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Transcription-induced deletions in plasmid vectors: M13 DNA replication as a source of instability
D Vilette1, S D Ehrlich, B Michel
1Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
Abstract:
We have previously shown that concurrent progression of pBR322 replication and pTac-directed transcription in opposite orientations induces illegitimate recombination events. We tested here the effects of M13 rolling circle replication on the incidence of plasmid deletions. The progression of the M13 replication fork leads to an increase of more than 300-fold in the frequency of transcription-dependent deletion events. pBR322 derivatives carrying the M13 replication origin and a 511 bp transcribed region under the control of the pTac promoter were used. Up to 12% of the plasmid population has sustained deletions within 4 h following the induction of pTac-directed transcription and M13 DNA replication, provided that the two proceed in opposite orientations. We observed that induction of transcription of the whole Escherichia coli lacZ gene (3244 bp) in the direction opposite to M13 replication leads to a fivefold decrease in plasmid copy number within 2 h, which is consistent with the proposal that deletions arise because replication fork progression is impeded. This decrease in parental plasmid copy number leads in turn to an enrichment in deleted plasmid forms. Our data confirm and extend the notion that simultaneous transcription and replication in opposite directions can efficiently promote deletion formation. In addition, this instability may be amplified when the rearranged molecules acquire a replicative advantage.
Insights
Concurrent M13 replication and transcription in opposite directions dramatically increases plasmid deletions by over 300-fold. This instability, driven by replication fork impediment, highlights risks in simultaneous DNA processes.
Area of Science:
- Molecular Biology
- Genetics
- Replication and Transcription Dynamics
Background:
- Previous work demonstrated illegitimate recombination during concurrent pBR322 replication and pTac-directed transcription in opposing orientations.
- The impact of M13 rolling circle replication on plasmid deletion frequency remained uninvestigated.
Purpose of the Study:
- To investigate the effect of M13 rolling circle replication on the frequency of plasmid deletions.
- To determine if concurrent transcription and replication in opposite directions enhance deletion formation.
Main Methods:
- Utilized pBR322 derivatives containing the M13 replication origin and a pTac-promoted transcribed region.
- Induction of pTac-directed transcription and M13 DNA replication in opposite orientations.
- Quantified plasmid deletions and copy number changes over time.
Main Results:
- M13 replication fork progression increased transcription-dependent deletion events by over 300-fold.
- Up to 12% of plasmids sustained deletions within 4 hours under opposing replication and transcription.
- Transcription opposite to M13 replication decreased plasmid copy number fivefold, suggesting replication fork impediment.
Conclusions:
- Simultaneous transcription and replication in opposite directions efficiently promote plasmid deletion formation.
- This DNA instability can be amplified if deleted forms gain a replicative advantage.
- Findings confirm and extend the understanding of replication-transcription conflicts in genome stability.