Colcemid inhibits the rejoining of the nucleotide excision repair of UVC-induced DNA damages in Chinese hamster ovary cells

In our previous study, we found that colcemid, an inhibitor of mitotic spindle, promotes UVC-induced apoptosis in Chinese hamster ovary cells (CHO.K1). In this study, a brief treatment of colcemid on cells after but not before UV irradiation could synergistically reduce the cell viability. Although colcemid did not affect the excision of UV-induced DNA damages such as [6–4] photoproducts or cyclobutane pyrimidine dimers, colcemid accumulated the DNA breaks when it was added to cells following UV-irradiation. This colcemid effect required nucleotide excision repair (NER) since the same accumulation of DNA breaks was barely or not detected in two NER defective strains of CHO cells, UV5 or UV24. Furthermore, the colcemid effect was not due to semi-conservative DNA replication or mitosis since the colcemid-caused accumulation of DNA breaks was also seen in non-replicating cells. Moreover, colcemid inhibited rejoining of DNA breaks accumulated by hydroxyurea/cytosine arabinoside following UV irradiation. Nevertheless, colcemid did not affect the unscheduled DNA synthesis as assayed by the incorporation of bromodeoxyuridine. Taken together, our results suggest that colcemid might inhibit the step of ligation of NER pathways.     

Recombinant expression of maize nucleotide excision repair protein Rad23 in Escherichia coli

Nucleotide excision is a highly conserved DNA repair pathway for correcting DNA lesions that cause distortion of the double helical structure. The protein heterodimer XPC-Rad23 is involved in recognition of and binding to such lesions. We have isolated full-length cDNAs encoding two different members of the maize Rad23 family. The deduced amino acid sequences of both maize orthologues show a high degree of homology to plant and animal Rad23 proteins. The cDNA encoding maize Rad23A was cloned as an in-frame C-terminal fusion of glutathione S-transferase. This chimera was expressed in Escherichia coli as a soluble protein and purified to homogeneity using glutathione-agarose followed by MonoQ column chromatography. Purified recombinant maize Rad23 protein was used to generate polyclonal antibodies that cross-react with a approximately 48-kDa protein in extracts from plant as well as mammalian cells. The purified recombinant protein and antibodies would be useful reagents to study the biochemistry of nucleotide excision repair in plants.     

Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair

The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4 h. However, RPA did not reach its maximum accumulation level until 3 h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8 h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.     

Active DNA damage eviction by HLTF stimulates nucleotide excision repair

1. Download : Download high-res image (178KB)
2. Download : Download full-size image

     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

Structural insights into the first incision reaction during nucleotide excision repair

Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3' incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 A resolution, which catalyzes the 3' incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond.     

Association between nucleotide excision repair gene polymorphisms and chromosomal damage in coke-oven workers

The associations between several genetic polymorphisms of nucleotide excision repair genes (NER) and chromosome damage level were studied among 140 coke-oven workers exposed to a high level of polyaromatic hydrocarbons (PAHs) and 66 non-exposed workers. Seven polymorphisms with functional potential in five NER genes (ERCC1, ERCC2, ERCC4, ERCC5 and ERCC6) were genotyped in the 206 study subjects. Multivariate analysis of covariance revealed that coke-oven workers with the ERCC1 19007 CC genotype had significantly higher cytokinesis-block micronucleus frequency (CBMN) (10.5±6.8‰) than those with CT (8.1±6.6‰, p=0.01) or TT (6.6±3.7‰, p=0.05) or CT+TT genotypes (7.5±6.3‰, p=0.004). The ERCC6 A3368G polymorphism was also associated with CBMN frequency among coke-oven workers. Subjects with the AA genotype have a significantly higher CBMN frequency (10.0±6.9‰) than those with AG (6.7±4.2‰, p=0.05) or AG+GG genotypes (6.6±4.1‰, p=0.02). Stratification analysis revealed the significant associations between ERCC1 C19007T and ERCC6 A3368G, and the CBMN frequencies were only found among older workers. In addition, a significant association between ERCC2 G23591A polymorphism and CBMN frequencies was also found among older coke-oven workers. The results suggest that polymorphisms of ERCC1 C19007T, ERCC6 A3368G and ERCC2 G23591A are associated with the CBMN frequencies among coke-oven workers     

Association between nucleotide excision repair gene polymorphisms and chromosomal damage in coke-oven workers

The associations between several genetic polymorphisms of nucleotide excision repair genes (NER) and chromosome damage level were studied among 140 coke-oven workers exposed to a high level of polyaromatic hydrocarbons (PAHs) and 66 non-exposed workers. Seven polymorphisms with functional potential in five NER genes (ERCC1, ERCC2, ERCC4, ERCC5 and ERCC6) were genotyped in the 206 study subjects. Multivariate analysis of covariance revealed that coke-oven workers with the ERCC1 19007 CC genotype had significantly higher cytokinesis-block micronucleus frequency (CBMN) (10.5±6.8‰) than those with CT (8.1±6.6‰, p=0.01) or TT (6.6±3.7‰, p=0.05) or CT+TT genotypes (7.5±6.3‰, p=0.004). The ERCC6 A3368G polymorphism was also associated with CBMN frequency among coke-oven workers. Subjects with the AA genotype have a significantly higher CBMN frequency (10.0±6.9‰) than those with AG (6.7±4.2‰, p=0.05) or AG+GG genotypes (6.6±4.1‰, p=0.02). Stratification analysis revealed the significant associations between ERCC1 C19007T and ERCC6 A3368G, and the CBMN frequencies were only found among older workers. In addition, a significant association between ERCC2 G23591A polymorphism and CBMN frequencies was also found among older coke-oven workers. The results suggest that polymorphisms of ERCC1 C19007T, ERCC6 A3368G and ERCC2 G23591A are associated with the CBMN frequencies among coke-oven workers     

Nucleosome positioning,nucleotide excision repair and photoreactivation in Saccharomyces cerevisiae

The position of nucleosomes on DNA participates in gene regulation and DNA replication. Nucleosomes can be repressors by limiting access of factors to regulatory sequences, or activators by facilitating binding of factors to exposed DNA sequences on the surface of the core histones. The formation of UV induced DNA lesions, like cyclobutane pyrimidine dimers (CPDs), is modulated by DNA bending around the core histones. Since CPDs are removed by nucleotide excision repair (NER) and photolyase repair, it is of paramount importance to understand how DNA damage and repair are tempered by the position of nucleosomes. In vitro, nucleosomes inhibit NER and photolyase repair. In vivo, nucleosomes slow down NER and considerably obstruct photoreactivation of CPDs. However, over-expression of photolyase allows repair of nucleosomal DNA in a second time scale. It is proposed that the intrinsic abilities of nucleosomes to move and transiently unwrap could facilitate damage recognition and repair in nucleosomal DNA.     

Organization of DNA damage,excision repair,and mutagenesis in chromatin: A genomic perspective

Genomic DNA is constantly assaulted by both endogenous and exogenous damaging agents. The resulting DNA damage, if left unrepaired, can interfere with DNA replication and be converted into mutations. Genomic DNA is packaged into a highly compact yet dynamic chromatin structure, in order to fit into the limited space available in the nucleus of eukaryotic cells. This hierarchical chromatin organization serves as both the target of DNA damaging agents and the context for DNA repair enzymes. Biochemical studies have suggested that both the formation and repair of DNA damage are significantly modulated by chromatin. Our understanding of the impact of chromatin on damage and repair has been significantly enhanced by recent studies. We focus on the nucleosome, the primary building block of chromatin, and discuss how the intrinsic structural properties of nucleosomes, and their associated epigenetic modifications, affect damage formation and DNA repair, as well as subsequent mutagenesis in cancer.     

Saccharomyces cerevisiae DNA repair processes: an update

The budding yeast Saccharomyces cerevisiae plays a central role in contributing to the understanding of one of the most important biological process, DNA repair, that maintains genuine copies of the cellular chromosomes. DNA lesions produce either spontaneously or by DNA damaging agents are efficiently repaired by one or more DNA repair proteins. While some DNA repair proteins function independently as in the case of base excision repair, others belong into three separate DNA repair pathways, nucleotide excision, mismatch, and recombinational. Of these pathways, nucleotide excision and mismatch repair show the greatest functional conservation between yeast and human cells. Because of this high degree of conservation, yeast has been regarded as one of the best model system to study DNA repair. This report therefore updates current knowledge of the major yeast DNA repair processes.     

The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA

ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the 'access, repair, restore' model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury.     

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.     

Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.