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Tripartite motif-containing protein 29 (TRIM29) is involved in DNA double-strand break (DSB) repair. However, the specific roles of TRIM29 in DNA repair are not clearly understood. To investigate the involvement of TRIM29 in DNA DSB repair, we disrupted TRIM29 in DT40 cells by gene targeting with homologous recombination (HR). The roles of TRIM29 were investigated by clonogenic survival assays and immunofluorescence analyses. TRIM29 triallelic knockout (TRIM29 À/À/À/+) cells were sensitive to etoposide, but resistant to camptothecin. Foci formation assays to assess DNA repair activities showed that the dissociation of etoposideinduced phosphorylated H2A histone family member X (ɣ-H2AX) foci was retained in TRIM29 À/À/À/+ cells, and the formation of etoposide-induced tumor suppressor p53-binding protein 1 (53BP1) foci in TRIM29 À/À/À/+ cells was slower compared with wild-type (WT) cells. Interestingly, the kinetics of camptothecin-induced RAD51 foci formation of TRIM29 À/À/À/+ cells was higher than that of WT cells. These results indicate that TRIM29 is required for efficient recruitment of 53BP1 to facilitate the nonhomologous end-joining (NHEJ) pathway and thereby suppress the HR pathway in response to DNA DSBs. TRIM29 regulates the choice of DNA DSB repair pathway by facilitating 53BP1 accumulation to promote NHEJ and may have potential for development into a therapeutic target to sensitize refractory cancers or as biomarker of personalized therapies.

Ring finger protein 43 (RNF43) is an E3 ubiquitin ligase which is well‐known for its role in negative regulation of the Wnt‐signaling pathway. However, the function in DNA double‐strand break repairs has not been investigated. In this study, we used a lymphoblast cell line, DT40, and mouse embryonic fibroblast as cellular models to study DNA double‐strand break (DSB) repairs. For this purpose, we created RNF43 knockout, RNF43−/− DT40 cell line to investigate DSB repairs. We found that deletion of RNF43 does not interfere with cell proliferation. However, after exposure to various types of DNA‐damaging agents, RNF43−/− cells become more sensitive to topoisomerase II inhibitors, etoposide, and ICRF193, than wild type cells. Our results also showed that depletion of RNF43 results in apoptosis upon etoposide‐mediated DNA damage. The delay in resolution of γH2AX and 53BP1 foci formation after etoposide treatment, as well as epistasis analysis with DNAPKcs, suggested that RNF43 might participate in DNA repair of etoposide‐induced DSB via non‐homologous end joining. Disturbed γH2AX foci formation in MEFs following pulse etoposide treatment supported the notion that RNF43 also functions DNA repair in mammalian cells. These findings propose two possible functions of RNF43, either participating in NHEJ or removing the blockage of 5′ topo II adducts from DSB ends.

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The inhibition of poly(ADP-ribose) polymerases (PARPs) and ataxia telangiectasia and Rad3-related (ATR) would be an alternative approach for cancer treatments. The aim of this study is to investigate the synergy of the different combinations of PARP inhibitors (olaparib, talazoparib, or veliparib) and ATR inhibitor AZD6738. A drug combinational synergy screen that combines olaparib, talazoparib, or veliparib with AZD6738 was performed to identify the synergistic interaction, and the combination index was calculated to verify synergy. TK6 isogenic cell lines with defects in different DNA repair genes were used as a model. Cell cycle analysis, micronucleus induction, and focus formation assays of serine-139 phosphorylation of the histone variant H2AX demonstrated that AZD6738 diminished G2/M checkpoint activation induced by PARP inhibitors and allowed DNA damage-containing cells to continue dividing, leading to greater increases in micronuclei as well as double-strand DNA breaks in mitotic cells. We also found that AZD6738 was likely to potentiate cytotoxicity of PARP inhibitors in homologous recombination repair deficiency cell lines. AZD6738 sensitized more genotypes of DNA repair-deficient cell lines to talazoparib than to olaparib and veliparib, respectively. The combinational approach of PARP and ATR inhibition to enhance response to PARP inhibitors could expand the utility of PARP inhibitors to cancer patients without BRCA1/2 mutations.

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

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