process of RepB-mediated strand-transf er reaction. Tl-Lis reaction occurred through a number of even steps b ecause the chirality of the phosphorothioate at the reaction site was retained after RepB-mediated strand transfer. This finding suggests th e existence of a covalent intermediate during the strand-transfer reac tion between the protein and its target DNA. By site-directed mutagene sis at the codon for Tyr99 of RepB, and purification and assay of acti vity of the mutant protein variants, we showed that the Tyr99 residue is involved in the nucleophilic attack of RepB to its cognate DNA. (C) 1997 Academic Press Limited.