Recombinant Saccharomyces cerevisiae DNA mismatch repair protein MSH3 (MSH3), partial

What is the primary role of MSH3 in Saccharomyces cerevisiae DNA mismatch repair?

MSH3 in S. cerevisiae functions as part of a heterodimeric complex with MSH2 (MSH2-MSH3) that recognizes and initiates repair of specific types of DNA mismatches. The MSH2-MSH3 complex primarily recognizes insertion/deletion loops (in/dels) up to approximately 17 nucleotides in length, playing a crucial role in maintaining genomic stability . This complex scans along the DNA, identifies errors, and recruits other proteins to assist in repair through the process of mismatch repair (MMR) .

The mispair binding domain (MBD) of MSH3 confers specific DNA-binding activity that allows recognition of these structural abnormalities in DNA . Recent research also suggests that MSH2-MSH3 has a previously unrecognized role in the repair of certain base-base mispairs, particularly GC to CG and AT to TA changes, expanding our understanding of its functional repertoire .

How does MSH2-MSH3 differ functionally from MSH2-MSH6 in the mismatch repair pathway?

The functional differences between these two complexes can be summarized in the following table:

Additionally, unlike MSH2-MSH6, the MSH2-MSH3 complex can bind to and participate in removing 3' non-homologous tails in DNA flap structures, with biochemical analysis showing it binds specifically at the double-strand/single-strand junction of these structures .

What types of DNA binding specificity does MSH3 exhibit?

MSH3's DNA binding specificity is distinct from that of other mismatch repair proteins:

• MSH2-MSH3 binds efficiently to 2-12 nucleotide loop mismatches, but shows limited binding to base-base mismatches .

• The critical residues involved in mismatch recognition differ between MSH3 and other MMR proteins. While MutS and MSH2-MSH6 use conserved phenylalanine and glutamate residues for mismatch recognition, these are not conserved in MSH3 . Instead, yeast MSH3 contains two lysine residues (Lys-187 and Lys-189) at equivalent positions, while human MSH3 contains a lysine and an arginine (Lys-246 and Arg-248) .

• When MSH2-MSH3 binds to DNA flap structures, it causes conformational changes in the DNA, specifically binding at the double-strand/single-strand junction .

• Domain I in MSH2 contributes non-specific DNA binding activity, while Domain I of MSH3 appears important for mismatch binding specificity and for suppressing non-specific DNA binding .

What is the role of MSH3 in trinucleotide repeat (TNR) expansions and neurodegenerative disease?

MSH3 plays a paradoxical role in trinucleotide repeat instability, particularly CAG/CTG repeat expansions that underlie several neurodegenerative disorders like Huntington's Disease and Myotonic Dystrophy Type 1 . The MSH2-MSH3 complex promotes TNR expansions through a mechanism that initially appears contradictory to its normal repair function .

The process works as follows:

Recent research challenges previous models that suggested MSH2-MSH3's DNA binding activity alone was sufficient to promote TNR expansions. Using a chimeric MSH complex that replaced the MBD of MSH6 with the MSH3 MBD, researchers demonstrated that DNA-binding activity alone is not sufficient . Current models propose that TNR expansions require fully functional MSH2-MSH3 including coordinated DNA binding, ATP binding and hydrolysis activities, and interactions with MLH complexes .

How do researchers experimentally distinguish between MSH3 and MSH6 pathways?

Researchers use several genetic and biochemical approaches to distinguish between MSH3 and MSH6 pathways:

• Genetic mutation analysis: Creating single and double mutants (msh3, msh6, msh3 msh6) and analyzing mutation rates and spectra. For example, mlh3 msh3 double mutants show similar mutation rates to single mutants, suggesting they function in the same pathway, while mlh3 msh6 double mutants show synergistic increases in mutation rates, indicating separate pathways .

