| The Three prime Repair Exonuclease (Trex) proteins, Trex1 and Trex2 are 3'→5' exonucleases that could be important for repairing DNA. Yet Trex1 processes cytosolic DNA to suppress an autoimmune response while Trex2 biological function is unclear.;To address this question, we generated trex2null ES cells and showed that TREX2 suppress spontaneous gross chromosomal rearrangements (GCRs) like: Robertsonian translocations (RbTs), duplications and translocations. To better understand TREX2's role in maintaining genomic stability, we perform a separation of function analysis by comparing Trex2 deleted mouse ES cells (trex2null) to those that express human wild type TREX2 (hTX2) or human TREX2 mutated in either the DNA binding domain (Trex2R167A) or exonuclease domain ( Trex2H188A). We found that TREX2 DNA binding domain is not critical for suppressing chromosomal breaks and that Trex2 H188A induces DSBs in a dominant-negative fashion. Surprisingly, RbTs phenotype is rescued by hTX2 and both mutant TREX2, suggesting that TREX2 may have some other biochemical function. So, TREX2 is important for maintaining spontaneous chromosomal stability through multiple mechanisms.;We also hypothesized that TREX2 is suppressing induced-genomic instability. Cisplatin, an anti-cancer drug, forms DNA interstrand crosslinks (ICLs) that interfere with replication. We showed that TREX2 is depleted in human cells derived from cancer after exposure to cisplatin but not other genotoxins including another crosslinking agent, mitomycin C (MMC). This indicates a potential role for TREX2 depletion in cisplatin-induced cytotoxicity. We showed that wt ES cells exposed to cisplatin exhibit RbTs, similar to trex2null ES. In contrast, another crosslinking agent, mitomycin C (MMC) did not deplete TREX2 and did not increase RbTs indicating RbTs are not caused by ICLs but instead by TREX2 depletion induced by cisplatin. Also, wt and trex2null cells exhibit a similar number of Cisplatin-induced chromosomal breaks while trex2 null cells exhibit many more MMC-induced chromosomal breaks. Taken together, our results demonstrate that cisplatin depletes TREX2 and causes genomic instability that is similarly observed in trex2 null ES. Thus, cisplatin has two potential cytotoxic activities: (1) the generation of ICLs, and (2) the depletion of TREX2.;To understand TREX2's relation with ICL repair, we used MMC, since MMC did not deplete TREX2. We found that trex2null and Trex2R167A cells were hypersensitive to MMC suggesting they were disabled for ICL repair while Trex2 H188A cells were resistant to MMC suggesting the exonuclease activity is cytotoxic in response to ICLs. Second, trex2null , Trex2R167A and Trex2H188A cells exhibited increased MMC-induced chromosomal breaks. Third, trex2null cellular extracts exhibited diminished ICL repair dependent on homologous recombination (HR) but heightened ICL repair dependent on nucleotide excision repair (NER). Forth, an epistatic analysis suggests TREX2 and the NER proteins Xpa and Xpc are in different ICL repair pathways. Thus, these data show that TREX2 facilitates HR-dependent ICL repair in a pathway parallel to NER-dependent ICL repair. We propose TREX2 is cytoprotective in response to ICLs, but its exonuclease activity can also be cytotoxic suggesting a dual biological function. |
