DNA transcription and repressor binding affect deletion formation in Escherichia coli plasmids.
Chimeric plasmids containing phage M13 and plasmid pBR322 sequences undergo deletions in Escherichia coli with a high frequency. In all plasmids one deletion endpoint is the M13 replication origin nick site. We examined the effects of transcription on the position of the other deletion end-point, by inserting in the plasmids an inducible promoter followed by a transcription terminator. Transcription dramatically affected deletions in an orientation-dependent way, such that greater than 95% of end-points were localized downstream from the inserted promoter when it faced the major plasmid transcripts. The end-points were not constrained to the transcribed region and were not affected by the orientation of pBR322 DNA replication. We propose that deletion events occur preferentially in a plasmid domain which is rendered positively supercoiled by convergent transcription. We also show that interaction of LacI repressor with the cognate operator generates a localized deletion hot spot. This hot spot is dependent on pBR322 replication, and therefore probably acts by arresting progression of DNA replication.
Vilette
D
D
Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
Uzest
M
M
Ehrlich
S D
SD
Michel
B
B
D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D008969:Molecular Sequence Data; D010957:Plasmids; D012097:Repressor Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004279:DNA, Viral; D012097:Repressor Proteins