Replication-coupled search positions MutH for strand incision in DNA mismatch repair

Technical Summary: Replication-coupled search positions MutH for strand incision in DNA mismatch repair

Problem Statement
DNA mismatch repair (MMR) is essential for maintaining genome stability by correcting replication errors. In Escherichia coli, the pathway relies on MutS and MutL to detect mismatches and activate MutH, an endonuclease that incises the newly synthesized strand at hemimethylated GATC sites. A central unresolved question is how these proteins coordinate across the vast distances (hundreds to thousands of base pairs) between a mismatch and a GATC site to ensure rapid and accurate incision before the transient strand-discrimination signal (hemimethylation) is lost. While in vitro studies have proposed mechanisms such as one-dimensional sliding or DNA looping, they fail to capture the native chromosomal organization and the simultaneous dynamics of replication, methylation, and repair within living cells. Specifically, it remains unclear whether MutH is recruited to the mismatch site by MutS–MutL complexes or if it binds GATC sites independently.

Methodology
The authors employed live-cell single-molecule tracking (SMT) in E. coli to resolve the target-search dynamics of MMR proteins.

• Strain Construction: They generated E. coli strains expressing HaloTag fusions of MutS, MutL, and MutH from their native chromosomal loci. These fusions were validated to retain full MMR functionality, as evidenced by wild-type spontaneous mutation frequencies (measured via rifampicin resistance).
• Imaging: Cells were labeled with cell-permeable fluorophores (Tetramethylrhodamine for short tracks; JFX650 for extended tracking) and imaged using a custom inverted microscope with Highly Inclined and Laminated Optical sheet (HILO) illumination.
• Perturbations: To dissect the dependencies of MutH binding, the authors utilized:
  • MMS treatment to increase mismatch load.
  • Deletion of mutS or mutL to test hierarchical recruitment.
  • Antibiotic treatment (chloramphenicol, rifampicin) and hupA deletion to alter DNA replication and nucleoid architecture.
  • Plasmid transformation (pUC19) to increase GATC site density.
  • dam deletion to eliminate hemimethylation asymmetry.
  • Co-imaging with YPet-DnaN (β-sliding clamp) to visualize replication forks.
• Analysis: Single-molecule trajectories were reconstructed to calculate diffusion coefficients. Molecules were classified into immobile, fast-diffusing, and slow-diffusing states using Gamma distribution fitting. Spatial correlations between MutH binding events and replication forks were quantified against randomized controls.

Key Results

1. Independent Recruitment of MutH: Contrary to models suggesting MutH is delivered to mismatches by MutS–MutL, MutH binds DNA independently of mismatch recognition. Treatment with MMS increased the immobile fraction of MutS and MutL but had no effect on MutH. Furthermore, MutH binding remained unchanged in ΔmutS and ΔmutL strains. MutH does not form stable foci at mismatch sites, unlike MutS and MutL.
2. Replication-Dependent Binding: MutH binding is strictly coupled to active DNA replication. In non-replicating cells (treated with antibiotics), the fraction of immobile MutH decreased significantly.
3. Dual Search Modes: Extended tracking with JFX650 revealed that MutH utilizes two distinct mobile search modes: a fast-diffusing state (1.7 µm²/s) exploring the cell volume and a slow-diffusing state (0.5 µm²/s) confined to the nucleoid. MutH transitions stochastically between these modes and an immobile state without a defined sequence.
4. Spatial Guidance by Replication Forks: MutH binding events and transitions from fast to slow diffusion occur preferentially near replication forks (marked by DnaN foci). In wild-type cells, MutH binding sites are significantly closer to DnaN foci than in randomized controls.
5. Role of Hemimethylation: The spatial guidance of MutH is dictated by hemimethylated GATC sites. In Δdam cells (where all GATC sites are unmethylated), MutH binding events are distributed randomly throughout the cell and are no longer enriched near replication forks. This confirms that MutH is guided by the methylation asymmetry created by replication, rather than by direct interaction with the replisome.

Significance and Claims
The paper proposes a "parallelized search mechanism" for MMR. Rather than a linear, hierarchical assembly where MutS recruits MutL, which then recruits MutH to a distant site, the authors demonstrate that MutH autonomously searches for and transiently occupies hemimethylated GATC sites near replication forks.

• Mechanism: MutH switches from a fast diffusion mode to a slow-search mode near replication forks, where it waits in a pre-bound state. Upon mismatch detection by MutS and recruitment of MutL, the MutS–MutL complex transiently activates the pre-bound MutH to incise the strand.
• Efficiency: This mechanism allows the MMR pathway to operate within the narrow temporal window before Dam methylase fully methylates the daughter strand. By pre-positioning MutH near the replication fork, the cell eliminates the need for long-range transport of a large protein complex, ensuring rapid incision.
• Specificity: The reliance on hemimethylation for both strand discrimination and spatial guidance ensures that incision occurs only on the newly synthesized strand and only in regions where the strand-discrimination signal is unambiguous.

The authors conclude that this replication-coupled, independent search strategy allows for rapid, targeted, and robust repair of replication errors in the crowded intracellular environment, challenging previous models that relied on long-lived ternary complexes or directed transport of MutH by MutL.