Fission yeast Swi1-Swi3 complex facilitates DNA binding of Mrc1

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Replication fork integrity is vital for DNA replication fidelity.
• The Swi1-Swi3 complex and Mrc1 protein are key regulators of replication fork stability and checkpoint signaling in *Schizosaccharomyces pombe*.
• The precise biochemical functions and interactions of these proteins remain incompletely understood.

Purpose of the Study:

• To biochemically characterize the DNA-binding properties and interactions of the Swi1-Swi3 complex and Mrc1 protein.
• To elucidate the molecular mechanisms underlying replication fork stabilization mediated by these factors.

Main Methods:

• Protein purification of Mrc1 and Swi1-Swi3 from *Schizosaccharomyces pombe*.
• In vitro DNA binding assays using various DNA structures.
• Analysis of protein-protein interactions using purified components and mutant forms.
• Assessment of mutant phenotypes related to genotoxic agent sensitivity and replication fork arrest.

Main Results:

• Mrc1 and Swi1-Swi3 bind DNA independently but synergistically in vitro.
• Mrc1 shows preferential binding to arrested fork and D-loop structures, while Swi1-Swi3 binds double-stranded DNA with higher affinity.
• A novel ternary complex forms in the presence of Swi1-Swi3, enhancing Mrc1's DNA binding affinity.
• Physical interaction between Mrc1 and Swi1-Swi3 was confirmed and found to be essential for their synergistic DNA binding.
• Mutations affecting this interaction disrupted synergistic binding and led to defects in polar fork arrest and genotoxic sensitivity.

Conclusions:

• The physical interaction between Mrc1 and Swi1-Swi3 is critical for their cooperative DNA binding and function in replication fork stabilization.
• These molecular interactions likely play a significant role in regulating replication pausing and maintaining genome stability during DNA replication.