MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Mismatch repair (MMR) corrects DNA replication errors.
• In Saccharomyces cerevisiae, MutS homolog (MSH) complexes initiate MMR by binding to mismatches.
• MSH2-MSH6 binds single nucleotide and insertion/deletion mispairs.

Purpose of the Study:

• To investigate the role of ATP and MutL homolog (MLH) complexes in MSH binding.
• To characterize the interaction between MSH complexes, MLH complexes, and proliferating cell nuclear antigen (PCNA).
• To elucidate the MMR pathway intermediates.

Main Methods:

• In vitro binding assays using purified proteins (MSH2-MSH6, MLH1-PMS1) and DNA substrates.
• ATP and non-hydrolyzable ATP analogs were used to study ATP's effect on MSH binding.
• Formation and disruption of ternary complexes were monitored.
• Experiments with streptavidin end-blocked mismatch substrates were performed.

Main Results:

• ATP binding to MSH2-MSH6 eliminates its mismatch binding specificity.
• Addition of MLH1-PMS1 to MSH2-MSH6 and ATP-bound mismatched DNA forms a stable ternary complex.
• This ternary complex is disrupted by proliferating cell nuclear antigen (PCNA).
• PCNA interacts with the MSH-MLH complex on DNA mispairs.

Conclusions:

• The stable ternary complex of MSH2-MSH6, MLH1-PMS1, and DNA represents a key intermediate in yeast MMR.
• PCNA likely interacts with this MSH-MLH complex, influencing downstream MMR steps.
• These findings provide insights into the sequential assembly of MMR factors on DNA.