Structure of the SSB-DNA polymerase III interface and its role in DNA replication

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Interactions between single-stranded DNA-binding proteins (SSBs) and DNA replication machinery are vital but poorly understood.
• In Escherichia coli, SSB protein interaction with the DNA polymerase III holoenzyme's χ subunit is hypothesized to stabilize the replisome and assist primer delivery.

Purpose of the Study:

• To identify the SSB-binding site on the χ subunit.
• To investigate the functional consequences of disrupting the χ/SSB interaction on DNA replication.

Main Methods:

• X-ray crystallography to determine the SSB-binding site on χ.
• Biochemical assays to assess DNA synthesis and protein interactions.
• Cellular studies to evaluate replication fidelity and cell cycle progression in vivo.

Main Results:

• The SSB-binding site on χ was identified crystallographically.
• Disrupting the χ/SSB interaction did not impair lagging-strand primer utilization.
• Mutations affecting the χ/SSB interface caused salt-dependent uncoupling of leading and lagging strand synthesis and inhibited leading-strand DNA polymerase in vitro.
• In vivo destabilization of the χ/SSB complex led to temperature-dependent cell cycle defects and replisome instability.

Conclusions:

• The χ/SSB interaction is not essential for lagging-strand primer utilization.
• The χ/SSB complex plays a critical role in replisome establishment and maintenance, influencing both leading- and lagging-strand synthesis.
• Disruption of the χ/SSB interaction leads to replisome instability and cell cycle defects, highlighting its importance in maintaining genome integrity during replication.