Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form...

Area of Science:

• Molecular Biology
• Genetics
• DNA Repair

Background:

• Genetic studies suggest MSH2-MSH3 stabilizes branched DNA intermediates during single strand annealing and gene conversion.
• Understanding the precise role of MSH2-MSH3 in recombination is crucial for elucidating DNA repair pathways.

Purpose of the Study:

• To experimentally validate the predicted binding of MSH2-MSH3 to branched recombination intermediates.
• To investigate the functional consequences of MSH2-MSH3 binding on DNA substrate conformation and repair signaling.

Main Methods:

• Construction of specific DNA substrates mimicking recombination intermediates.
• Electrophoretic mobility shift assays (EMSA) to detect MSH2-MSH3 binding.
• Chemical footprinting to map MSH2-MSH3 binding sites and conformational changes.

Main Results:

• Saccharomyces cerevisiae MSH2-MSH3 specifically binds branched DNA substrates with 3' single-stranded DNA.
• ATP binding promotes the release of MSH2-MSH3 from these DNA substrates.
• MSH2-MSH3 binds the double-strand/single-strand junction, altering DNA conformation and opening the junction.

Conclusions:

• MSH2-MSH3 binding to branched DNA creates a distinct nucleoprotein structure.
• This structure likely signals downstream DNA repair events, involving mismatch repair and nucleotide excision repair factors.