Rescuing the bacterial replisome at a nick requires recombinational repair and helicase reloading

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• DNA damage is a constant threat to genome integrity, necessitating efficient repair mechanisms.
• Unrepaired DNA lesions, particularly single-strand discontinuities, can be converted into double-strand breaks (DSBs) when encountered by replication forks.
• Investigating the impact of DNA repair sites on replication machinery has been challenging due to the stochastic nature of DNA damage.

Purpose of the Study:

• To investigate the fate of the DNA replication machinery (replisome) at site-specific single-strand discontinuities.
• To elucidate the mechanisms by which DNA replication restarts after encountering DNA repair intermediates.

Main Methods:

• Utilized Cas9 nickases in Bacillus subtilis to create specific single-strand discontinuities on the bacterial chromosome.
• Employed genetic, biochemical, and single-cell analyses to observe replication dynamics.
• Examined the role of recombinational repair and PriA in enabling replication restart.

Main Results:

• A nick in either the leading or lagging strand effectively arrested DNA replication.
• The behavior of the replicative helicase differed depending on which DNA strand was nicked.
• Replisome encounters with nicks generated single-end DSBs, necessitating recombinational repair for restart.
• PriA-dependent pathways were crucial for reinitiating DNA synthesis.

Conclusions:

• DNA replication fork inactivation at single-strand discontinuities leads to DSB formation.
• Bacillus subtilis employs a specific pathway involving recombinational repair and PriA for replication restart.
• This study defines the physiological pathway for reinitiating DNA synthesis following replication fork arrest at DNA repair sites.