TREX2 deficiency suppresses spontaneous and genotoxin-associated mutagenesis.

TREX2, a 3'-5' exonuclease, is a part of the DNA damage tolerance (DDT) pathway that stabilizes replication forks (RFs) by ubiquitinating PCNA along with the ubiquitin E3 ligase RAD18 and other DDT factors. Mismatch repair (MMR) corrects DNA polymerase errors, including base mismatches and slippage. Here we demonstrate that TREX2 deletion reduces mutations in cells upon exposure to genotoxins, including those that cause base lesions and DNA polymerase slippage. Importantly, we show that TREX2 generates most of the spontaneous mutations in MMR-mutant cells derived from mice and people. TREX2-induced mutagenesis is dependent on the nuclease and DNA-binding attributes of TREX2. RAD18 deletion also reduces spontaneous mutations in MMR-mutant cells, albeit to a lesser degree. Inactivation of both MMR and TREX2 additively increases RF stalls, while it decreases DNA breaks, consistent with a synthetic phenotype.

The RAD51 S181P mutation shortens lifespan of female mice.

RAD51 is critical to the homologous recombination (HR) pathway that repairs DNA double strand breaks (DSBs) and protects replication forks (RFs). Previously, we showed that the S181P (SP) mutation in RAD51 causes defective RF maintenance but is proficient for DSB repair. Here we report that SP/SP female mice exhibit a shortened lifespan compared to +/+ females but not males. Histological analysis found that most mice in this study died from lymphoma, independent of genotype and sex. We propose that a potential cause for shortened lifespan in SP/SP females is due to the RF defect.

RAD51 separation of function mutation disables replication fork maintenance but preserves DSB repair.

Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.