Fig. 3

Decreased activity of the nucleotide excision repair (NER) pathway in cells with prolonged p21^WAF1/Cip1 expression. a RNAseq analysis showed that essential factors of the NER pathway were statistically significantly down-regulated (p ≤ 0.05; see also Additional file 1: Figure S3 for specific real-time RT-PCR validation) in 96-h induced Saos2- (i) and Li-Fraumeni- (ii) p21^WAF1/Cip1 Tet-ON cells. Selective immunoblots for DDB1, ERCC1, ERCC4-XPF, XPC, and ERCC5-XPG, key factors in NER [22], confirming the specificity of the RT-PCR results. α-Tubulin served as loading control; the same protein extracts were used as in Fig. 2. b Decreased repair capacity of N-alkylpurine monoadducts in induced Saos2- (i) and Li-Fraumeni- (ii) p21^WAF1/Cip1 Tet-ON cells and treated with monohydroxymelphalan, an inducer of specific NER substrates [69,70,71]. The data shown are based on five independent experiments with at least two analyses for independent experiment/independent experiment experiment (* p < 0.05, error bars indicate SDs). The middle panel depicts the components and function of NER. The NER pathway is responsible for repair of bulky lesions, especially UV-induced thymine dimers and 6,4-photoproducts, as well as non-bulky ones. Following DNA damage recognition, a short single-stranded DNA fragment that contains the lesion is removed. The remaining undamaged single-stranded DNA segment is used by DNA polymerase as a template to synthesize the complementary sequence. Final ligation to complete NER and formation of a double-stranded DNA is carried out by DNA ligase. Depending on how the DNA damage is recognized, NER can be divided into two subpathways: transcription coupled NER (TC-NER) and global genome NER (GG-NER). While the two subpathways differ in how they recognize DNA damage, they share the same process for lesion incision, repair, and ligation. RBX1 Ring-box 1, Cul4 Cullin 4, DDB1/2 Damage specific DNA binding protein 1/2, ERCC8 (CSA) ERCC excision repair 8, CSA ubiquitin ligase complex subunit, ERCC6 (CSB) ERCC excision repair 6, chromatin remodeling factor, USP7 Ubiquitin-specific peptidase 7; ERCC4-XPF Excision repair 4, endonuclease, ERCC5-XPG ERCC excision repair 5, endonuclease, XPA XPA, DNA damage recognition and repair factor, XAB2 XPA binding protein 2, RPA Replication protein A, HMGN1 High mobility group nucleosome binding domain 1; XPC XPC complex subunit, DNA damage recognition and repair factor, RAD23B RAD23 homolog B, CETN2 Centrin 2, CDK7 Cyclin-dependent kinase 7, MNAT1 CDK activating kinase assembly factor, CCNH Cyclin H, TFIIH1–4 Transcription/repair factor IIH 1–4, ERCC3 ERCC excision repair 3, TFIIH core complex helicase subunit, ERCC2 ERCC excision repair 2, TFIIH core complex helicase subunit, TTDA (GTF2H5/TFB5) General transcription factor IIH subunit 5