Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Yeast Genetics

Background:

• Msh2-Msh3 complex in Saccharomyces cerevisiae mediates both mismatch repair (MMR) of insertion/deletion loops and 3' non-homologous tail removal (3' NHTR) during double-strand break repair.
• Both pathways involve Msh2-Msh3 binding to double-strand/single-strand junctions in an ATP-dependent manner.
• MMR is rapid, coordinating with replication forks, while 3' NHTR is a slower process.

Purpose of the Study:

• To investigate the in vivo molecular requirements of conserved Msh3 residues for MMR and 3' NHTR.
• To elucidate the role of Msh3 residues in DNA-binding and ATPase domain communication or nucleotide binding/exchange within the Msh2-Msh3 complex.

Main Methods:

• In vivo analysis of well-conserved Msh3 residues in Saccharomyces cerevisiae.
• Mutagenesis of predicted Msh3 residues involved in nucleotide binding and inter-domain communication.
• Assessing the impact of mutations on Msh2-Msh3-mediated MMR and 3' NHTR activities.

Main Results:

• Aromatic residues within the Msh3 FLY motif are essential for Msh2-Msh3-mediated MMR but not for 3' NHTR.
• Mutations in other Msh3 regions exhibited similar effects on both MMR and 3' NHTR.
• Distinct requirements for ATP binding and positioning within Msh3 were observed for the two repair pathways.

Conclusions:

• The Msh2-Msh3-mediated MMR and 3' NHTR pathways possess distinct molecular requirements, particularly concerning ATP binding within Msh3.
• Differences in ATP positioning requirements are likely linked to the differing kinetics of MMR (rapid) and 3' NHTR (slower).
• Efficient MMR necessitates precise ATP binding for rapid conformational changes at replication forks, whereas 3' NHTR allows for more flexibility due to its slower pace.