Fig 2: Unified working model: role of NER proteins in 8-oxoguanine repair.Treatment of cells expressing FAP-TRF1 with dye (100 nM, 15 min) plus light (660 nm, 10 min) introduces 8-oxoG lesions at telomeres. In the DDB2-dependent repair pathway, DDB2 recognizes 8-oxoG lesions and facilitates chromatin relaxation through chromatin decompaction allowing the recruitment of XPC and OGG1 to the damage site. OGG1 recruitment facilitates the dissociation of DDB2. In the absence of downstream repair, DDB2 is retained longer at 8-oxoG sites requiring DDB1-Cul4A-RBX1 (CRL) mediated DDB2 dissociation. At actively transcribed regions, OGG1 can access the lesion independent of DDB2. 8-oxoG processing can lead to toxic BER intermediates that can act as a transcription block. Transcription-coupled repair (TCR) proteins, including XPA, participate in the repair of these BER intermediates. (See also Supplementary Movie 3).

Fig 3: Multiple DDR and DNA repair pathway genes targeted by radiation sensitizing miRNAs. (A) Western blot analyses of MAD2L2, WEE1, XPC, KU80, RAD23B, XLF and MCL1 48 h after LNCaP transfection with 20 nM of miR-890 (left) and miR-744–3p (right). Asterisk represents a nonspecific band. ACTB was utilized as a loading reference. (B) Normalized percent protein knock-down (mean ± SE, n = 3) by miR-890 (left) and miR-744–3p (right), relative to control miRNA (from three separate experiments). (C) Luciferase activities following transfection with indicated 3′UTR reporters and miR-890 or control mimetics in 293T cells. (D) Luciferase activities following transfection with indicated 3′UTR reporters and miR-744–3p or control mimetics in 293T cells. Relative luciferase activity (mean ± SE, n =12) is normalized to control miRNA for each reporter. *, P < 0.05 relative to control miRNAs.

Fig 4: Schematic diagram demonstrating the inhibitory effects of TPL on CBP induced upregulation of NER pathway. TPL synergistically enhances CBP-induced apoptosis by selectively inhibiting the NER activity and overexpressing p53 in melanoma. The platinum induced DNA damage is identified and primarily repaired by the NER pathway. The damaged DNA can be recognized by XPC, and then XPA and RPA are recruited and act as another damage recognition complex that might participate in lesion verification. The DNA around the lesion site is opened asymmetrically in an ATP-dependent manner, employing the TFIIH complex with its two DNA helicases, ERCC3/XPB and ERCC2/XPD (Evans et al., 1997). TFIIH is involved in both transcription and NER processes. Subsequently, ERCC4/XPF-ERCC1 complex and ERCC5/XPG, two structure-specific endonucleases, incise on the 5′and 3′sides of the DNA adduct, respectively, to remove the damaged single-stranded DNA that contains the lesion. Finally, Pol δ/ε resynthesize the removed oligonucleotides and successfully repair the DNA damage by DNA ligase.

Fig 5: Effect of TPL and CBP on the mRNA and protein expressions of the NER pathway components. A375 cells were treated with TPL or CBP for 48 h with indicated concentrations. TPL abrogates the (A) mRNA and (B) protein expression levels of the rate-limiting components (ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG, XPA, and XPC) of the NER pathway, while CBP increases the (C) mRNA and (D) protein expression levels. (E) Effect of TPL (30 nM) and CBP (50 μM) alone or in combination on the levels of the NER pathway proteins in melanoma cells.