DNA end resection and its role in DNA replication and DSB repair choice in mammalian cells

DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3′-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice.Subject terms: Double-strand DNA breaks, Cell signalling     

Multiple pathways of DNA repair in bacteria and their roles in mutagenesis.

Abstract— In bacteria, three processes of DNA repair are known: photoreactivation, excision repair, and postreplication repair. Photoreactivation, the enzymatic splitting of cyclobutyl pyrimidine dimers in situ, is mediated by exposure of the enzyme-dimer complex to near-UV and visible light. This repair process appears to be error free. The excision repair of UV-induced DNA base damage has been divided into two major pathways on the basis of both physiological requirements and genetic control. The major pathway requires a functional pol A gene, does not require complete growth medium. and appears to be largely error-free and to produce short patches during repair. The second pathway requires complete growth medium and functional recA, recB, recC, lexA, uvrD, and polC genes, and appears to be mutagenic and to produce long patches during repair. There exists a second type of excision repair in which the modified base is removed by an N-glycosidase, and the chain is then nicked by an apurinic (apyrimidinic) acid endonuclease. Subsequent events are presumed to be similar to the above excision repair process. The postreplication repair system has been divided into at least four separate pathways. Three of these are dependent upon functional recB, lexA, and uvrD genes, respectively, and appear to be error free. A fourth pathway depends upon the above gene products, but is blocked by postirradiation treatment with chloramphenicol, and may be the UV-inducible, errorprone, mutagenic pathway of repair (“SOS repair”). A possible fifth pathway depends upon a functional recF gene, and is independent of the recB⁺-dependent pathway. Mutagenesis appears to be the result of error-prone DNA repair, and there is growing evidence that carcinogenesis is also the result of error-prone DNA repair.     

Shuttle vectors for studying mutagenesis in mammalian cells

Shuttle vectors are DNA plasmids able to replicate in both mammalian cells and bacteria. They have been used to examine rapidly various aspects of DNA repair, recombination and mutagenesis. Three main classes of shuttle vector have been developed. The transiently replicating vectors are usually based on Simian Virus 40 replication origin. The episomal vectors based on the Epstein-Barr virus replication replicate almost permanently in host cells. Different biological systems, including retroviral vectors, allow the integration of a target gene into the chromosomal structure of the infected cells. In all cases, low molecular weight DNA can be recovered from mammalian cells and shuttled back to bacteria for mutagenesis screening. The advantages and disadvantages of these different types of shuttle vectors are discussed with a special emphasis on their use for a rapid analysis of mutation spectra in mammalian cells.     

Context-dependent mutagenesis by DNA lesions.

BACKGROUND: Detailed analyses of mutational hotspots following DNA damage provide an understanding of oncogene activation and tumor suppressor gene inactivation, and hence provide an insight into the earliest steps in the induction of cancer. A mutational hotspot might be created by preferential lesion formation, decreased lesion repair, or increased misinsertion past the lesion during DNA replication. The respective contribution of these factors might be influenced by the DNA sequence context of the hotspot. RESULTS: As a prelude to addressing the contribution of all possible nearest-neighbor contexts on the replication past O6-methylguanine (m6G) and repair of m6G in vivo, we have devised a mutation frequency (MF) detection strategy on the basis of the properties of type IIs restriction enzymes. We also report a method for constructing site-specific single-stranded viral DNA genomes that should yield identical ligation efficiencies regardless of the lesion or its surrounding sequence context. Using repair-deficient Escherichia coli, we discovered that m6G in three sequence contexts was nearly 100% mutagenic in vivo, showing that the DNA polymerase holoenzyme almost always placed a thymine base opposite m6G during replication. In partially repair-proficient cells, the Ada O6-methylguanine-DNA methyltransferase repair protein was twice as efficient on m6G when a guanine base rather than an adenine base was 5' to the lesion. CONCLUSIONS: The system allows the mutagenic potential of, theoretically, any DNA lesion that exhibits point mutations, in any varied local sequence context, to be rapidly determined. The assay demonstrates low background, high throughput, and does not require phenotypic selection, making it possible to discern the effects of sequence context on the processing of m6G.     

Analysis of metal-induced oxidative DNA damage in cultured mammalian cells

Reactive oxygen species are continuously generated during oxygen metabolism, and a measurable amount of oxidative DNA damage exists in aerobic organisms. By the determination of Fpg-sensitive sites in mammalian cells in culture, we assessed the background level of oxidative DNA damage and its potential increase by extracellularly applied complexes of iron(III). In V79 Chinese hamster cells the endogenous level of Fpg-sensitive modifications is detectable, but the extent is much lower as compared with results derived from other analytical methods. In V79 cells, the frequency of Fpg-sensitive modifications is considerably enhanced by Fe-NTA in a time- and dose-dependent manner, while no increase is observed after treatment with Fe-citrate. These results indicate that the ability of transition metals to generate oxidative DNA damage in intact cells strongly depends on factors like uptake and intracellular distribution, which will affect the intracellular availability of redox-active metal ions close to critical targets.     

Analysis of metal-induced oxidative DNA damage in cultured mammalian cells

Reactive oxygen species are continuously generated during oxygen metabolism, and a measurable amount of oxidative DNA damage exists in aerobic organisms. By the determination of Fpg-sensitive sites in mammalian cells in culture, we assessed the background level of oxidative DNA damage and its potential increase by extracellularly applied complexes of iron(III). In V79 Chinese hamster cells the endogenous level of Fpg-sensitive modifications is detectable, but the extent is much lower as compared with results derived from other analytical methods. In V79 cells, the frequency of Fpg-sensitive modifications is considerably enhanced by Fe-NTA in a time- and dose-dependent manner, while no increase is observed after treatment with Fe-citrate. These results indicate that the ability of transition metals to generate oxidative DNA damage in intact cells strongly depends on factors like uptake and intracellular distribution, which will affect the intracellular availability of redox-active metal ions close to critical targets.     

DNA repair inhibition and cancer therapy.

The DNA repair process in mammalian cells is a multi-pathway mechanism that protects cells from the plethora of DNA damaging agents that are known to attack nuclear DNA. Moreover, the majority of current anticancer therapies (e.g. ionising radiation and chemotoxic therapies) rely on this ability to create DNA lesions, leading to apoptosis/cell death. A cells natural ability to repair such DNA damage is a major cause of resistance to these existing antitumour agents. It seems logical, therefore, that by modulating these repair mechanisms, greater killing effect to anticancer agents would occur. Experimental data support this, either through knockout studies or by the use of pharmacological inhibitors which target some of the key regulatory proteins involved in the DNA repair process. Several of these key DNA repair proteins which are actively under investigation as novel sites for intervention in cancer biology are discussed.     

Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells

The heat-shock response is a cellular defence mechanism against environmental stresses that is evolutionarily conserved from bacteria to man. Numerous reports demonstrate the beneficial effects of heat-shock protein induction on cell survival under toxic or oxidative stress, e.g., in cardiac and cerebral ischemia or prior to organ transplantation. However, there is little data on the effects of heat treatment on damage caused by UV irradiation. Applying three independent techniques, we have tested the influence of thermal pretreatment of skin cells (1 h, 43 degrees C) on the initial extent of UV-B-induced DNA damage and its subsequent repair. For cultured human epidermal keratinocytes and dermal fibroblasts we can show reduced levels of nucleotide-excision-repair-associated DNA strand incision in the comet assay. Moreover, immunostaining and flow cytometric quantitation of thymidine dimers immediately and one day after irradiation, respectively, reveal that the initial DNA damage is not (keratinocytes) or only moderately (fibroblasts) lower in heat-shocked cells as compared to untreated controls. However, excision repair of dimers is significantly attenuated during the first 24 h in both cell types. Furthermore, using a modified host-cell reactivation assay, we are able to demonstrate that repair of UV-B-damaged plasmid DNA is lower if the transfected cells are previously heat shocked. In summary, heat treatment (1 h, 43 degrees C) inducing heat-shock proteins reduces nucleotide excision repair of UV-B-mediated DNA lesions in fibroblasts and keratinocytes during the following 24 h. This is not necessarily caused by elevated heat-shock protein levels themselves. Possibly the direct thermal damage of repair enzymes is more severe than the potential protective effects of heat-shock proteins.     

DNA methylation and mammalian epigenetics

Epigenetic modifications of DNA such as methylation are important for genome function during development and in adults. DNA methylation has central importance for genomic imprinting and other aspects of epigenetic control of gene expression, and during development methylation patterns are largely maintained in somatic lineages. The mammalian genome undergoes major reprogramming of methylation patterns in the germ cells and in the early embryo. Some of the factors that are involved both in maintenance and in reprogramming, such as methyltransferases, are being identified. Epigenetic changes are likely to be important in animal cloning, and influence the occurrence of epimutations and of epigenetic inheritance. Environmental factors can alter epigenetic modifications and may thus have long lasting effects on phenotype. Epigenetic engineering is likely to play an important role in medicine in the future.     

Influence of astaxanthin, zeaxanthin and lutein on DNA damage and repair in UVA-irradiated cells

In order to gain more knowledge about the antioxidant role of the predominant carotenoids (lutein and zeaxanthin) of the human retina, this study investigated their antioxidant activity and capacity. Astaxanthin was also studied, because its structure is very close to that of lutein and zeaxanthin. The antioxidant activity of these molecules was evaluated using chemiluminescence techniques, with lucigenin and luminol as chemiluminogenic probes for the superoxide radical and hydrogen peroxide, respectively. It was found that all three carotenoids have similar superoxide-scavenging activity. The effect on the reduction of H(2)O(2)-luminol chemiluminescence was present in the following order, zeaxanthin>astaxanthinlutein. Possible antioxidant capacity of these three compounds was sought using a biological system consisting of SK.N.SH human neuroblastoma and rat trachea epithelial cells subjected to oxidative stress from exposure to UVA radiation. In particular, we determined whether these compounds were capable of minimizing DNA damage and influencing the kinetics of DNA repair. DNA damage was assessed using the Comet assay, a rapid and sensitive single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. Neuroblastoma cells appeared more resistant to oxidative irradiation insult. The presence of carotenoids reduced DNA damage when rat epithelial cells were exposed to UVA radiation for 2min. A different result was obtained in experiments performed on neuroblastoma cells; in this case, the presence of carotenoid during UVA exposition increased the damage. The addition of carotenoids to epithelial cells after 2min of UVA exposition did not seem to improve the kinetics of DNA repair; on the contrary, zeaxanthin (after 60' incubation) and lutein (after 180' incubation) showed a genotoxic effect. The addition of carotenoids to neuroblastoma cells after 30' UVA exposition positively influences the kinetics of DNA repair in the first 15min of incubation. At longer exposition times, while the behaviour measured was not constant, a genotoxic effect was not observed. The data from this study provide additional information on the antioxidant and pro-oxidant activities of the predominant macular pigment carotenoids of the human retina.     

High frequency induction of CC to TT tandem mutations in DNA repair-proficient mammalian cells

The CC to TT tandem mutation is induced by UV radiation exposure, though at relatively low frequencies when compared with the more commonly induced C to T mutation. Induction of the tandem mutation by UV is enhanced in mammalian cells with certain genetic deficiencies; however, conditions have not been described in which the frequency of this mutation is enhanced in DNA repair-proficient mammalian cells. For this study, an integrated construct that detects C to T and CC to TT mutations at a single codon in mouse Aprt was used to examine UVB mutagenesis under various conditions. Oxidative stress, in the form of intracellular hydrogen peroxide, increased the frequency of UVB-induced CC to TT mutations. Surprisingly, exposure of the cells to two antioxidants (N-acetylcysteine and trolox), either alone or in combination, also enhanced UVB induction of CC to TT tandem mutations. These results demonstrate, for the first time, that the frequency of UVB-induced CC to TT tandem mutations can be enhanced dramatically in DNA repair-proficient mammalian cells, and suggest that the enhancing effect does not require direct damage to DNA.     

Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells

The ability of non-viral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common non-viral gene delivery system. Solid-phase synthesis was used to produce three libraries of polyamine-based cationic lipids with diverse hydrophobic tails. These were characterised, and structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. Two of the cationic lipids produced high-efficiency transfection of human cells. Surprisingly, these two compounds were from the library with two headgroups and one aliphatic tail, a compound class regarded as detergent-like and little investigated for transfection. These cationic lipids are promising reagents for gene delivery and illustrate the potential of solid-phase synthesis methods for lipoplex discovery.     

Chemistry and biology of DNA repair

Numerous agents of endogenous and exogenous origin damage DNA in our genome. There are several DNA-repair pathways that recognize lesions in DNA and remove them through a number of diverse reaction sequences. Defects in DNA-repair proteins are associated with several human hereditary syndromes, which show a marked predisposition to cancer. Although DNA repair is essential for a healthy cell, DNA-repair enzymes counteract the efficiency of a number of important antitumor agents that exert their cytotoxic effects by damaging DNA. DNA-repair enzymes are therefore also targets for drug design. DNA-repair processes differ greatly in their nature and complexity. Whereas some pathways only require a single enzyme to restore the original DNA sequence, others operate through the coordinated action of 30 or more proteins. Our understanding of the genetic, biochemical, and structural basis of DNA repair and related processes has increased dramatically over the past decade. This review summarizes the latest developments in this field.     

