Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K.

Abstract

Plasmid pMM3 is a pBR322 derivative carrying the .gamma. origin of replication of the naturally occurring plasmid R6K. A .gamma.-origin mutant bank of this plasmid was produced using the single-strand-specific mutagen sodium bisulfite. Members of this bank contain single or multiple mutations in the 7 direct repeats and the flanking sequences in the .gamma. origin. Three mutants with defective .gamma. origins were isolated from this mutant bank. Two of these direct repeat mutants, .gamma.117 and .gamma.120, are unable to replicate and also have lost the ability to bind the R6K initiation protein .pi. in vitro at 1 of the 7 22-base-pair direct repeats within their respective origins. Precise deletion of the damaged repeat of either of these mutants restores origin function, suggesting that the primary defect of these mutants involves a disruption of the normal spacing of .pi. binding and flanking sequences within the .gamma. origin. The 3rd mutant, .gamma.111, binds .pi. normally but replicates at a greatly reduced copy number due to a mutation near the 7th repeat. This mutation falls within a short sequence that appears to be conserved among a number of other plasmids that contain direct repeats within their origins of replication.