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Updated: Jul 11, 2025

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Published on: March 31, 2010
Elevated MSH2 MSH3 expression interferes with DNA metabolism in vivo
Melisa Medina-Rivera1, Samantha Phelps1, Madhumita Sridharan2
1Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo NY, 14203, USA.
Abstract:
The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.
Insights
Elevated levels of the Msh2-Msh3 mismatch repair (MMR) complex disrupt DNA replication and repair in yeast. This excess MMR protein causes genomic instability through an active mechanism requiring its ATPase activity.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae plays a dual role in genome stability.
- It repairs insertion/deletion loops (IDLs) and interacts with various DNA structures, influencing homologous recombination and DNA damage response.
- However, Msh2-Msh3 can also promote genome instability, particularly through trinucleotide repeat (TNR) expansions.
Purpose of the Study:
- To investigate the in vivo consequences of elevated Msh2-Msh3 levels on DNA replication and repair.
- To elucidate the mechanistic details underlying Msh2-Msh3-mediated genomic instability.
- To determine the role of Msh2-Msh3 abundance and activity in these processes.
Main Methods:
- In vivo studies in Saccharomyces cerevisiae assessing DNA replication and base excision repair.
- Analysis of cell cycle arrest phenotypes, including dependence on RAD9 and ELG1, and PCNA modification.
- Investigation of the requirement for Msh2-Msh3 ATPase activity and downstream MMR proteins.
Main Results:
- Elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo.
- Increased Msh2-Msh3 induced a cell cycle arrest dependent on RAD9 and ELG1, and caused PCNA modification.
- These effects were dependent on Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active disruption mechanism.
Conclusions:
- Excess Msh2-Msh3 actively disrupts DNA replication and repair pathways.
- Msh2-Msh3 protein abundance is a critical factor in Msh2-Msh3-mediated genomic instability.
- This study provides new mechanistic insights into how MMR complex levels impact genome stability.
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