A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Clamp loaders, like replication factor C (RFC), are essential for DNA replication, repair, and synthesis.
• RFC facilitates the loading of sliding clamp proteins onto DNA.
• Existing models suggest RFC's primary DNA binding site is sterically hindered by nicked or gapped DNA, common in DNA repair.

Purpose of the Study:

• To investigate the mechanism of RFC binding to nicked or gapped DNA substrates.
• To identify novel DNA binding sites or mechanisms within RFC.
• To understand RFC's role in DNA repair pathways involving damaged DNA intermediates.

Main Methods:

• Biochemical assays to study DNA binding and clamp loading.
• Structural analysis of yeast RFC.
• Genetic analysis of RFC mutants, specifically deletion of the BRCT domain.

Main Results:

• A second, external DNA binding site on yeast RFC was discovered, accessible in its open conformation.
• This external site facilitates initial binding to nicked/gapped DNA, enabling access to the central binding site.
• The external site can unwind DNA, creating a larger single-stranded gap for enhanced RFC binding and ATPase activation.
• Deletion of the BRCT domain, part of the external site, impairs yeast growth under DNA damaging conditions.

Conclusions:

• Yeast RFC possesses an external DNA binding site that enhances its interaction with nicked and gapped DNA.
• This secondary binding site is crucial for efficient clamp loading at DNA repair sites.
• The discovery provides new insights into the adaptability of clamp loaders in managing genomic integrity during DNA repair.