• Reporter assays: Specific genetic reporters like hom3-10 and lys2-Bgl are sensitive to defects in MMR. The hom3-10 reversion assay measures deletion of a single T in a run of 7 Ts, which is particularly affected by MSH3 function .

• Substrate specificity testing: In vitro biochemical assays using purified proteins and DNA substrates with specific mismatches demonstrate that MSH2-MSH3 binds efficiently to 2-12 nucleotide loop mismatches, while MSH2-MSH6 prefers base-base mismatches and very small loops .

• Chimeric protein approaches: Creating fusion proteins that contain domains from different MSH proteins helps determine which domains are responsible for specific functions, as demonstrated in the study with the chimeric Msh complex containing the MSH3 MBD in MSH6 .

What mutation patterns arise in MSH3-deficient yeast strains?

MSH3-deficient strains show distinctive mutation patterns that differ from other MMR-deficient strains:

How does Domain I contribute to MSH3 function and specificity?

Domain I plays a critical role in the function and specificity of MSH3:

• Domain I in MSH2 contributes non-specific DNA binding activity, providing general affinity for DNA substrates .

• In contrast, Domain I of MSH3 appears crucial for mismatch binding specificity and for suppressing non-specific DNA binding . This suggests a regulatory role in ensuring appropriate target selection.

• Mutations in Domain I of MSH3 lead to defects in MSH2-MSH3-mediated MMR and recombination functions, while having minimal impact on MSH2-MSH6-mediated functions .

• The distinct requirements for Domain I between MSH3 and MSH6 indicate that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that are fundamentally different from those required for MSH2-MSH6 mismatch binding .

• Unlike the conserved phenylalanine and glutamate residues in MSH6 and MutS that are critical for mismatch recognition, MSH3 employs different amino acids (two lysine residues in yeast, Lys-187 and Lys-189) for substrate recognition .

What is the relationship between MSH3 and MLH3 in mismatch repair pathways?

MSH3 and MLH3 work together in a specific branch of the mismatch repair pathway:

• Genetic studies show that mutations in MLH3 increase the rate of frameshift mutations, particularly deletions of a single T in runs of repeated Ts, similar to the phenotype observed in msh3 mutants .

• The mlh3 msh3 double mutant has essentially the same mutation rate as either single mutant for reversion of lys2-Bgl and hom3-10, suggesting they function in the same pathway .

• In contrast, combining mutations in MLH3 and MSH6 causes a synergistic increase in mutation rates, indicating they function in separate pathways .

• MLH3, like MSH3, appears to be involved in repair of specific types of DNA damage, including certain frameshift mutations and potentially some base-base mispairs .

• The S. cerevisiae genome encodes four MutL homologs (MLH1, PMS1, MLH2, and MLH3), with MLH3 being most closely related to human PMS1, showing 20% sequence identity and 47% similarity .

What experimental approaches are used to study recombinant MSH3 binding to DNA substrates?

Several experimental approaches are employed to study MSH3 DNA binding:

• Mispair binding analysis: Purified MSH2-MSH3 protein is tested with DNA substrates derived from sequences found to be mutated in vivo, allowing correlation between in vitro binding and in vivo function .

• Chimeric protein studies: By creating chimeric proteins that replace specific domains (such as the MSH3 MBD with the MSH6 MBD), researchers can determine which domains are necessary and sufficient for specific functions .

• Molecular dynamics simulations: As seen in lung cancer studies with MSH3 polymorphisms, molecular dynamics simulations can provide insights into how specific mutations affect protein structure and function, with standard procedures including 100-ns long MD simulations using tools like GROMACS .

• DNA binding assays with structural variants: Testing MSH2-MSH3 binding to various DNA structures including loops, flaps, and junction structures reveals that it specifically binds at double-strand/single-strand junctions and causes conformational changes in DNA structure .

• ATP binding and hydrolysis studies: Since MSH3 function in TNR expansions requires coordinated ATP binding and hydrolysis activities, assays measuring these parameters help elucidate the complete mechanism .