Two Replication Fork Maintenance Pathways Fuse Inverted Repeats to Rearrange Chromosomes

Replication fork (RF) maintenance pathways preserve chromosomes, but their faulty application at nonallelic repeats could generate rearrangements causing cancer, genomic disorders and speciation(1-3). Potential causal mechanisms are homologous recombination (HR) and error-free postreplication repair (EF-PRR). HR repairs damage induced DNA double strand breaks (DSBs) and single-ended DSBs within replication. To facilitate HR, the recombinase RAD51 and mediator BRCA2 form a filament on the 3’ DNA strand at a break to enable annealing to the complementary sister chromatid(4) while the RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to prevent recombination(5). HR also stabilizes(6,7) and restarts(8,9) RFs without a DSB(10,11). EF-PRR bypasses DNA incongruities that impede replication by ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6/RAD18 and UBC13/MMS2/RAD5 ubiquitin ligase complexes(12). Some components are common to both HR and EF-PRR like RAD51 and RAD18(13,14). Here we delineate two pathways that spontaneously fuse inverted repeats to generate unstable chromosomal rearrangements in wild type mouse embryonic stem (ES) cells. Gamma-radiation induced a BLM-regulated pathway that selectively fused identical, but not mismatched repeats. By contrast, UV light induced a RAD18-dependent pathway that efficiently fused mismatched repeats. Furthermore, TREX2 (a 3’→5’ exonuclease) suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly separating these pathways. TREX2 associated with UBC13 and enhanced PCNA ubiquitination in response to UV light, consistent with it being a novel member of EF-PRR. RAD18 and TREX2 also suppressed RF stalling in response to nucleotide depletion. Interestingly, RF stalling induced fusion for identical and mismatched repeats implicating faulty replication as a causal mechanism for both pathways.