RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory...

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Replication accessory helicases Rep and UvrD, and recombination protein RecF are crucial for bacterial growth under certain conditions.
• Cells lacking these proteins exhibit cold sensitivity (cryo-sensitivity) in rich media.
• Understanding genetic suppressors of this cryo-sensitivity can reveal mechanisms of DNA replication and repair.

Purpose of the Study:

• To identify and characterize mutations that suppress the cryo-sensitivity of rep uvrD recF mutant bacteria.
• To elucidate the role of RNA polymerase (RNAP) in DNA replication stress and its interaction with accessory helicases.
• To investigate how RNAP mutations can compensate for the loss of replication accessory helicases.

Main Methods:

• Isolation and genetic mapping of spontaneous mutations suppressing Luria-Bertani (LB)-cryo-sensitivity in rep uvrD cells.
• Characterization of identified mutations in the rpoB and rpoC genes, encoding RNAP subunits.
• Phenotypic analysis of mutant strains, including promoter activation, suppression of auxotrophy, and R-loop formation assays.

Main Results:

• Five suppressor mutations were identified in the rpoB and rpoC genes.
• Specific rpoB mutations (H447R, D444G) inhibited Prrn promoter activation and rpoB(H447R) suppressed stringent-like phenotypes.
• rpoC(H113R) suppressed thermo-sensitivity in greA greB mutants, suggesting destabilization of stalled elongation complexes. Most mutations prevented R-loop formation.

Conclusions:

• The identified rpoB and rpoC mutations suppress cryo-sensitivity by destabilizing RNAP during replication-transcription collisions, aiding replication in the absence of Rep and UvrD.
• These RNAP mutations facilitate replication fork progression across transcribed sequences, particularly when DinG is present.
• The study demonstrates that replication forks arrested by unstable transcription complexes can restart without Rep, UvrD, and DinG in a recF mutant background.