CAG repeat expansion in the HTT gene drives Huntington’s disease (HD) pathogenesis and is modulated by
DNA damage repair pathways. In this context, the interaction between FAN1, a DNA-structure-specific
nuclease, and MLH1, member of the DNA mismatch repair pathway (MMR), is not defined. Here, we identify
a highly conserved SPYF motif at the N terminus of FAN1 that binds to MLH1. Our data support a model where
FAN1 has two distinct functions to stabilize CAG repeats. On one hand, it binds MLH1 to restrict its recruitment by MSH3, thus inhibiting the assembly of a functional MMR complex that would otherwise promote CAG
repeat expansion. On the other hand, it promotes accurate repair via its nuclease activity. These data highlight a potential avenue for HD therapeutics in attenuating somatic expansion