Initiation signals for complementary strand DNA synthesis on single-stranded plasmid DNA

Abstract

The bacteriophage 0X174 origin for (+) strand DNA synthesis, when inserted in a plasmid, is in vivo a substrate for the initiator A protein, that is produced by infecting phages. The result of this interaction is the packaging of single-stranded plasmid DNA into preformed phage coats. These plasmid particles can transduce 0X-sensitive cells; however, the transduction efficiency depends strongly on the presence in the packaged DNA strand of an initiation signal for complementary strand DNA synthesis. A plasmid with the complementary (-) strand origin of 0X inserted in the same strand as the viral (+) origin transduces 50-100 times more efficient than the same plasmid without the (-) origin of 0X. The transduction efficiency of such a particle is comparable to the infection efficiency of the phage particle. It is shown that in this system the 0X (-) origin can be replaced by the complementary strand origins of the bacteriophages G4 and M13. We have used this system to isolate sequences, from E. coli plasmids (pACYC177, CloDF13, miniF and OriC) and from the E. coli chromosome that can function as initiation signals for the conversion of single-stranded plasmid DNA to double-stranded DNA. All isolated origins were found to be dependent for their activity on the dnaB, dnaC and dnaG proteins. We conclude that these signals were all primosome-dependent origins and that primosome priming is the major mechanism for initiation of the lagging strand DNA synthesis in E. coli. The assembly of the primosome depends on the sequence-specific interaction of the n' protein with single-stranded DNA. We have used the isolated sequences to deduce a consensus recognition sequence for the n' protein. The role of a possible secondary structure in this sequence is discussed.

Identification of single‐strand initiation signals in the terC region of the Escherichia coli chromosome

“…First, homologies to the consensus sequence of the n' protein recognition site [26], 5'-GAAGCGG-3', are observed within both of the sit signal sequences. This type of sequence homology has been found in the initiation signals from plasmids [1] and the E. coli chromosome [3]. These observations suggest that these sit signals can function as n'-dependent primosome assembly sites, as do other ssi signals from plasmids and phages.…”

“…Sequence homologies, the variations of the 5'-GAAGCGG-3' sequence, were found ( fig.lA). This consensus sequence, 5'-GAAGCGG-3', has been observed in the n' protein recognition sites in ~X174 DNA [26] and in the n'-dependent initiation signals found in plasmids [1,3]. Judging from the nucleotide sequences, a stable stem-loop structure was predicted in sitB ( fig.…”

“…A stem-loop structure has been predicted in many ssi signals [1,3,8,18,19]. Judging from the nucleotide sequence of sitB, a stable stem-loop structure is possible as shown in fig.…”

“…Some efforts have been made to identify the primosome assembly sites in the oriC region of the E. coil chromosome. However, these efforts were in vain because no DNA sequences within the oriC region or its close vicinity support initiation of DNA synthesis [1,2]. In contrast, some n' protein recognition sequences which promote DNA initiation are located within and clockwise to the asnA gene [2,3].…”

DNA replication of single-stranded Escherichia coli DNA phages

“…The stem of the different recognition sites has a stretch of 7 nucleotides in common: ~AAGCGG. This 7-nucleotide stretch may be important in the recognition of the DNA by the n' protein [46]. DNAase foot-printing and methylation protection experiments have indicated that factor Y binds to the entire length of the effector site with three preferential sites, at the top, in the middle and at the bottom of the hairpin [47].…”