Trex2 Enables Spontaneous Sister Chromatid Exchanges Without Facilitating DNA Double-Strand Break Repair

Abstract

Trex2 is a 39 / 59 exonuclease that removes 39-mismatched sequences in a biochemical assay; however, its biological function remains unclear. To address biology we previously generated trex2 null mouse embryonic stem (ES) cells and expressed in these cells wild-type human TREX2 cDNA (Trex2 hTX2 ) or cDNA with a single-amino-acid change in the catalytic domain (Trex2 H188A ) or in the DNA-binding domain (Trex2 R167A ). We found the trex2 null and Trex2 H188A cells exhibited spontaneous broken chromosomes and trex2 null cells exhibited spontaneous chromosomal rearrangements. We also found ectopically expressed human TREX2 was active at the 39 ends of I-SceI-induced chromosomal double-strand breaks (DSBs). Therefore, we hypothesized Trex2 participates in DNA DSB repair by modifying 39 ends. This may be especially important for ends with damaged nucleotides. Here we present data that are unexpected and prompt a new model. We found Trex2-altered cells (null, H188A, and R167A) were not hypersensitive to camptothecin, a type-1 topoisomerase inhibitor that induces DSBs at replication forks. In addition, Trex2-altered cells were not hypersensitive to g-radiation, an agent that causes DSBs throughout the cell cycle. This observation held true even in cells compromised for one of the two major DSB repair pathways: homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Trex2 deletion also enhanced repair of an I-SceI-induced DSB by both HDR and NHEJ without affecting pathway choice. Interestingly, however, trex2 null cells exhibited reduced spontaneous sister chromatid exchanges (SCEs) but this was not due to a defect in HDR-mediated crossing over. Therefore, reduced spontaneous SCE could be a manifestation of the same defect that caused spontaneous broken chromosomes and spontaneous chromosomal rearrangements. These unexpected data suggest Trex2 does not enable DSB repair and prompt a new model that posits Trex2 suppresses the formation of broken chromosomes.

RAD51 Mutants Cause Replication Defects and Chromosomal Instability

“…Unlike the chromatid breaks, the isochromatid breaks and fragments were usually in the pericentromere, a highly repetitive region composed of major satellite repeats (21). This observation is interesting compared to trex2 null cells that also exhibited spontaneous isochromatid breaks and fragments in the pericentromere (16). TREX2 is a 3=¡5= exonuclease that removes 3= mismatches from single-stranded DNA (40).…”

“…For this study, both lines were mutated at the hypoxanthine phosphoribosyltransferase (Hprt) gene (51). A random retroviral insertion mutated the Hprt gene in AB2.2 cells, while an Hprt targeting vector mutated Hprt exon 3 in the Lex1 cells by a standardized protocol (16) that includes the use of the puro⌬tk doubleselection cassette (10) with subsequent removal of puro⌬tk. Lex1 and AB2.2 ES cells were maintained in M15 medium: high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum, 100 mol/liter ␤-mercaptoethanol, 1 mmol/liter glutamine, 3 mg/ml penicillin, 5 mg/ml streptomycin, and 1,000 U/ml ESGRO leukemia inhibitory factor (LIF).…”

The ameliorative effect ofCetraria islandicaagainst diabetes-induced genetic and oxidative damage in human blood

“…Also, intracellular calcium levels increase as a result of oxidative damage to cell membranes, calcium then enters the nucleus where it can activate nucleases which cause DNA strand breaks (McConkey et al, 1989). DNA damage and defective DNA repair cause SCEs (Dumitrache et al, 2011). In this study, the abnormal signs of T1D on blood cells were dramatically and dose-dependently unchanged by C. islandica.…”

A mechanism for 1,4-Benzoquinone-induced genotoxicity

“…These include cells defective for FA ( Fancb ), nonhomologous end joining (NHEJ, Ku70 ) and interstrand crosslink repair (ICLR)/HR ( Ercc1 ). By comparison, cells with a mutation in a lesion bypass gene, Trex2 , caused BQ-resistance supporting the notion that DSB repair and replication fork maintenance are important for correcting BQ-induced lesions since Trex2 -deletion is known to increase HR and NHEJ [23, 24]. The Brca2 -mutant cells did not exhibit profound hypersensitivity even though BRCA2 is important for DSB repair and replication fork maintenance [25].…”