UV Irradiation Of Human Cells Research Articles

Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation

UV light induces DNA lesions, which are removed by nucleotide excision repair (NER). Exonuclease 1 (EXO1) is highly conserved from yeast to human and is implicated in numerous DNA metabolic pathways, including repair, recombination, replication, and telomere maintenance. Here we show that hEXO1 is involved in the cellular response to UV irradiation in human cells. After local UV irradiation, fluorescent-tagged hEXO1 localizes, together with NER factors, at the sites of damage in nonreplicating cells. hEXO1 accumulation requires XPF-dependent processing of UV-induced lesions and is enhanced by inhibition of DNA repair synthesis. In nonreplicating cells, depletion of hEXO1 reduces unscheduled DNA synthesis after UV irradiation, prevents ubiquitylation of histone H2A, and impairs activation of the checkpoint signal transduction cascade in response to UV damage. These findings reveal a key role for hEXO1 in the UV-induced DNA damage response linking NER to checkpoint activation in human cells.

Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila

BackgroundWe observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process.MethodsWe explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP.ResultsWe first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.ConclusionOur results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.

New Histone Incorporation Marks Sites of UV Repair in Human Cells

Chromatin organization is compromised during the repair of DNA damage. It remains unknown how and to what extent epigenetic information is preserved in vivo. A central question is whether chromatin reorganization involves recycling of parental histones or new histone incorporation. Here, we devise an approach to follow new histone deposition upon UV irradiation in human cells. We show that new H3.1 histones get incorporated in vivo at repair sites. Remarkably we find that H3.1, which is deposited during S phase, is also incorporated outside of S phase. Histone deposition is dependent on nucleotide excision repair (NER), indicating that it occurs at a postrepair stage. The histone chaperone chromatin assembly factor 1 (CAF-1) is directly involved in the histone deposition process in vivo. We conclude that chromatin restoration after damage cannot rely simply on histone recycling. New histone incorporation at repair sites both challenges epigenetic stability and possibly contributes to damage memory.

Low levels of NPM gene expression in UV-sensitive human cell lines

Nucleophosmin (NPM) is a major nuclear matrix protein associated with neoplastic growth in various cell types. We recently suggested that expression of the NPM gene is involved in an increased resistance to UV irradiation in human cells against the cell-killing effects of UV (mainly 254nm wavelength far-ultraviolet ray) [Y. Higuchi, K. Kita, H. Nakanishi, X–L. Wang, S. Sugaya, H. Tanzawa, H. Yamamori, K. Sugita, A. Yamaura, N. Suzuki, Biochem. Biophys. Res. Commun. 248 (1998) 597–602]. In the present study, expression levels of the NPM gene were examined in human cell lines with a high sensitivity to UV cell-killing. Cockayne syndrome patient-derived cell lines, CSAI and CSBI, and the Xeroderma pigmentosum patient-derived cell line, XP2OS(SV), XP13KY, XP3KA, XP6BE(SV), XP101OS and XP3BR(SV), have been investigated for their NPM mRNA expression with Northern blotting analysis. All of these UV-sensitive cells demonstrated lower expression levels compared with those of normal fibroblast cells, FF, or an UV-resistant cell line, UH r-10; quite a lower level of expression in XP205(SV) cells after UV irradiation in contrast to a distinguishable increase in the expression in UV r- cells. These results confirmed an intimate correlation between degree of UV sensitivity and expression levels of the NPM gene in human cells.

Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells.

The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.

Mutagenesis at putative apurinic sites in alkylated single-stranded DNA of parvovirus H-1 propagated in human cells

The treatment of parvovirus H-1, a single-stranded DNA virus, with ethylnitrosourea immediately prior to infection of human cells, resulted in both virus mutagenesis and lethality (immediate hits). The incubation of treated virus, prior to inoculation, under conditions promoting the release of alkylated bases, slightly reduced the mutagenicity of ethylnitrosourea but significantly increased its killing effect (delayed hits). In untreated cells, the appearance of one apurinic/apyrimidinic site in viral DNA correlated with the formation of approximately one delayed lethal hit per virus. Cells which had been sublethally UV irradiated prior to infection, were able to overcome about 20% of the delayed lethal hits inflicted to ehthylnitrosourea-treated H-1. This UV-enhanced reactivation was accompanied by viral mutagenesis and was not observed for immediate lethal hits. Therefore, UV irradiation of human cells appears to trigger a conditioned recovery response which might alleviate a block to the replication of single-stranded DNA containing apurinic sites, allowing these noncoding lesions to direct mutagenesis. UV-irradiated cells also displayed a mutator phenotype towards untreated parvovirus H-1. In contrast, ethylnitrosourea failed to induce human cells to cause mutagenesis of undamaged viral DNA, although it enhanced their ability to reactivate damaged virus.