DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The DNA damage checkpoint protein DisA and branch migration translocase RecG are crucial for genome integrity in Bacillus subtilis spores.
• DisA synthesizes cyclic 3', 5'-diadenosine monophosphate (c-di-AMP), a second messenger, with synthesis suppressed during replication stress.

Purpose of the Study:

• To investigate the interplay between DisA and RecG in preserving genome integrity.
• To elucidate the regulatory roles of DisA and RecG in response to DNA replication perturbations.

Main Methods:

• In vitro biochemical assays using purified DisA and RecG proteins.
• Analysis of DisA-mediated c-di-AMP synthesis in the presence of various DNA structures (gapped forks, Holliday junctions).
• Assessment of RecG's activity on branched DNA intermediates under different conditions (ATP concentration, protein pre-binding).

Main Results:

• DisA binding to stalled or reversed replication forks inhibits its own c-di-AMP synthesis.
• RecG unwinds branched DNA intermediates and blocks DisA-mediated c-di-AMP synthesis under specific conditions.
• DisA pre-bound to fork structures limits RecG's ATP hydrolysis and unwinding activity, while RecG pre-binding does not affect DisA.

Conclusions:

• DisA acts as a molecular switch, regulating RecG's fork remodeling activities (reversal and restoration).
• The coordinated action of DisA and RecG provides a temporal window for processing perturbed replication forks, thus preventing genome breakage.
• RecG-mediated fork remodeling is proposed as a significant in vivo activity regulated by DisA.