Mechanisms of genetic instability caused by (CGG)_n repeats in an experimental mammalian system

We developed an experimental system for studying genome instability caused by fragile X (CGG)_n repeats in mammalian cells. Our method uses a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with (CGG)_n repeats in its 5' UTR, which is integrated into the unique RL5 site in murine erythroid leukemia cells. Carrier-size (CGG)_n repeats markedly elevated the frequency of reporter inactivation, making cells ganciclovir resistant. These resistant clones had a unique mutational signature: a change in repeat length concurrent with mutagenesis in the reporter gene. Inactivation of genes implicated in break-induced replication, including Pold3, Pold4, Rad52, Rad51, and Smarcal1, reduced the frequency of ganciclovir-resistant clones to the baseline level that was observed in the absence of (CGG)_n repeats. We propose that replication fork collapse at carrier-size (CGG)_n repeats can trigger break-induced replication, which results in simultaneous repeat length changes and mutagenesis at a distance.