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.     

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.     

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.     

When DNA repair goes wrong: BER-generated DNA-protein crosslinks to oxidative lesions

Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3′-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase β (Polβ) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide (“long-patch”) BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polβ cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polβ and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polβ-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5′-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polβ-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15–30 min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polβ-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60–70% of the trapped ubiquitin depends on Polβ. The mechanism of ubiquitin targeting to Polβ-DPC, the subsequent processing of the expected 5′-peptidyl-dL, and the biological consequences of unrepaired DPC are important to assess. Many other lyase enzymes that attack dL can also be trapped in DPC, so the processing mechanisms may apply quite broadly.     

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.     

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral introduction of UV damage can potentially compromise genetic interpretations.     

The Repeat Expansion Diseases: The dark side of DNA repair

DNA repair normally protects the genome against mutations that threaten genome integrity and thus cell viability. However, growing evidence suggests that in the case of the Repeat Expansion Diseases, disorders that result from an increase in the size of a disease-specific microsatellite, the disease-causing mutation is actually the result of aberrant DNA repair. A variety of proteins from different DNA repair pathways have thus far been implicated in this process. This review will summarize recent findings from patients and from mouse models of these diseases that shed light on how these pathways may interact to cause repeat expansion.     

Interaction of PARP-2 with DNA structures mimicking DNA repair intermediates and consequences on activity of base excision repair proteins

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Among the 17 members of the PARP family, PARP-1 and PARP-2 are described as enzymes whose catalytic activity is stimulated by some types of DNA damages.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.     

A new perspective on oxidation of DNA repair proteins and cancer

Reactive oxygen and nitrogen species (RONS) are formed as byproducts of many endogenous cellular processes, in response to infections, and upon exposure to various environmental factors. An increase in RONS can saturate the antioxidation system and leads to oxidative stress. Consequently, macromolecules are targeted for oxidative modifications, including DNA and protein. The oxidation of DNA, which leads to base modification and formation of abasic sites along with single and double strand breaks, has been extensively investigated. Protein oxidation is often neglected and is only recently being recognized as an important regulatory mechanism of various DNA repair proteins. This is a review of the current state of research on the regulation of DNA repair by protein oxidation with emphasis on the correlation between inflammation and cancer.     

A comparison of two DNA base excision repair glycosylases from Arabidopsis thaliana

Plants contain the genes for both formamidopyrimidine-DNA glycosylase (FPG) and oxoguanine glycosylase (OGG). These enzymes play analogous roles in the base excision repair pathways of bacteria (FPG) and archaea, yeast, and mammals (OGG). Why have plants retained both genes? We tested one hypothesis by comparing the specificities of Arabidopsis FPG and OGG purified from Escherichia coli expression clones. Using depurinated DNA as substrate, the specific activity of Arabidopsis FPG was higher than that of Arabidopsis OGG. Using DNA oxidized by treatment with light in the presence of methylene blue, the specific activities of Arabidopsis FPG and OGG were equal. Using an oligonucleotide containing one oxoguanine (paired with C) and labeled with fluorescein, the specific activity of Arabidopsis OGG was greater than that of either FPG. The results support the hypothesis that genes for the two enzymes have been retained during evolution of plants for their specialized enzyme activities.     

Comet assay-based methods for measuring DNA repair <Emphasis Type="Italic">in vitro</Emphasis>; estimates of inter- and intra-individual variation

