DNA polymerase III protein, HolC, helps resolve replication/transcription conflicts

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• DNA polymerase III holoenzyme is essential for DNA replication in *Escherichia coli*.
• The *holC* gene encodes the accessory protein HolC (χ), crucial for the clamp loader complex and interaction with single-strand DNA binding protein (SSB).
• While not essential for viability, *holC* mutants exhibit growth defects, genetic instability, and sensitivity to DNA damaging agents.

Purpose of the Study:

• To identify genetic suppressors of *holC* deletion mutants in *E. coli*.
• To investigate the relationship between transcription and DNA replication in *holC* mutant strains.
• To elucidate the role of transcription termination and specific factors in *holC* mutant phenotypes.

Main Methods:

• Isolation and whole genome sequencing of spontaneous suppressor mutants in a *holC*Δ strain.
• Utilizing a conditional *holC* plasmid to study transcription elongation and termination.
• Phenotypic analysis of *holC* mutants under various conditions, including treatment with bicyclomycin and manipulation of transcription factors.

Main Results:

• Suppressor mutations were identified in RNA polymerase subunits (RpoA, RpoB, RpoC) and *sspA* (stringent starvation protein A).
• Mutations in transcription factors DksA and NusA exacerbated *holC* mutant inviability.
• HolC mutants showed increased sensitivity to bicyclomycin, an inhibitor of Rho-dependent termination, and this drug reversed suppression by *rpoA*, *rpoC*, and *sspA*.
• Inversion of the *rrnA* operon worsened *holC* mutant growth defects.

Conclusions:

• Transcription complexes likely impede replication in *holC* mutants, leading to genetic instability.
• Rho-dependent transcriptional termination and DksA function are critical for maintaining viability and chromosome integrity in *holC* mutant strains.
• The findings underscore the intricate interplay between transcription and replication, particularly under conditions of DNA replication stress.