DNA damage: air-breaks?

Cells deficient in repairing DNA double-strand breaks have an increased level of spontaneous chromosomal aberrations. Modulating the level of molecular oxygen and its reactive metabolites demonstrates that oxygen metabolism is a major source of genomic instability.     

Does vitamin D protect against DNA damage?

Vitamin D is a secosteroid best known for its role in maintaining bone and muscle health. Adequate levels of vitamin D may also be beneficial in maintaining DNA integrity. This role of vitamin D can be divided into a primary function that prevents damage from DNA and a secondary function that regulates the growth rate of cells. The potential for vitamin D to reduce oxidative damage to DNA in a human has been suggested by clinical trial where vitamin D supplementation reduced 8-hydroxy-2'-deoxyguanosine, a marker of oxidative damage, in colorectal epithelial crypt cells. Studies in animal models and in different cell types have also shown marked reduction in oxidative stress damage and chromosomal aberrations, prevention of telomere shortening and inhibition of telomerase activity following treatment with vitamin D. The secondary function of vitamin D in preventing DNA damage includes regulation of the poly-ADP-ribose polymerase activity in the DNA damage response pathway involved in the detection of DNA lesions. It is also able to regulate the cell cycle to prevent the propagation of damaged DNA, and to regulate apoptosis to promote cell death. Vitamin D may contribute to prevention of human colorectal cancer, though there is little evidence to suggest that prevention of DNA damage mediates this effect, if real. Very limited human data mean that the intake of vitamin D required to minimise DNA damage remains uncertain.     

Melanogenesis: a photoprotective response to DNA damage?

Exposure to ultra violet radiation (UVR) is associated with significant long-term deleterious effects such as skin cancer. A well-recognised short-term consequence of UVR is increased skin pigmentation. Pigmentation, whether constitutive or facultative, has widely been viewed as photoprotective, largely because darkly pigmented skin is at a lower risk of photocarcinogenesis than fair skin. Research is increasingly suggesting that the relationship between pigmentation and photoprotection may be far more complex than previously assumed. For example, photoprotection against erythema and DNA damage has been shown to be independent of level of induced pigmentation in human white skin types. Growing evidence now suggests that UVR induced DNA photodamage, and its repair is one of the signals that stimulates melanogenesis and studies suggest that repeated exposure in skin type IV results in faster DNA repair in comparison to skin type II. These findings suggest that tanning may be a measure of inducible DNA repair capacity, and it is this rather than pigment per se which results in the lower incidence skin cancer observed in darker skinned individuals. This evokes the notion that epidermal pigmentation may in fact be the mammalian equivalent of a bacterial SOS response. Skin colour is one of most conspicuous ways in which humans vary yet the function of melanin remains controversial. Greater understanding of the role of pigmentation in skin is vital if one is to be able to give accurate advice to the general public about both the population at risk of skin carcinogenesis and also public perceptions of a tan as being healthy.     

WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH^−/−) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.     

A special issue on DNA damage responses and genome maintenance

There is nothing more fundamental than the genome for the existence and maintenance of all living beings. The importance of the genome is increasingly appreciated as recent discoveries have revealed that changes in the human genome, regardless of being inherited or induced, can result in diseases that either significantly shorten lives （as seen in cancer） or dramatically affect the quality of lives （often seen in neurodegenerative diseases）. Therefore, maintaining genome integrity is critical for not only the continuation of a species in evolution （although mutations may be occasionally beneficial during evolution） but also for longevity and general health.     

