Construction and characterization of recombinant plasmid DNAs containing sequences of the origin of bacteriophage ?X174 DNA replication

摘要

The synthetic DNA fragment (corresponding to nucleotides 4299–4317 of the ?X DNA sequence) was cloned into either the AmpR gene or the KmR gene of plasmid pACYC 177. The DNA sequence of the KmR gene around the insertion site was determined by nucleotide sequence analysis of the pACYC 177 Fnud∥ Irestriction DNA fragment N₆ (345 b.p.). Of five selected plasmid DNAs, which contained inserted DNA sequences in the antibiotic resistance genes, the nucleotide sequences at and around these insertions were determined. Two recombinant plasmids (pFH 704 and pFH 614) contain the hexadecamer sequence in tandem (tail-to-tail and tail-to-head). In the recombinant plasmids pFH 812, pFH 903 and pFH 807 the DNA sequence homology with the ?X o rigin region was 14 (No. 4300–4313), 16 (No. 4299–4314) and 20 nucleotides (No. 4299–4318), respectively. None of the supercoiled recombinant plasmid DNAs is nicked upon incubation with ?X gene A protein. Moreover, the recombinant plasmid RFI DNAs cannot act as substitutes for ?X RFI DNA in the in vitro (+) strand synthesizing system. It has been shown earlier that single-stranded DNA, which contains the decamer sequence CAACTTGATA is efficiently nicked by the ?X gene A protein. The present results indicate that for nicking of double-stranded supercoiled DNA nucleotide sequence homology with the ?X origin region of more than 20 nucleotides is required. These results suggest a model for initiation of ?X RF DNA replication, which involves the presence of the recognition sequence CAACTTGATA of the ?X gene A protein as well as a second specific nucleotide sequence which is required for the binding of the ?X gene A protein. This binding causes local unwinding of the DNA double helix and exposure of the recognition sequence in a single-stranded form, which then can be nicked by the ?X gene A protein.