Mismatch Recognition by Saccharomyces cerevisiae Msh2-Msh6: Role of Structure and Dynamics.

Yan Li,Manju M. Hingorani,Meera Joshi,Zane Lombardo,Ishita Mukerji

doi:10.3390/ijms20174271

Abstract

The mismatch repair (MMR) pathway maintains genome integrity by correcting errors such as mismatched base pairs formed during DNA replication. In MMR, Msh2–Msh6, a heterodimeric protein, targets single base mismatches and small insertion/deletion loops for repair. By incorporating the fluorescent nucleoside base analog 6-methylisoxanthopterin (6-MI) at or adjacent to a mismatch site to probe the structural and dynamic elements of the mismatch, we address how Msh2–Msh6 recognizes these mismatches for repair within the context of matched DNA. Fluorescence quantum yield and rotational correlation time measurements indicate that local base dynamics linearly correlate with Saccharomyces cerevisiae Msh2–Msh6 binding affinity where the protein exhibits a higher affinity (KD ≤ 25 nM) for mismatches that have a significant amount of dynamic motion. Energy transfer measurements measuring global DNA bending find that mismatches that are both well and poorly recognized by Msh2–Msh6 experience the same amount of protein-induced bending. Finally, base-specific dynamics coupled with protein-induced blue shifts in peak emission strongly support the crystallographic model of directional binding, in which Phe 432 of Msh6 intercalates 3′ of the mismatch. These results imply an important role for local base dynamics in the initial recognition step of MMR.

Highlights

The mismatch repair (MMR) system ensures the integrity of the genome by scanning, identifying and correcting errors made in DNA when the replication machinery fails
Many other types of repair processes exist in the cell, such as double strand break repair, non-homologous end joining and base-excision repair; our study is based on the Msh2–Msh6 protein, mainly involved in MMR; we focus on this process
This study investigates specific properties of mismatched DNA substrates to determine those elements that influence recognition and repair through examination of S. cerevisiae Msh2–Msh6 binding affinity, global DNA bending and local DNA base dynamics of mismatched duplexes with and without protein bound

Summary

Introduction

The mismatch repair (MMR) system ensures the integrity of the genome by scanning, identifying and correcting errors made in DNA when the replication machinery fails. Active MMR reduces the post replicative error frequency by roughly a thousand-fold and prevents recombination between homologous (similar but non-identical) sequences [1,2,3,4]. Many other types of repair processes exist in the cell, such as double strand break repair, non-homologous end joining and base-excision repair; our study is based on the Msh2–Msh protein, mainly involved in MMR; we focus on this process. MutS changes conformation and recruits MutL in an ATP-dependent manner, and subsequently signals excision of the nascent error-containing strand. Re-synthesis of the excised strand and ligation of the nick completes the repair process

Results

Discussion

Conclusion