Condensation of DNA—A putative obstruction for repair process in abasic clustered DNA damage

Clustered DNA damages are defined as two or more closely located DNA damage lesions that may be present within a few helical turns of the DNA double strand. These damages are potential signatures of ionizing radiation and are often found to be repair resistant. Types of damaged lesions frequently found inside clustered DNA damage sites include oxidized bases, abasic sites, nucleotide dimers, strand breaks or their complex combinations. In this study, we used a bistranded two-lesion abasic cluster DNA damage model to access the repair process of DNA in condensate form.Oligomer DNA duplexes (47 bp) were designed to have two deoxyuridine in the middle of the sequences, three bases apart in opposite strands. The deoxyuridine residues were converted into abasic sites by treatment with UDG enzyme creating an abasic clustered damage site in a precise position in each of the single strand of the DNA duplex. This oligomer duplex having compatible cohesive ends was ligated to pUC19 plasmid, linearized with HindIII restriction endonuclease. The plasmid–oligomer conjugate was transformed into condensates by treating them with spermidine. The efficiency of strand cleavage action of ApeI enzyme on the abasic sites was determined by denaturing PAGE after timed incubation of the oligomer duplex and the oligomer–plasmid conjugate in presence and absence of spermidine. The efficiency of double strand breaks was determined similarly by native PAGE. Quantitative gel analysis revealed that rate of abasic site cleavage is reduced in the DNA condensates as compared to the oligomer DNA duplex or the linear ligated oligomer–plasmid conjugates. Generation of double strand break is significantly reduced also, suggesting that their creation is not proportionate to the number of abasic sites cleaved in the condensate model. All these suggest that the ApeI enzyme have difficulty to access the abasic sites located deep into the condensates leading to repair refractivity of the damages. In addition, we found that presence of a polyamine such as spermidine has no notable effect in the incision activity of ApeI enzyme in linear oligomer DNA duplexes in our experimental concentration.     

The thyroid hormone receptor β induces DNA damage and premature senescence

There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate–activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism.     

Measurement of 60Co-λ ray-induced DNA damage by capillary electrophoresis

Capillary electrophoresis was employed in this study to monitor ⁶⁰Co-γ-ray-induced damage to a 1 kb DNA ladder which consists of restriction fragments ranging from 75 to 12 000 bp. DNA samples (0.5 mg/ml) were exposed to 0–60 Gy of γ-radiation in the presence and absence of 110 μmol/l ethidium bromide (EB). The analysis showed peak broadening without significant changes in the size distribution of irradiated fragments. Radiation-induced conformational changes may account for this peak broadening. EB addition caused small increases in the retention times of DNA fragments without affecting the overall DNA damage. This indicates that the presence of intercalated EB during radiation will not stabilize the DNA against ⁶⁰Co-γ ray-induced damage.     

Who's on first in the cellular response to DNA damage?

Cellular DNA-repair pathways involve proteins that have roles in other DNA-metabolic processes, as well as those that are dedicated to damage removal. Several proteins, which have diverse functions and are not known to have roles in DNA repair, also associate with damaged DNA. These newly discovered interactions could either facilitate or hinder the recognition of DNA damage, and so they could have important effects on DNA repair and genetic integrity. The outcome for the cell, and ultimately for the organism, might depend on which proteins arrive first at sites of DNA damage.     

Repair of idarubicin-induced DNA damage: A cause of resistance?

Idarubicin, a widely used anticancer drug inhibits topoisomerase (topo) IIalpha and induces DNA double strand breaks. The finding that idarubicin-induced DNA damage is repaired before cell death is initiated encouraged us to examine the role of DNA repair for the cytotoxicity of idarubicin in human promyelocytic HL60 leukaemia cells. We found that DNA double strand breaks induced by a 90 min transient exposure to 0.5 microgml(-1) idarubicin were rapidly repaired throughout the whole population, while topo IIalpha itself was degraded. In spite of DNA repair, the vast majority of cells died within 40 h. Using differential staining of the chromatids and microscopic evaluation of DNA break points, we found evidence for a high number of false ligations of loose DNA strands arising from the inhibition of topo IIalpha action by idarubicin. If mainly actively transcribed genes are affected, this results in a disruption of vital genetic information, of regulatory sequences and, ultimately, in induction of the cell death pathway. Our results confirm the hypothesis that misrepair of DNA damage is a decisive event in idarubicin-induced cell death. They are discussed in the context of topo IIalpha-function and the currently known mechanisms of DNA double strand break repair.     

