MLH1 (mutL homolog 1) is a core component of the post-replicative DNA mismatch repair (MMR) system that heterodimerizes with PMS2 to form MutLα complex 1. This complex is recruited to mismatches detected by MutSα or MutSβ and activates PMS2 endonuclease activity to introduce single-strand breaks, enabling exonuclease EXO1 to degrade the error-containing strand 2. MLH1 also heterodimerizes with MLH3 to form MutLγ, involved in meiotic recombination. MLH1 protein stability is dynamically regulated through ubiquitin-proteasome degradation, with E3 ligase UBR4 promoting degradation while deubiquitylase USP5 stabilizes the protein; PMS2 binding protects MLH1 from degradation 3. Beyond repair function, MLH1 participates in DNA damage signaling and is associated with longevity 4. Clinically, MLH1 dysfunction is paramount in Lynch syndrome and colorectal cancer pathogenesis. Reduced MLH1 gene expression correlates with increased colorectal cancer stage and progression 5. MLH1 promoter methylation-mediated silencing causes microsatellite instability in endometrial and colorectal cancers 6. Functional characterization reveals that missense variants predominantly affect the ATP-binding pocket and C-terminal domain 1, with MLH1 disruption now targetable for improved prime editing genome therapy 7.