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

We describe a new experimental system to study genome instability caused by fragile X (CGG)(n) repeats in mammalian cells. It is based on a selectable cassette carrying the HyTK gene under the control of the FMR1 promoter with (CGG)(n) repeats in its 5′-UTR, which was integrated into the unique RL5 site in murine erythroid leukemia cells. Carrier-size (CGG)(n) repeats dramatically elevate the frequency of the reporter’s inactivation making cells ganciclovir-resistant. These resistant clones have a unique mutational signature: a change in the 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 result in simultaneous repeat length changes and mutagenesis at a distance.