Is the repair of oxidative DNA base modifications inducible by a preceding DNA damage induction?

In mammalian cells, 7,8-dihydro-8-oxoguanine (8-oxoG) and some other oxidative guanine modifications are removed from the DNA by base excision repair, which is initiated by OGG1 protein. We have tested whether this repair is inducible in mouse embryonic fibroblasts (MEFs), MCF-7 breast cancer cells and primary human fibroblasts by a pretreatment with the photosensitizer Ro19-8022 plus light, which generates predominantly 8-oxoG, or with methyl methanesulfonate (MMS), which generates alkylated bases and abasic sites (AP sites). The results indicate that the repair rate of the oxidative guanine modifications induced by the photosensitizer was not increased if a priming dose of the oxidative or alkylating agent was applied 6 or 18h prior to a challenging dose, although pretreatments with both agents resulted in two-fold elevated glutathione levels as an indication for an adaptive response. Similarly, the activity of total protein extracts of the cells to incise at a single 8-oxoG residue in an oligonucleotide was unchanged. It has to be concluded that the repair of 8-oxoG is not inducible by oxidative or alkylation damage.     

Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA–PK

DNA double-strand breaks (DSB) are considered as the most deleterious DNA lesions, and their repair is further complicated by increasing damage complexity. However, the molecular effects of clustered lesions are yet not fully understood. As the locally restricted phosphorylation of H2AX to form γH2AX is a key step in facilitating efficient DSB repair, we investigated this process after localized induction of clustered damage by ionizing radiation. We show that in addition to foci at damaged sites, H2AX is also phosphorylated in undamaged chromatin over the whole-cell nucleus in human and rodent cells, but this is not related to apoptosis. This pan-nuclear γH2AX is mediated by the kinases ataxia telangiectasia mutated and DNA-dependent protein kinase (DNA–PK) that also phosphorylate H2AX at DSBs. The pan-nuclear response is dependent on the amount of DNA damage and is transient even under conditions of impaired DSB repair. Using fluorescence recovery after photobleaching (FRAP), we found that MDC1, but not 53BP1, binds to the nuclear-wide γH2AX. Consequently, the accumulation of MDC1 at DSBs is reduced. Altogether, we show that a transient dose-dependent activation of the kinases occurring on complex DNA lesions leads to their nuclear-wide distribution and H2AX phosphorylation, yet without eliciting a full pan-nuclear DNA damage response.     

USP45 deubiquitylase controls ERCC1–XPF endonuclease-mediated DNA damage responses

Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF–ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1–XPF. Together, these results establish USP45 as a new regulator of XPF–ERCC1 crucial for efficient DNA repair.     

What role for DNA damage and repair in the bystander response?

The radiation-induced bystander effect challenges the accepted paradigm of direct DNA damage in response to energy deposition driving the biological consequences of radiation exposure. With the bystander response, cells which have not been directly exposed to radiation respond to their neighbours being targeted. In our own studies we have used novel targeted microbeam approaches to specifically irradiate parts of individual cells within a population to quantify the bystander response and obtain mechanistic information. Using this approach it has become clear that energy deposited by radiation in nuclear DNA is not required to trigger the effect, with cytoplasmic irradiation required. Irradiated cells also trigger a bystander response regardless of whether they themselves live or die, suggesting that the phenotype of the targeted cell is not a determining factor. Despite this however, a range of evidence has shown that repair status is important for dealing with the consequences of a bystander signal. Importantly, repair processes involved in the processing of dsb appear to be involved suggesting that the bystander response involves the delayed or indirect production of dsb-type lesions in bystander cells. Whether these are infact true dsb or complexes of oxidised bases in combination with strand breaks and the mechanisms for their formation, remains to be elucidated.     

Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress

Relocalization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein reorganization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by subcellular destination enables the identification of pathways that respond to replication stress. We analysed pairwise combinations of GFP fusions and gene deletion mutants to define and order two previously unknown DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways.