Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by...

Area of Science:

• Cellular Biology
• Molecular Biology
• DNA Replication and Repair

Background:

• Minichromosome maintenance (MCM) helicase is crucial for DNA replication.
• Rad51 and Rad52 are key proteins involved in DNA recombination and repair.
• Interactions between MCM helicase and recombination proteins are conserved across species.

Purpose of the Study:

• To investigate the physical interactions between MCM helicase, Rad51, and Rad52 in Saccharomyces cerevisiae.
• To elucidate the role of these interactions in DNA replication and repair processes.
• To understand the regulation of these complexes during the cell cycle and in response to DNA damage.

Main Methods:

• Yeast genetics and molecular biology techniques.
• Co-immunoprecipitation assays to detect protein interactions.
• Microscopy to visualize protein localization within nuclear scaffolds.
• Analysis of a rad51 mutant with impaired MCM binding.

Main Results:

• MCM helicase physically interacts with Rad51 and Rad52 in a nuclease-insoluble scaffold.
• Rad51 accumulates independently of MCM and DNA binding, interacting with MCM outside replication origins/forks.
• MCM, Rad51, and Rad52 accumulate in scaffolds in G1 and are released in S phase.
• Cdc7 prevents release from scaffolds in the presence of replication-blocking lesions.
• A rad51 mutant defective in MCM binding shows impaired ssDNA gap filling and fork progression.

Conclusions:

• Cells form MCM/Rad51/Rad52 complexes at nuclear scaffolds in G1.
• These complexes assist stressed replication forks through non-recombinogenic functions.
• Cdc7 plays a role in regulating the release of these complexes during S phase.
• The MCM/Rad51 interaction is important for replication fork stability and DNA repair.