IN-VITRO RECOGNITION OF THE REPLICATION ORIGIN OF PLS1 AND OF PLASMIDS OF THE PLS1 FAMILY BY THE REPB INITIATOR PROTEIN

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid -encoded RepB protein, which has nicking-closing (site-specific DNA st rand transferase) enzymatic activity, The leading-strand origin of pLS 1 contains two regions, (i) the RepB-binding site, constituted by thre e directly repeated sequences (iterons or the bind region), and (ii) t he sequence where RepB introduces the nick to initiate replication (th e nic region), A series of plasmids, belonging to the pLS1 family, sho w features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region, In addition, they all share homologies at the level of their Rep proteins, However, the bind regions of these plasmi ds are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1, The RepB protein has a temperature-dependen t nicking-closing action on supercoiled pLS1, as well as on recombinan t plasmid DNAs harboring the pLS1 nic region, The DNA strand transfera se activity of pLS1-encoded RepB was also assayed on two plasmids of t he pLS1 family, namely, pE194 and pFX2, DNAs from both plasmids were r elaxed by RepB, provided they had a proper degree of supercoiling; i.e ., it was necessary to modulate the supercoiling of pE194 DNA to achie ve RepB-mediated DNA relaxation, Single-stranded oligonucleotides cont aining the nic regions of various plasmids belonging to the pLS1 famil y, including those of pE194 and pFX2, were substrates for RepB, In vit ro, the RepB protein does not need to bind to the iterons for its nick ing-closing activity.