DNA repair is one of the important determinants of susceptibility to cancer. It is therefore useful to be able to measure DNA repair capacity in samples from population studies. Our aim was, first, to develop a simple comet-based in vitro assay for nucleotide excision repair (NER), similar to that already in use for base excision repair (BER), and then to apply these in vitro assays to lymphocyte samples collected on several occasions from healthy subjects, to gain an impression of the degree of intra- and inter-individual variability. The in vitro assay consists of an incubation of lymphocyte extract with substrate nucleoid DNA from cells pretreated with specific damaging agent; either photosensitiser plus light to induce 8-oxoguanine, for BER, or short wavelength ultraviolet light irradiation for NER. In the new NER assay, which requires magnesium but not adenosine triphosphate, there was significant accumulation of UV-dependent incisions during a 30-min incubation of extract with DNA. We found significant correlations between individual repair rates from samples taken on different occasions; i.e. individuals have a characteristic repair capacity. There was also significant variation between individuals, to the extent of about fourfold for BER and tenfold for NER. There was no correlation between BER and NER rates. The BER and NER assays are simple to perform and can provide valuable information in molecular epidemiological studies in which DNA instability is an endpoint.     

DNA repair profiles of disease-associated isolates of Neisseria meningitidis

Neisseria meningitidis, or the meningococcus, is the source of significant morbidity and mortality in humans worldwide. Even though mutability has been linked to the occurrence of outbreaks of epidemic disease, meningococcal DNA repair pathways are poorly delineated. For the first time, a collection of meningococcal disease-associated isolates has been demonstrated to express constitutively the DNA glycosylases MutY and Fpg in vivo. DNA sequence analysis showed considerable variability in the deduced amino acid sequences of MutS and Fpg, while MutY and RecA were highly conserved. Interestingly, multi-locus sequence typing demonstrated a putative link between the pattern of amino acid substitutions and levels of spontaneous mutagenicity in meningococcal strains. These results provide a basis for further studies aimed at resolving the genotype/phenotype relationships of meningococcal genome variability and mutator activity.     

DNA 去甲基化机制的研究进展*

DNA甲基化作为动植物体内一种重要的表观遗传修饰形式,在调控基因表达、维持基因组的稳定性等方面发挥重要的生物学作用。固有DNA甲基化水平和模式的变化会导致生物的表型异常甚至死亡。而5-甲基胞嘧啶的水平和模式是由DNA甲基化和去甲基化共同决定的。DNA去甲基化可以分为主动去甲基化与被动去甲基化,而基因组甲基化模式的形成主要依赖于主动去甲基化。本文综述了生物体内DNA主动去甲基化五种潜在机制:DNA转葡糖基酶参与的碱基切除修复途径、脱氨酶参与的碱基切除修复途径、核苷酸切除修复途径、氧化作用去甲基化与水解作用去甲基化。     

Non-enzymatic fast repair of DNA oxidative damage might also exist in cells

Many studies have shown that in a chemical system natural polyphenols can rapidly repair DNA oxidative damage. In this paper we report that in a cellular system the non-enzymatic fast repair activities of two natural polyphenols might also exist. The viability of a Chinese hamster ovary cell line (AA8) highly expressing the XRCC1 gene (a DNA repairing protein) treated with H2O2 is significantly higher than that of a normal Chinese hamster ovary cell line (CHO). Following inhibition of the enzymatic repair system by different inhibitors--methoxyamine (MX), 3-aminobenzamide (3AB) or nicotinamide (NIC)--DNA oxidative damage by H2O2 increased 2-5-fold in both cell lines. However, when natural polyphenols--rosmarinic acid (RA) or verbascoside (VER)--were added, DNA oxidative damage was significantly reduced. This decrease of DNA oxidative damage by RA or VER is not due to their scavenging activity for reactive oxygen species (ROS) because cells suffered from heavy ROS throughout the whole experimental process. Therefore, the decrease of DNA damage might be due to their non-enzymatic fast repair mechanisms. Further investigation showed that H2O2 induced a drop in the mitochondrial membrane potential (MMP), and that RA and VER were able to attenuate the drop. Previous studies have shown that H2O2 initiates a chain of events in cells, involving mtDNA damage, a drop in MMP and loss of repair activity. These results, taken together with our present results, suggest that the non-enzymatic fast repair mechanism exists not only in chemical systems but also might exist in cells.     

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.     

Repair of endogenous DNA base lesions modulate lifespan in mice

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O⁶-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm^−/− mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity.