A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His(6)-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni(2+)-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs

Phosphorylation of XPB helicase regulates TFIIH nucleotide excision repair activity

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. The xeroderma pigmentosum group B (XPB) helicase subunit of TFIIH functions in NER and transcription. The serine 751 (S751) residue of XPB was found to be phosphorylated in vivo. This phosphorylation inhibits NER and the microinjection of a phosphomimicking XPB-S751E mutant is unable to correct the NER defect of XP-B cells. Conversely, XPB-S751 dephosphorylation or its substitution with alanine (S751A) restores NER both in vivo and in vitro. Surprisingly, phospho/dephosphorylation of S751 spares TFIIH-dependent transcription. Finally, the phosphorylation of XPB-S751 does not impair the TFIIH unwinding of the DNA around the lesion, but rather prevents the 5′ incision triggered by the ERCC1-XPF endonuclease. These data support an additional role for XPB in promoting the incision of the damaged fragment and reveal a point of NER regulation on TFIIH without interference in its transcription activity

A DNA damage repair gene‐associated signature predicts responses of patients with advanced soft‐tissue sarcoma to treatment with trabectedin

Predictive biomarkers of trabectedin represent an unmet need in advanced soft‐tissue sarcomas (STS). DNA damage repair (DDR) genes, involved in homologous recombination or nucleotide excision repair, had been previously described as biomarkers of trabectedin resistance or sensitivity, respectively. The majority of these studies only focused on specific factors (ERCC1, ERCC5, and BRCA1) and did not evaluate several other DDR‐related genes that could have a relevant role for trabectedin efficacy. In this retrospective translational study, 118 genes involved in DDR were evaluated to determine, by transcriptomics, a predictive gene signature of trabectedin efficacy. A six‐gene predictive signature of trabectedin efficacy was built in a series of 139 tumor samples from patients with advanced STS. Patients in the high‐risk gene signature group showed a significantly worse progression‐free survival compared with patients in the low‐risk group (2.1 vs 6.0 months, respectively). Differential gene expression analysis defined new potential predictive biomarkers of trabectedin sensitivity (PARP3 and CCNH) or resistance (DNAJB11 and PARP1). Our study identified a new gene signature that significantly predicts patients with higher probability to respond to treatment with trabectedin. Targeting some genes of this signature emerges as a potential strategy to enhance trabectedin efficacy

The AHR represses nucleotide excision repair and apoptosis and contributes to UV-induced skin carcinogenesis

Ultraviolet B (UVB) radiation induces mutagenic DNA photoproducts, in particular cyclobutane pyrimidine dimers (CPDs), in epidermal keratinocytes (KC). To prevent skin carcinogenesis, these DNA photoproducts must be removed by nucleotide excision repair (NER) or apoptosis. Here we report that the UVB-sensitive transcription factor aryl hydrocarbon receptor (AHR) attenuates the clearance of UVB-induced CPDs in human HaCaT KC and skin from SKH-1 hairless mice. Subsequent RNA interference and inhibitor studies in KC revealed that AHR specifically suppresses global genome but not transcription-coupled NER. In further experiments, we found that the accelerated repair of CPDs in AHR-compromised KC depended on a modulation of the p27 tumor suppressor protein. Accordingly, p27 protein levels were increased in AHR-silenced KC and skin biopsies from AHR-/- mice, and critical for the improvement of NER. Besides increasing NER activity, AHR inhibition was accompanied by an enhanced occurrence of DNA double-strand breaks triggering KC apoptosis at later time points after irradiation. The UVB-activated AHR thus acts as a negative regulator of both early defense systems against carcinogenesis, NER and apoptosis, implying that it exhibits tumorigenic functions in UVB-exposed skin. In fact, AHR-/- mice developed 50% less UVB-induced cutaneous squamous cell carcinomas in a chronic photocarcinogenesis study than their AHR+/+ littermates. Taken together, our data reveal that AHR influences DNA damage-dependent responses in UVB-irradiated KC and critically contributes to skin photocarcinogenesis in mice