Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins

Abstract

The functional R6K alpha origin is composed of two DNA elements, one of 580 bp carrying the alpha origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previously identified as also required for gamma and beta origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for alpha origin activity. The function of the alpha origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the alpha origin depends on the R6K replication initiation protein pi. Within the 580 bp of the alpha origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the beta replicon. Deletion of the 96 bp or 98 bp results in inactivation of the alpha and the beta origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the alpha and the beta origins of R6K. DNA homology analysis performed on alpha, beta and gamma origin sequences, also reveals 10-23 bp sequences in the alpha and the beta origins that are related to the family of 22 bp direct repeats in the gamma origin which were shown previously to be binding sites for the pi protein.