Photoimmunology, DNA repair and photocarcinogenesis

In recent years major progress has been made in identifying the molecular mechanisms by which UV radiation modulates the immune system of the skin. From these studies it appears that the generation of DNA damage and the subsequent activation of DNA repair enzymes play a critical role in the generation of UV-B-induced immunosuppression. These studies have made use of cells from both nucleotide excision repair (NER)-deficient individuals and mice. Results obtained from these studies have important clinical implications for DNA-repair-deficient patients in particular and for effective photoprotection of human skin in general.     

DNA polymerase I-mediated repair of 365 nm-induced single-strand breaks in the DNA of Escherichia coli.

Abstract. Irradiation of closed circular phage Λ DNA in vivo at 365 nm results in the induction of single-strand breaks and alkali-labile lesions at rates of 1.1 × 10^-14, and 0.2 × 10^-14/dalton/J/m², respectively. The sum of the induction rates is similar to the rate of induction of single-strand breaks plus alkali-labile lesions (1 × 10^-14/dalton/J/m²) observed in the E. coli genome. Postirradiation incubation of wild-type cells in buffer results in rapid repair of the breaks (up to 80% repaired in 10 min). No repair was observed in a DNA polymerase I-deficient mutant of E. coli.     

Probing the mechanism of transport and compartmentalisation of polyamines in mammalian cells.

BACKGROUND: Many mammalian cells possess an active polyamine uptake system but little is known about the molecular mechanism of this transporter. The fate of polyamines taken up from the medium and the relationship to polyamine homeostasis remains to be fully established. The aim of this study was to develop a range of modified polyamines, particularly ligands incorporating a fluorophore, to explore the structural tolerances of the polyamine transport system and to probe the intracellular location of polyamines acquired from the medium. RESULTS: We synthesised a wide range of polyamine analogues incorporating cytotoxic agents, fluorescent chromophores and bulky substituents. All of these analogues have been shown to be good competitive inhibitors of spermidine uptake in a range of mammalian cells. Direct evidence for uptake of the fluorescent polyamine analogues and their subcellular distribution was obtained from confocal laser scanning fluorescence microscopy, which showed that they accumulated in granular structures within the cytoplasm and not in the nucleus. We demonstrated that their uptake is through the polyamine transport system by showing that pretreatment with DFMO, a potent inhibitor of polyamine biosynthesis, led to enhanced uptake, and cells deficient in the polyamine transport system did not accumulate these polyamine analogues. CONCLUSIONS: The polyamine transport system has a surprisingly broad structural tolerance. Fluorophore-containing polyamine analogues derived from the extracellular pool are located in granular structures within the cytoplasm and not to any great extent in the nuclei of mammalian cells. These observations might be consistent with a mechanism involving receptor-mediated endocytosis, and the granular 'structures' seen might reflect polyamine compartmentalisation within vesicles.     

Fluorometric analysis of DNA unwinding (FADU) as a method for detecting repair-induced DNA strand breaks in UV-irradiated mammalian cells

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).     

New aspects of the repair and genotoxicity of psoralen photoinduced lesions in DNA.

Several approaches are described aiming at a better understanding of the genotoxicity of psoralen photoinduced lesions in DNA. Psoralens can photoinduce different types of photolesions including 3,4- and 4',5'-monoadducts and interstrand cross-links, oxidative damage (in the case of 3-carbethoxypsoralen (3-CPs)) and even pyrimidine dimers (in the case of 7-methylpyrido(3,4-c)psoralen (MePyPs)). The characterization and detection of different types of lesions has been essential for the analysis of their possible contributions to genotoxicity. For example, oxidative damage photoinduced by 3-CPs can be detected by the formamidopyrimidine glycosylase (FPG) protein. Furthermore, it is shown how the presence of MePyPs induced monoadducts may interfere with the photoreactivation of concomitantly induced pyrimidine dimers, how the ratio of monoadducts and interstrand cross-links (CL) affects the occurrence of double-strand breaks during the repair of photolesions and genotoxicity. In vitro treatment of yeast plasmids, followed by transformation, also indicates that the repair of photoadducts on exogenous DNA differs for 8-methoxy-psoralen (8-MOP) induced mono- and diadducts and for monoadducts alone. The recombinational rad52 dependent pathway is not needed for the repair of 8-MOP induced monoadducts. The results obtained suggest that the genotoxic effects of psoralens are conditioned by the nature, number, ratio and sequence distribution of the photolesions induced in DNA.