When a cut makes the difference : DNA damage incision from human cells to C. elegans

The DNA damage response (DDR) is a complex network of DNA repair processes and associated signaling mechanisms that maintains genome integrity by removing DNA lesions that are continuously induced by endogenous and exogenous sources. The intricate cooperation of all DNA repair and signaling mechanisms involved in the DDR determines how cells cope with DNA damage. In this thesis, particular attention is given to two major DNA repair pathways: the first is nucleotide excision repair (NER), which removes DNA-helix distorting lesions such as those induced by UV light. The second is interstrand crosslink repair (ICLR), which removes covalent linkages between bases on opposing DNA strands induced by DNA crosslinking chemicals and antitumor agents, such as cisplatin, psoralens and mitomycin C. The structure specific endonuclease ERCC1-XPF is essential to incise DNA next to lesions during both NER and ICLR. In humans, mutations in this complex give rise to hereditary DNA repair syndromes such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), xeroderma pigmentosum-Cockayne syndrome complex (XPCS), and Fanconi anemia (FA). Most of the XPF patients carry heterozygous mutations, which makes it difficult to dissect the link between each XPF allele and its phenotypic consequences. Here, we investigate how XPF mutations reported in XP and XPCS patients functionally affect XPF activity in NER. By means of confocal imaging, genetic and proteomics approaches on human cells in culture we show that XP-causing mutations diminish XPF recruitment to DNA damage and only mildly affect repair capacity. In contrast, XPCS-causing mutation impair repair capacity, and are associated with persistent binding or continuous recruitment of XPF and the core NER machinery to UV-induced DNA damage. Moreover, our results suggest that different patient phenotypes associated with XPF mutations are dependent on the ability of the other XPF allele to function in NER. While it is well known that engagement of ERCC1-XPF in NER is facilitated by binding to the key factor XPA, how its recruitment in ICLR is regulated is less clear. Therefore, we investigated how in h

C. elegans TFIIH subunit GTF-2H5/TTDA is a non-essential transcription factor indispensable for DNA repair

The 10-subunit TFIIH complex is vital to transcription and nucleotide excision repair. Hereditary mutations in its smallest subunit, TTDA/GTF2H5, cause a photosensitive form of the rare developmental disorder trichothiodystrophy. Some trichothiodystrophy features are thought to be caused by subtle transcription or gene expression defects. TTDA/GTF2H5 knockout mice are not viable, making it difficult to investigate TTDA/GTF2H5 in vivo function. Here we show that deficiency of C. elegans TTDA ortholog GTF-2H5 is, however, compatible with life, in contrast to depletion of other TFIIH subunits. GTF-2H5 promotes TFIIH stability in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of TFIIH to DNA damage. Strikingly, when transcription is challenged, gtf-2H5 embryos die due to the intrinsic TFIIH fragility in absence of GTF-2H5. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy and establish C. elegans as model for studying pathogenesis of this disease.</p

ERCC1–XPF targeting to psoralen–DNA crosslinks depends on XPA and FANCD2

The effectiveness of many DNA-damaging chemotherapeutic drugs depends on their ability to form monoadducts, intrastrand crosslinks and/or interstrand crosslinks (ICLs) that interfere with transcription and replication. The ERCC1–XPF endonuclease plays a critical role in removal of these lesions by incising DNA either as part of nucleotide excision repair (NER) or interstrand crosslink repair (ICLR). Engagement of ERCC1–XPF in NER is well characterized and is facilitated by binding to the XPA protein. However, ERCC1–XPF recruitment to ICLs is less well u

Tissue-Specific DNA Repair Activity of ERCC-1/XPF-1

Sabatella et al. image the DNA repair endonuclease ERCC-1/XPF-1 in C. elegans to show that nucleotide excision repair exhibits tissue-specific activity. DNA lesions are very rapidly removed from the entire genome in oocytes but only from transcribed genes in somatic cells. Neurons are more sensitive to DNA damage than muscle cells.Hereditary DNA repair defects affect tissues differently, suggesting that in vivo cells respond differently to DNA damage. Knowledge of the DNA damage response, however, is largely based on in vitro and cell culture studies, and it is currently unclear whether DNA repair changes depending on the cell type. Here, we use in vivo imaging of the nucleotide excision repair (NER) endonuclease ERCC-1/XPF-1 in C. elegans to demonstrate tissue-specific NER activit

Neuropeptide signaling regulates the susceptibility of developing C. elegans to anoxia

Inadequate delivery of oxygen to organisms during development can lead to cell dysfunction/death and life-long disabilities. Although the susceptibility of developing cells to low oxygen conditions changes with maturation, the cellular and molecular pathways that govern responses to low oxygen are incompletely understood. Here we show that developing Caenorhabditis elegans are substantially more sensitive to anoxia than adult animals and that this sensitivity is controlled by nervous system generated hormones (e.g., neuropeptides). A screen of neuropeptide genes identified and validated nlp-40 and its receptor aex-2 as a key regulator of anoxic survival in developing worms. The survival-promoting action of impaired neuropeptide signaling does not rely on five known stress resistance pathways and is specific to anoxic insult. Together, these data highlight a novel cell non-autonomous pathway that regulates the susceptibility of developing organisms to anoxia

Base and nucleotide excision repair facilitate resolution of platinum drugs-induced transcription blockage

Sensitivity and resistance of cells to platinum drug chemotherapy are to a large extent determined by activity of the DNA damage response (DDR). Combining chemotherapy with inhibition of specific DDR pathways could therefore improve treatment efficacy. Multiple DDR pathways have been implicated in removal of platinum-DNA lesions, but it is unclear which exact pathways are most important to cellular platinum drug resistance. Here, we used CRISPR/Cas9 screening to identify DDR proteins that protect colorectal cancer cells against the clinically applied platinum drug oxaliplatin. We find that besides the expected homologous recombination, Fanconi anemia and translesion synthesis pathways, in particular also transcription-coupled nucleotide excision repair (TC-NER) and base excision repair (BER) protect against platinum-induced cytotoxicity. Both repair pathways are required to overcome oxaliplatin- and cisplatin-induced transcription arrest. In addition to the generation of DNA crosslinks, exposure to platinum drugs leads to reactive oxygen species production that induces oxidative DNA lesions, explaining the requirement for BER. Our findings highlight the importance of transcriptional integrity in cells exposed to platinum drugs and suggest that both TC-NER and BER should be considered as targets for novel combinatorial treatment strategies

Repair protein persistence at DNA lesions characterizes XPF defect with Cockayne syndrome features

The structure-specific ERCC1-XPF endonuclease plays a key role in DNA damage excision by nucleotide excision repair (NER) and interstrand crosslink repair. Mutations in this complex can either cause xeroderma pigmentosum (XP) or XP combined with Cockayne syndrome (XPCS-complex) or Fanconi anemia. However, most patients carry compound heterozygous mutations, which confounds the dissection of the phenotypic consequences for each of the identified XPF alleles. Here, we analyzed the functional impact of individual pathogenic XPF alleles on NER. We show that XP-causing mutations diminish XPF recruitment to DNA damage and only mildly affect global genome NER. In contrast, an XPCS-complex-specific mutation causes persistent recruitment of XPF and the upstream core NER machinery to DNA damage and severely impairs both global genome and transcription-coupled NER. Remarkably, persistence of NER factors at DNA damage appears to be a common feature of XPCS-complex cells, suggesting that this could be a determining factor contributing to the development of additional developmental and/or neurodegenerative features in XP patients

Studies on the Depth of Crown of Rice, Wheat and Barly Plant 4 : Effects of oxidation reduction potential in soil and soil humidity on the length of subcrown-internode and the depth of crown

textabstractXPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of SUMOylated XPC after DNA damage. However, the exact regulatory function of these modifications in vivo remains elusive. Here we show that RNF111 is required for efficient repair of ultraviolet-induced DNA lesions. RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER initiation, and is needed for stable incorporation of the NER endonucleases XPG and ERCC1/XPF. Our data suggest that RNF111, together with the CRL4^DDB2 ubiquitin ligase complex, is responsible for sequential XPC ubiquitylation, which regulates the recruitment and release of XPC and is crucial for efficient progression of the NER reaction, thereby providing an extra layer of quality control of NER

Binding of Carbonic Anhydrase IX to 45S rDNA Genes Is Prevented by Exportin-1 in Hypoxic Cells

Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45S rDNA gene promoters