DNA Replication Initiation Is Blocked by a Distant Chromosome-Membrane Attachment.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Chromosome structure is known to influence DNA replication origin initiation.
• Mechanisms by which cells actively regulate chromosome structure to control initiation remain largely unknown.

Purpose of the Study:

• To investigate if actively regulating chromosome structure can block replication initiation.
• To characterize the effects of a localized protein tether on DNA replication and cellular processes in E. coli.

Main Methods:

• Inducible protein tethers composed of trans-membrane and transcription repressor fusion proteins were used.
• Tethers were bound to operator sequences on the chromosome, positioned up to 1 Mb from replication origins.
• Whole-genome and site-specific fluorescent DNA labeling, RNA sequencing, and assessment of cell viability were performed.

Main Results:

• Localized protein tethers rapidly and completely blocked replication origin initiation.
• Tethering did not affect elongation at existing replication forks or cause cell/DNA damage.
• Disruption of global nucleoid structure, chromosome organization, and induction of supercoiling changes were observed.
• Gene expression patterns were globally altered, suggesting incompatibility with replication initiation.

Conclusions:

• Active regulation of chromosome structure via protein tethers can effectively block DNA replication initiation.
• Tether-induced changes in chromosome organization and supercoiling are key mechanisms for controlling replication.
• This study provides a novel tool to probe the relationship between chromosome structure and replication control.