Isolation and Characterization of Functional Replication Origin from Streptomyces avermitilis

Most eubacteria contain highly conservative gene clusters in the adjacent regions
of oriC. According to this principle, a 1.4 kb DNA fragment containing
parts of dnaA and dnaN genes of Streptomyces avermitilis
was cloned by degenerate PCR. Sequence analysis of this fragment indicated that
it encoded two partial genes in the order dnaA (the putative initiator
protein) and dnaN (the β subunit of DNA polymerase III). The intergenic
non-coding region between dnaA and dnaN was found to contain 19
putative DnaA boxes, i.e. 9 nt long DnaA protein recognition sequences. It was
confirmed that the location, orientation and spacing of DnaA boxes in this intergenic
region are conserved among Streptomyces. The consensus sequence of DnaA
box identified is (T/C)(T/C)(G/A/C)TCCACA (preferred
bases underlined). When this fragment was cloned into Escherichia coli
plasmid pQC156, which is otherwise non-replicative in Streptomyces, it
exhibited autonomous replication activity in Streptomyces lividans, a
closely related Streptomyces strain. Different parts of the oriC
contribute unequally to the stability and transformation efficiency. The 3′
region of oriC may contain features that support stable autonomous replication.
The implications of these results for the understanding of the S. avermitilis
oriC replication initiation process and its future application are discussed.

Key
words Streptomyces avermitilis; replication origin oriC;
DnaA box; replication

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