Identification and characterization of the functional α origin of DNA replication of the R6K plasmid and its relatedness to the R6K β and γ 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.

Genes involved in conjugative DNA processing of plasmid R6K

“…1. ␣-oriV was confined to a 639 bp region between R6K co-ordinates 9.4 kb and 10.0 kb (and thus contains ␣-LIR) (Shafferman et al, 1987). With respect to ␤-oriV, there is conflict among published data.…”

“…1). When this sequence was merged with the previously published sequence around oriT␣ (co-ordinates 9.4 kb to 10.1 kb) (Shafferman et al, 1987), analysis of the merged sequence uncovered two potential open reading frames (ORFs). The first ORF was located next to the palindromic sequence containing oriT␣ (Avila et al, 1996), in the trailing region of the transferred strand (co-ordinates 9.8 kb to 10.4 kb in Fig.…”

“…DNA sequence of the ␣-oriV-oriT␣-taxA region. Nucleotides 1-485 of the sequence shown were taken from the 3Ј end of the complementary strand of sequence X05644 that contains R6K ␣-oriV (Shafferman et al, 1987). Nucleotides 474-813 are from sequence X95535 (Avila et al, 1996).…”

“…A site containing ssi activity located at ␣-oriV is a binding site for protein DnaG (Masai et al, 1990) and, consequently, it is probable that the G site would be the site of initiation of replication in ␣-oriV. The ␣ and ␤ oriVs of R6K map 5.3 kb apart, and are physically localized to segments of DNA containing a pair of palindromic sequences (long inverted repeats (LIR)) that are 94/98 bp identical (Shafferman et al, 1987). As the LIR sequences lay within the limits of the experimentally determined oriVs, another set of data assumed the LIR sequences to be essential for oriV function (Shafferman et al, 1987;Flashner and Shafferman, 1990).…”

“…The ␣ and ␤ oriVs of R6K map 5.3 kb apart, and are physically localized to segments of DNA containing a pair of palindromic sequences (long inverted repeats (LIR)) that are 94/98 bp identical (Shafferman et al, 1987). As the LIR sequences lay within the limits of the experimentally determined oriVs, another set of data assumed the LIR sequences to be essential for oriV function (Shafferman et al, 1987;Flashner and Shafferman, 1990). In particular, two landmarks of the LIR sequences were thought to reflect their activity as replication origins (Flashner and Shafferman, 1990): a primary distortion characterized both by a hypersite and a specific footprint upon in vivo dimethyl sulphate (DMS) treatment, and a secondary distortion which created a cluster of breakpoints upon in vivo treatment with KMnO 4 .…”