Transcription leads to pervasive replisome instability in bacteria

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• The established model of DNA replication posits a continuous, highly processive genome duplication process.
• This model is primarily derived from in vitro and ensemble in vivo experiments.

Purpose of the Study:

• To investigate the stoichiometry and dynamics of replisome complexes at the single-molecule level within bacterial cells.
• To challenge the canonical model of DNA replication by examining its in vivo behavior.

Main Methods:

• Single-molecule imaging and analysis of replisome complexes in live bacterial cells.
• Characterization of complex stoichiometry, dynamics, and lifetimes.
• Assessment of replication-transcription conflict effects on replisome stability.

Main Results:

• A significant fraction of bacterial cells (>40%) exhibit only one active replisome, contrary to expectations.
• Replisome complexes show short lifetimes (<8 min), indicating frequent disassembly and reassembly.
• Inhibition of transcription stabilizes replisomes and restores the presence of a second replisome, highlighting conflict-induced instability.

Conclusions:

• DNA replication in vivo appears to be a discontinuous process, diverging from the canonical continuous model.
• Replication-transcription conflicts are a major factor contributing to replisome instability and disassembly.
• The findings necessitate a revised understanding of DNA replication dynamics in living cells.