DNA excision repair at telomeres

DNA damage is caused by either endogenous cellular metabolic processes such as hydrolysis, oxidation, alkylation, and DNA base mismatches, or exogenous sources including ultraviolet (UV) light, ionizing radiation, and chemical agents. Damaged DNA that is not properly repaired can lead to genomic instability, driving tumorigenesis. To protect genomic stability, mammalian cells have evolved highly conserved DNA repair mechanisms to remove and repair DNA lesions. Telomeres are composed of long tandem TTAGGG repeats located at the ends of chromosomes. Maintenance of functional telomeres is critical for preventing genome instability. The telomeric sequence possesses unique features that predispose telomeres to a variety of DNA damage induced by environmental genotoxins. This review briefly describes the relevance of excision repair pathways in telomere maintenance, with the focus on base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). By summarizing current knowledge on excision repair of telomere damage and outlining many unanswered questions, it is our hope to stimulate further interest in a better understanding of excision repair processes at telomeres and in how these processes contribute to telomere maintenance.     

Influence of cellular differentiation on repair of ultraviolet-induced DNA damage in murine proadipocytes

The effects of cellular differentiation on the repair of DNA damage induced by uv radiation were investigated in the murine 3T3-T proadipocyte cell culture system. Upon exposure to human plasma, actively cycling 3T3-T cells (stem cells) undergo growth arrest, which is followed by terminal differentiation into lipid-laden adipocytes. In response to uv irradiation, the level of unscheduled DNA synthesis is significantly lower in adipocytes as compared to stem cells. The alkaline elution assay was used to monitor the appearance of repair-induced strand breaks in 3T3-T cells after uv irradiation. DNA strand breaks were detected in stem cells by 4 min post-uv with essentially no further increase after 8 min. When terminally differentiated adipocytes were irradiated and allowed to repair, however, more strand breaks were present at 4 min and, in marked contrast to stem cells, continued to accumulate in adipocytes for at least 16 min post-uv. Inhibition of repair-replication with hydroxyurea and cytosine arabinoside significantly increased accumulation of repair-induced strand breaks in stem cells, yet had little effect on this accumulation in adipocytes. For stem cells and adipocytes, incision activity was linear out to at least 10 Jm-2 without saturation. These data suggested that 3T3-T cell differentiation is accompanied by a defect in some postincision process of the excision-repair pathway.     

Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm

Semen cryopreservation is widely used in clinical medicine, agriculture, aquaculture and biomedical research, but it is an inefficient technique that induces extensive cytoplasmic damage and loss of fertilising ability. Whether any genetic damage (i.e. DNA strand breakage or mutation) is also induced is still unclear. However, previous data has indicated that this is likely. The present study was designed to explore this possibility further by using inhibitors of the DNA repair system to block DNA repair in embryos derived from cryopreserved spermatozoa. If cryopreservation causes strand breaks in sperm DNA it might be expected that inhibition of a repair enzyme such as poly(ADP-ribose) polymerase (PARP) would enhance any such negative effect of cryopreservation. To check this hypothesis 3-aminobenzamide (3-AB) was used as an inhibitor of PARP. Weather loach (Misgurnus fossilis) eggs were fertilised using cryopreserved as well as fresh spermatozoa. Embryos derived from cryopreserved spermatozoa were exposed to 10 mM 3-AB for 2 h after fertilisation. The experiments were carried out using 43,544 embryos from 5 females and 10 males. Embryo survival was evaluated at different stages until the hatching stage. Sperm cryopreservation significantly decreased embryo survival (53.6+/-2.79% compared to 76.97+/-2.79% of control; P<0.01). The addition of 3-AB to the medium with embryos derived from cryopreserved sperm further decreased embryo survival from 53.6+/-2.79% to 46.1+/-2.79% (P<0.01) whereas there was no adverse effect of 3-AB exposed embryos derived from fresh sperm (76.97+/-2.79% of control compared to 74.8+/-2.79% of control+3-AB). The effect of 3-AB provides indirect evidence that cryopreservation might induce instability in sperm DNA, and that such damage can be repaired by the oocyte repair system after fertilisation.     

Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans

Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.     

Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Syrian baby hamster kidney cells

The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective.     

Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Synrian baby hamster kidney cells

The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G₂ block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G₂ phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective.     

Base excision repair of DNA: glycosylases

The review considers the role of base excision repair in maintaining the constancy of genetic information in the cell. The genetic control and biochemical mechanism are described for the first stage of base excision repair, which is catalyzed by specific enzymes, DNA glycosylases.     

The time course of repair of ultraviolet-induced DNA damage; implications for the structural organisation of repair

Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the "random collision" model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a "processive" mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. These two models give different predictions concerning the time course of repair. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate of repair until scanning is complete. We have examined the time course of DNA repair in human fibroblasts given low (generally sublethal) doses of UV light. Using 3 distinct assays, we find no sign of a constant repair rate after 4 J/m2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. The implications of this finding for the structural organisation of repair are discussed.     

Effect of the uvrD mutation on excision repair.

A pair of related Escherichia coli K-12 strains, one of which contains the uvrD101 mutation, were constructed and compared for ability to perform various steps in the excision repair of deoxyribonucleic acid damage inflicted by ultraviolet radiation. The results of this study indicated: (i) ultraviolet sensitivity in the uvrD101 mutant was greater than that of wild type but less than that measured in an incision-deficient uvrA mutant; (ii) host cell reactivation paralleled the survival data; (iii) postirradiation deoxyribonucleic acid degradation was virtually identical in the two strains; (iv) incision, presumably at the sites of pyrimidine dimers, proceeded normally in the uvrD101 strain; (v) excision of pyrimidine dimers was markedly reduced in both rate and extent in the uvrD101 mutant; (vi) the amount of repair resynthesis was the same in both strains, and there was no evidence of abnormally long repair patches in the uvrD mutant; and (vii) rejoining of incision breaks was slow and incomplete in the uvrD strain. These data suggest that the ultraviolet sensitivity conferred by the uvrD mutation arises from inefficient removal of pyrimidine dimers or from failure to close incision breaks. The data are compatible with the notion that the uvrD+ gene produce affects the conformation of incised deoxyribonucleic acid molecules.     

Inhibition by metals of X-ray and ultraviolet-induced DNA repair in human cells

A number of metals have been shown to be involved in the etiology of animal and human neoplasms. The molecular mechanisms have not yet been determined, but the observed plethora of genetic effects observed following treatment of mammalian cells with metals clearly indicates the possibility that metals can exert their effects at least partially at the level of DNA metabolism. Several studies have suggested that metal treatment may inhibit normal DNA repair processes in procaryotic and eucaryotic cells but a systematic study of this question has not previously been conducted. The present study surveyed the ability of 15 metal salts to interfere with repair of X-ray or UV-induced DNA damage in HeLa cells. Hg⁺⁺, As⁺⁺⁺, Cu⁺⁺, Ni⁺⁺, Co⁺⁺, and Cd⁺⁺ were shown to inhibit the excision of pyrimidine dimers from DNA and to do so in a dose-dependent fashion. Inhibition of repair by only Ni⁺⁺ and Co⁺⁺ resulted in the accumulation of long-lived DNA strand breaks suggestive of a block in the gap-filling stage of repair. Ability to inhibit repair was not correlated with cytotoxicity. X-ray repair was sensitive to Hg⁺⁺, Ni⁺⁺, As⁺⁺⁺, Ga⁺⁺, Zn⁺⁺, and Mo(VI). All inhibitory metals inhibited closure of single strand DNA breaks. Ga⁺⁺ appeared, in addition, to inhibit a later step involving chromatin reconstitution. These findings support the notion that interference of DNA repair processes may be a consequence of exposure of mammalian cells to certain metals. This may be a factor in the etiology of metal-associated carcinogenesis.     

DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridization analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed in non-proliferating tissue such as the mature leaves and elongation zone of root. The removal of DNA damage, cyclobutane pyrimidine dimers and (6–4) photoproducts, in both mature leaves and the root apical meristem (RAM) was observed after UV irradiation under light. In the dark, DNA damage in mature leaves was not repaired efficiently, but that in the RAM was removed rapidly. Using a rice 22K custom oligo DNA microarray, we compared global gene expression patterns in the shoot apical meristem (SAM) and mature leaves. Most of the excision repair genes were more strongly expressed in SAM. These results suggested that photoreactivation is the major DNA repair pathway for the major UV-induced damage in non-proliferating cells, while both photoreactivation and excision repair are active in proliferating cells.     

Envisioning the molecular choreography of DNA base excision repair

Recent breakthroughs integrate individual DNA repair enzyme structures, biochemistry and biology to outline the structural cell biology of the DNA base excision repair pathways that are essential to genome integrity. Thus, we are starting to envision how the actions, movements, steps, partners and timing of DNA repair enzymes, which together define their molecular choreography, are elegantly controlled by both the nature of the DNA damage and the structural chemistry of the participating enzymes and the DNA double helix.     

Damage recognition in nucleotide excision DNA repair

Nucleotide excision repair (NER) is a highly versatile DNA repair process. Its ability to repair a large number of different damages with the same subset of recognition factors requires structural tools for damage recognition that are both broad and very accurate. Over the past few years detailed structural information on damage recognition factors from eukaryotic and prokaryotic NER has emerged. These structures shed light on the toolkit utilized in the damage recognition process and help explain the broad substrate specificity of NER.     

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

Using permeable diploid human fibroblasts, we have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent Km values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 microM. For UV-induced DNA repair synthesis, the apparent Km values were substantially lower, ranging from 0.11 to 0.44 microM for AG1518 cells and from 0.06 to 0.24 microM for IMR-90 cells. Control experiments established that these values were not significantly influenced by nucleotide degradation during the permeable cell incubations or by the presence of residual endogenous nucleotides within the permeable cells. Recent data implicate DNA polymerase delta in UV-induced repair synthesis and suggest that DNA polymerases alpha and delta are both involved in semiconservative replication. We measured Km values for dGTP and dTTP for polymerases alpha and delta, for comparison with the values for replication and repair synthesis. Km values for polymerase alpha were 2.0 microM for dGTP and 5.0 microM for dTTP. For polymerase delta, the Km values were 2.0 microM for dGTP and 3.5 microM for dTTP. The deoxyribonucleotide Km values for DNA polymerase delta are much greater than the Km values for UV-induced repair synthesis, suggesting that when polymerase delta functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase.

An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans. Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol. Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol. The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins. These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein.     

Damage recognition in nucleotide excision repair of DNA

Nucleotide excision repair (NER) is found throughout nature, in eubacteria, eukaryotes and archaea. In human cells it is the main pathway for the removal of damage caused by UV light, but it also acts on a wide variety of other bulky helix-distorting lesions caused by chemical mutagens. An ongoing challenge is to understand how a site of DNA damage is located during NER and distinguished from non-damaged sites. This article reviews information on damage recognition in mammalian cells and the bacterium Escherichia coli. In mammalian cells the XPC-hHR23B, XPA, RPA and TFIIH factors may all have a role in damage recognition. XPC-hHR23B has the strongest affinity for damaged DNA in some assays, as does the similar budding yeast complex Rad4-Rad23. There is current discussion as to whether XPC or XPA acts first in the repair process to recognise damage or distortions. TFIIH may play a role in distinguishing the damaged strand from the non-damaged one, if translocation along a DNA strand by the TFIIH DNA helicases is interrupted by encountering a lesion. The recognition and incision steps of human NER use 15 to 18 polypeptides, whereas E. coli requires only three proteins to obtain a similar result. Despite this, many remarkable similarities in the NER mechanism have emerged between eukaryotes and bacteria. These include use of a distortion-recognition factor, a strand separating helicase to create an open preincision complex, participation of structure-specific endonucleases and the lack of a need for certain factors when a region containing damage is already sufficiently distorted.