Nuclear PI3P produced by the Beclin-1/Vps34 complex regulates DNA mismatch repair

Genome integrity relies on DNA mismatch repair (MMR) to correct replication errors, yet whether non-protein cofactors regulate this pathway remains unexplored. Here, we identify nuclear phosphatidylinositol-3-phosphate (PI3P) as a lipid regulator of MMR. Using biosensors, lipid pulldown, and proximity ligation assays, we show that PI3P forms discrete nuclear puncta in close proximity to the MutSα (MSH2:MSH6) and MutSβ (MSH2:MSH3) MMR recognition complexes. Pharmacological or genetic depletion of the class III PI3-kinase Vps34 impaired MutSα and MutSβ heterodimer assembly without altering MMR protein nuclear abundance, compromised DNA substrate association of MMR components in nuclear extracts, and elevated microsatellite instability at mononucleotide repeats. Exogenous PI3P enhanced MMR recognition complex assembly and DNA association in PI3P-deficient nuclear extracts, supporting a role for PI3P in promoting MMR. We further show that a nuclear Beclin-1/Vps34 complex produces this PI3P pool through an autophagy-independent mechanism. Functionally, loss of nuclear PI3P blunts MMR-dependent DNA damage signaling and confers 6-thioguanine resistance in cultured cells and in Beclin-1-deficient zebrafish in vivo. These findings reveal an autophagy-independent nuclear function for the Beclin-1/Vps34 complex in genome maintenance and identify PI3P as a lipid mediator of MMR, thereby expanding the functional repertoire of nuclear phosphoinositide signaling.