Tetracycline promoter mutations decrease non-B DNA structural transitions, negative linking differences and deletions...

Main Methods:

• Cloning of various DNA sequences, with differing non-B DNA forming potentials, into three distinct loci (EcoRI, BamHI, PvuII) of the pBR322 plasmid.
• Modification of the tetracycline resistance gene promoter by altering unique HindIII or ClaI sites.
• Analysis of insert stability (deletions) and plasmid negative linking differences in Escherichia coli.

Main Results:

• Inserts were generally stable in the EcoRI site but showed deletions in the BamHI site (tetracycline resistance gene) if prone to Z-DNA or triplex formation.
• Altering the tetracycline resistance gene promoter prevented deletions in the BamHI site, concurrently reducing negative linking differences.
• Z-DNA forming sequences were deleted in the PvuII site (replication origin vicinity) but less so when the tetracycline promoter was mutated.
• Sequences forming cruciforms were stable in all tested conditions and sites.
• Deletion frequency correlated with the propensity of sequences to form Z-DNA or triplex structures, not with random sequences or less structured inserts.

Conclusions:

• The formation of non-B DNA structures, specifically Z-DNA and triplexes, in vivo is a primary cause of DNA insert deletions in certain pBR322 plasmid sites.
• DNA supercoiling and the cellular environment play a complex role in mediating insert deletions, particularly in relation to non-B DNA formation.
• Insert stability is sequence-dependent and site-specific, highlighting the intricate interplay between DNA structure, plasmid topology, and genetic recombination processes in living cells.