Mechanism of trinucleotide repeat expansion by MutSβ-MutLγ and contraction by FAN1

Triplet repeat expansion underlies multiple pathologies, including Huntington's disease, often arising in somatic non-dividing tissues such as the brain. Despite identification of genetic modifiers, mechanistic insights remain limited. Using purified human proteins, we show that MutLγ (MLH1-MLH3), stimulated by MutSβ (MSH2-MSH3), incises DNA opposite an extrahelical loop on the 5' side. This activity, with a moderate sequence preference, generates DNA nicks enabling Polδ-mediated displacement synthesis with the loop as a template, leading to expansion. PCNA confines these MutLγ incisions near the loop. FAN1, instead, preferentially targets the looped strand. RFC-PCNA stimulate and direct FAN1 nuclease to the 3' boundary of the loop while restricting its exonuclease activity. No pre-existing nick is required. Following FAN1-RFC-PCNA action, Polδ removes the loop and resynthesizes DNA, causing contraction. FAN1 also directly inhibits MutLγ, preventing its activation by MutSβ. Our study illuminates both repeat expansion and contraction mechanisms and reveals the protective function of FAN1.