ENAGenomicsInstitute of Research in BiomedicineIRBhttps://www.ebi.ac.uk/ena/browser/view/PRJEB91651Triplet repeat expansions underlie multiple pathologies, including Huntington’s disease, often aris-ing 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 MutL (MLH1-MLH3), stimulated by MutS (MSH2-MSH3), incises DNA opposite an extrahelical loop on the 5' side. This activity, with moderate sequence preference, generates DNA nicks enabling Pol-mediated displacement synthesis with the loop as a template, leading to expansions. PCNA con-fines these MutL 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, Pol re-moves the loop and resynthesizes DNA, causing contraction. FAN1 also directly inhibits MutL, preventing its activation by MutS. Our study illuminates both repeat expansion and contraction mechanisms and reveal the protective function of FAN1.ENAfalseMechanism of trinucleotide repeat expansion by MutSβ-MutL and contraction by FAN1Triplet repeat expansions underlie multiple pathologies, including Huntington’s disease, often aris-ing 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 MutL (MLH1-MLH3), stimulated by MutS (MSH2-MSH3), incises DNA opposite an extrahelical loop on the 5' side. This activity, with moderate sequence preference, generates DNA nicks enabling Pol-mediated displacement synthesis with the loop as a template, leading to expansions. PCNA con-fines these MutL 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, Pol re-moves the loop and resynthesizes DNA, causing contraction. FAN1 also directly inhibits MutL, preventing its activation by MutS. Our study illuminates both repeat expansion and contraction mechanisms and reveal the protective function of FAN1.2025-07-032025-07-03PRJEB91651