Cell-type specific concentration regulation of the basal transcription factor TFIIH in XPBy/y mice model

Background: The basal transcription/repair factor TFIIH is a ten sub-unit complex essential for RNA polymerase II (RNAP2) transcription initiation and DNA repair. In both these processes TFIIH acts as a DNA helix opener, required for promoter escape of RNAP2 in transcription initiation, and to set the stage for strand incision within the nucleotide excision repair (NER) pathway. Methods: We used a knock-in mouse model that we generated and that endogenously expresses a fluorescent version of XPB (XPB-YFP). Using different microscopy, cellular biology and biochemistry approaches we quantified the steady state levels of this protein in different cells, and cells imbedded in tissues. Results: Here we demonstrate, via confocal imaging of ex vivo tissues and cells derived from this mouse model, that TFIIH steady state levels are tightly regulated at the single cell level, thus keeping nuclear TFIIH concentrations remarkably constant in a cell type dependent manner. Moreover, we show that individual cellular TFIIH levels are proportional to the speed of mRNA production, hence to a cell's transcriptional activity, which we can correlate to proliferation status. Importantly, cancer tissue presents a higher TFIIH than normal healthy tissues. Conclusion: This study shows that TFIIH cellular concentration can be used as a bona-fide quantitative marker of transcriptional activity and cellular proliferation

Coordination of dual incision and repair synthesis in human nucleotide excision repair

Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5′ to the lesion by ERCC1-XPF and 3′ to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5′ incision by ERCC1-XPF precedes the 3′ incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a ‘cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity

Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4

International audienceDNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not DNA-PK(CS). We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV

Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4

DNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends w

First reported patient with human ERCC1 deficiency has cerebro-oculo-facio- skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure

Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1