TREX2 Exonuclease Causes Spontaneous Mutations and Stress-Induced Replication Fork Defects in Cells Expressing RAD51K133A

Abstract

Highlights d In RAD51 K133A cells, TREX2's exonuclease causes mutations and stalled forks d RAD18 and PCNA ubiquitination influence mutation levels in RAD51 K133A cells d TREX2 H188A rescues strand degradation in cells defective for PARP1 or FANCB d TREX2 H188A binds to UBC13 and rescues PCNA ubiquitination

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

“…Furthermore, a miniHPRT loss-of-function (LOF) assay was performed to assess the mutation rate in RAD51 variants by measuring cell survival in TG. 31 KR, SP, and KR/SP exhibited a spontaneous increase of 82-, 22-, and 3-fold, respectively, in TG-resistant colonies compared to WT ( Figure 5 C). Interestingly, the KR/SP mutant displays a lower mutation level than either KR or SP, suggesting that serine 181 promotes mutagenesis in KR cells, while lysine 133 likely plays a similar, but less prominent role in SP cells.…”

“…To assess the cellular phenotype of human RAD51 WT and SP, a knockout-knockin protocol in mouse embryonic stem (ES) cells (AB2.2) was used to introduce corresponding cDNAs adjacent to the mouse RAD51 promoter while leaving the remaining mouse allele intact. 31 RAD51 KR and KR/SP cDNAs were also included. An essential component of this protocol is the miniHPRT gene, which can be selected for or against expression using HAT (hypoxanthine, aminopterin, thymidine) and 6-thioguanine (TG), respectively 32 , 33 ( Figure S3 A).…”

“…Previously we showed that RAD51 and BRCA2 were responsible for the fusion of identical inverted repeats, while DDT was responsible for the fusion of mismatched inverted repeats. 54 Although we have previously shown that RAD51 K133A mutation relies upon TREX2 function to induce nearly all the mutations in ES cells, 31 the effect of KR and SP mutants on these fusion events remains unknown. Further investigation of these mutants may provide valuable insight into distinguishing between strand exchange and RF protection and shed light on their utilization by alternative pathways.…”

Exonucleases: Degrading DNA to Deal with Genome Damage, Cell Death, Inflammation and Cancer

“…In artificial settings, chromosomal and genomic instability were reported using Trex2 -null and mutated embryonic stem cells [ 232 , 233 ]; however, TREX2 deficiency in mice ( Table 2 ) does not lead to a tumour prone phenotype [ 22 , 224 ]. Depending on the DNA repair status of embryonic stem cells, TREX2 may display a dual function in the DDR pathway, dependent and independent of its exonuclease activity, facilitating or avoiding replication fork instability and mutations [ 234 , 235 ]. However, neither genomic nor chromosomal instability are observed in cells from Trex2 knockout mice or in keratinocytes, where Trex 2 is highly expressed, nor in embryonic stem cells, in which TREX 2 expression is not detectable [ 224 ].…”

Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci

“…TREX2 is a non-processive 3 -5 exonuclease that has multiple functions: removes 3 mismatches form DNA [18,19] and alters replication fork stability and mutation levels in cells defective for homologous recombination [20]. TREX 2 is most closely related to TREX1 that is a component of the SET complex that degrades 3 ends of nicked DNA during the programmed cell death pathway in mitochondrion [21].…”