TREX2 exonuclease defective cells exhibit double-strand breaks and chromosomal fragments but not Robertsonian translocations

Abstract

TREX2 is a 3'-->5' exonuclease that binds to DNA and removes 3' mismatched nucleotides. By an in vitro structure function analysis, we found a single amino acid change (H188A) completely ablates exonuclease activity and impairs DNA binding by about 60% while another change (R167A) impairs DNA binding by about 85% without impacting exonuclease activity. For a biological analysis, we generated trex2null cells by deleting the entire Trex2 coding sequences in mouse embryonic stem (ES) cells. We found Trex2 deletion caused high levels of Robertsonian translocations (RbTs) showing Trex2 is important for chromosomal maintenance. Here we evaluate the exonuclease and DNA binding domains by expressing in trex2(null) cells coding sequences for wild type human TREX2 (Trex2hTX2) or human TREX2 with the H188A change (Trex2H188A) or the R167A change (Trex2R167A). These cDNAs are positioned adjacent to the mouse Trex2 promoter by Cre-mediated knock-in. By observing metaphase spreads, we found Trex2H188A cells exhibited high levels of double-strand breaks (DSBs) and chromosomal fragments. Therefore, TREX2 may suppress spontaneous DSBs or exonuclease defective TREX2 may induce them in a dominate-negative manner. We also found Trex2hTX2, hTrex2H188A and hTrex2R167A cells did not exhibit RbTs. Thus, neither the exonuclease nor DNA binding domains suppress RbTs suggesting TREX2 possesses additional biochemical activities.

Fig. 1

Knock-in of human TREX2 cDNA variants. (A) The HPRT minigene, expressed by the PGK promoter [25,29] is used for selection and contains exons 1 and 2 (box labeled 1&2), exons 3–8 + polyadenylation sequences (box labeled 3–8) separated by an intron (straight line). Select for minigene expression in HAT. A RE mutant loxP (black arrow head) [26] is 5′ to PGK and another RE mutant loxP is in the intron. An FRT (open arrow) is located 3′ to miniHPRT. Upon targeting the entire known mouse TREX2 open reading frame (rectangle) is deleted as previously described [12]; this sequence corresponds to the human short isoform [21]. PRO, mouse Trex2 promoter. (B) Removal of the 5′ half of miniHPRT by Cre recombination as previously described [12]. (C) Knock-in of the short isoform of human TREX2 cDNA (hTX2). A Cre-mediated knock-in plasmid is cotransfected along with a Cre-expression plasmid. The knock-in vector contains the 5′ half of miniHPRT, a left element (LE) mutant loxP (Grey arrow head) and the cDNA with bovine growth hormone polyadenylation sequences as previously described [23]. Cells are selected in HAT for restoration of miniHPRT. The knock-in corrects miniHPRT, generates an RE LE mutant loxP (left, black grey arrow), a wild type loxP (right, grey black arrow) and places the cDNA adjacent to the mouse TREX2 promoter. (D) Verification of knock-in. Due to the stringent selection, all HAT resistant clones are positive for knock-in as verified by PCR (left) using Cre1 and hTX2Rev primers. Ku80 (80) was used as a loading control for PCR as previously described [12]. In addition, human TREX2 expression is confirmed by RT-PCR (right) using primers specific to human TREX2 (hTX2For, hTX2Rev). Note the primers are specific for human TREX2 since mouse Trex2 is not amplified in the AB2.2 control. PCR was performed on RNA with and without reverse transcriptase (+/−) to ensure there is no DNA contamination. Rad51 (51) was used as loading control for RT-PCR as previously described [12].

Fig. 2

Trex2^H188A cells exhibit chromosome DSBs and fragments. Chromosomes are stained with DAPI (blue), a telomeric probe (green) and a MSR probe from the pericentromere (red). In mice the short arms are very small causing the telomeric and MSR probes to overlap resulting in a bleached yellow dot. (A) Metaphase spread with a chromosome DSB/fragment (arrow). (B) Enlargement of the chromosome with a DSB. Note the DSB occurs at the junction of the long arms and the pericentromere such that some of the pericentromere is still attached to the long arms. (C) Graph that shows the Trex2^H188A cells exhibit a greater number of chromosome DSBs compared to AB2.2, trex2^null, Trex2^hTX2 and Trex2^R167A cells. The numbers for both clones shown in Table 1 is averaged.