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N(1)-methyladenine (m(1)A) and N(3)-methylcytosine (m(3)C) are major toxic and mutagenic lesions induced by alkylation in single-stranded DNA. In bacteria and mammals, m(1)A and m(3)C were recently shown to be repaired by AlkB-mediated oxidative demethylation, a direct DNA damage reversal mechanism. No AlkB gene homologues have been identified in Archaea. We report that m(1)A and m(3)C are repaired by the AfAlkA base excision repair glycosylase of Archaeoglobus fulgidus, suggesting a different repair mechanism for these lesions in the third domain of life. In addition, AfAlkA was found to effect a robust excision of 1,N(6)-ethenoadenine. We present a high-resolution crystal structure of AfAlkA, which, together with the characterization of several site-directed mutants, forms a molecular rationalization for the newly discovered base excision activity.

It was established several decades ago that it is crucial for all organisms to repair their DNA to maintain genome integrity and numerous proteins are dedicated to this purpose. However, it is becoming increasingly clear that it is also important to prevent and repair lesions in the macromolecules encoded by the DNA, i.e. RNA and protein. Many neurological disorders such as Alzheimer's disease and Parkinson's disease are associated with the aggregation of defective, misfolded proteins, and several mechanisms exist to prevent such aggregation, both through direct protein repair and through the elimination and repair of faulty or damaged RNAs. A few years ago, it was discovered that the E. coli AlkB protein represented an iron and 2-oxoglutarate dependent oxygenase capable of repairing methyl lesions in DNA by a novel mechanism, termed oxidative demethylation. Furthermore, it was found that both human and bacterial AlkB proteins were able to demethylate lesions also in RNA, thus representing the first example of RNA repair. In the present review, recent findings on the AlkB mechanism, as well as on RNA damage in general, will be discussed.

Nat. Chem. Biol. 10, 487–488 (2014); published online 17 June 2014; corrected after print 30 June 2014 In the version of this article initially published, the surname of the first author in reference 2 was misspelled as Pfaffender. The correct spelling is Pfaffeneder. The error has been corrected inthe HTML and PDF versions of the article.

Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1-methyladenine (1meA) and 3-methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene-targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2-deficient mice are unable to remove methyl methane sulfate (MMS)-induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double-stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.

RNA interference (RNAi) has become an invaluable tool for functional genomics. A critical use of this tool depends on an understanding of the factors that determine the specificity and activity of the active agent, small interfering RNA (siRNA). Several studies have concluded that tolerance of mutations can be considerable and hence lead to off-target effects. In this study, we have investigated in vivo the toleration of wobble (G:U) mutations in high activity siRNAs against Flap Endonuclease 1 (Fen1) and Aquaporin-4 (Aqp4). Mutations in the central part of the antisense strand caused a pronounced decrease in activity, while mutations in the 5′ and 3′ends were tolerated very well. Furthermore, based on analysis of nine different mutated siRNAs with widely differing intrinsic activities, we conclude that siRNA activity can be significantly enhanced by wobble mutations (relative to mRNA), in the 5′ terminal of the antisense strand. These findings should facilitate design of active siRNAs where the target mRNA offers limited choice of siRNA positions. © The Author 2005. Published by Oxford University Press. All rights reserved The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org

In order to eliminate the possibility that diet may influence urinary oxidative DNA lesion levels, in our experiments we used a recently developed technique involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection. This methodology was applied for the determination of the lesions: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 5-(hydroxymethyl)uracil (5HMUra) in the urine of mice fed with nucleic acid free diet and normal, unrestricted diet. The mean levels of 8-oxoGua, 8-oxodGuo and 5HMUra of the animals fed the normal diet reached the mean values of 15.6 +/- 3.5, 2.0 +/- 0.53 and 16.8 +/- 10.4 nmol/kg/24 h, After feeding the mice for 12 days with nucleic acid free diet the respective values were 18.8 +/- 4.6, 1.6 +/- 0.3 and 25.4 +/- 10.5 nmol/kg/24 h, respectively. The results clearly demonstrate that irrespective of the diet, the excretion rates were not statistically different during the course of feeding. The respective p values for the differences between lesions in the two types of diets were: 0.13 (8-oxoGua), 0.16 (8-oxodGuo), 0.18 (5-HMUra). Our results clearly indicate that diet does not contribute to urinary excretion of the lesions in mouse model.

Genome integrity is maintained via removal (repair) of DNA lesions and an increased load of such DNA damage has been linked to numerous pathological conditions, including carcinogenesis and ageing. 8-Oxo-7,8-dihydroguanine is one of the most critical lesions of this type. The free 8-oxo-7,8-dihydroguanine produced by the action of a specific DNA glycosylase is a potential source of this compound in urine. To date, there has been no direct, experimental evidence demonstrating that urinary 8-oxo-7,8-dihydroguanine is produced by the base excision repair pathway. For clarification of this issue, we applied a recently developed methodology which involved high performance liquid chromatography pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection to compare the urinary excretion rate of 8-oxo-7,8-dihydroguanine in wild type and OGG1 glycosylase knock out mice. Our study revealed a 26% reduction in urinary level of 8-oxo-7,8-dihydroguanine in OGG1 deficient mice in comparison with the wild type strain. This clearly indicates that the mouse OGG1 glycosylase contributes significantly to the generation of urinary 8-oxo-7,8-dihydroguanine. Therefore, urinary measurements of 8-oxo-7,8-dihydroguanine may be attributed to DNA damage and repair, which in turn suggests that they may be useful in studying associations between DNA repair and disease.

Using recently developed methodology, which includes HPLC prepurification followed by GC/MS with isotope dilution, we analyzed urinary excretion of possible repair products of oxidative DNA damage—8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), and 5-(hydroxymethyl)uracil (5—HMUra)-in mammalian species that substantially differ in metabolic rate and longevity, namely, mice, rats, rabbits, dogs, pigs, and humans. We found highly significant, positive correlations between specific metabolic rates of the animals studied and their excretion rates for all the modifications analyzed with respective r values for the lesions of (8-oxoGua) r = .891, p < .01; (8-oxodG) r = .998, p < .001; and (5-HMUra) r = .949, p < .005. However, only 8-oxoGua significantly correlates negatively with maximum life span (MLSP) (r = −.928, p < .01). Despite substantial differences in MLSP between humans and pigs (120 and 27 years, respectively), the rates of excretion of all measured modifications were very similar. The urinary levels of all measured modifications found in our study for mouse and humans account respectively for about 34,000 and 2800 repaired events per average cell, per 24 h. It is therefore possible that the high metabolic rate in mice (or other short-lived animals) may be responsible for severe everyday oxidative DNA insults that may be accumulated faster than in long-lived species.

We have investigated repair of DNA containing 8-oxoguanine and certain mismatches in cell-free extracts from mouse embryonic fibroblasts (MEFs) using a plasmid substrate with a single lesion at a defined position. Repair synthesis was monitored in a small restriction fragment with different size single strands in order to follow the fate of repair reactions in both strands at the same time. An important part of the study was to assess the role of OGG1 in various repair reactions and the experiments were carried out with extracts from mouse embryonic fibroblasts diploid for a mogg1 deletion (Ogg1(-/-)) as well as wild type. In wild type, DNA containing 8-oxoG:C was repaired in the expected fashion predominantly through short-patch repair. Overall repair was reduced to 20% in the Ogg1(-/-) extracts and to 40% if only long-patch repair was considered. The 8-oxoG:A pair was processed similarly in wild type and Ogg1(-/-) extracts and repair synthesis at A as well as at 8-oxoG could be demonstrated, however, to the same extent in Ogg1(-/-) and wild type for both strands. Extracts from Ogg1(-/-) behaved normally in the correction of A:C and C:C mismatches, with a strong bias for correction of A for A:C and no significant strand discrimination for C:C. Similar experiments with extracts from Escherichia coli showed a 50% reduction in the repair of 8-oxoG:C in fpg extracts and an increase to 50% above wild type in mutY. These results show that the mouse OGG1 is the major enzyme for 8-oxoG repair in the MEF cells and does not participate in mismatch repair of A:C or C:C. Furthermore, 8-oxoG opposite A appears to be repaired by a two-step repair pathway with sequential removal of A and 8-oxoG mediated by enzymes different from OGG1.

Faithful maintenance of the genomic information is crucial for the survival of a species. Consequently, DNA repair processes must have evolved early during evolution. DNA damage left unrepaired might cause mutations leading to cell death, increased cancer incidence and severe syndromes.In 1968, for the first time a link was found between a human syndrome, xeroderma pigmentosum, and a defect in the machinery for DNA repair. These patients develop skin cancer at an early age if not completely protected against sunlight. More recently, several other DNA repair syndromes with cancer predisposition and premature aging have been identified.A number of DNA repair genes causing such defects have now been cloned and characterised. These genes represent different DNA repair pathways and some of them are involved in the coupling between DNA repair and DNA transcription.It is now possible to produce mice models with defects identical to those identified in humans. During the last 5 years, more than 100 mice models with DNA repair deficiency have been produced. Further characterisation of such mice will provide a unique opportunity for understanding the clinical picture caused by altered DNA repair capacity, and also elucidate the complex interaction of different DNA repair genes.

Hydrolytic deamination of DNA-cytosines into uracils is a major source of spontaneously induced mutations, and at elevated temperatures the rate of cytosine deamination is increased. Uracil lesions are repaired by the base excision repair pathway, which is initiated by a specific uracil DNA glycosylase enzyme (UDG). The hyperthermophilic archaeon Archaeoglobus fulgidus contains a recently characterized novel type of UDG (Afung), and in this paper we describe the over-expression of the afung gene and characterization of the encoded protein. Fluorescence and activity measurements following incubation at different temperatures may suggest the following model describing structure-activity relationships: At temperatures from 20 to 50 degrees C Afung exists as a compact protein exhibiting low enzyme activity, whereas at temperatures above 50 degrees C, the Afung conformation opens up, which is associated with the acquisition of high enzyme activity. The enzyme exhibits opposite base-dependent excision of uracil in the following order: U>U:T>U:C>>U:G>>U:A. Afung is product-inhibited by uracil and shows a pronounced inhibition by p-hydroxymercuribenzoate, indicating a cysteine residue essential for enzyme function. The Afung protein was estimated to be present in A. fulgidus at a concentration of approximately 1000 molecules per cell. Kinetic parameters determined for Afung suggest a significantly lower level of enzymatic uracil release in A. fulgidus as compared to the mesophilic Escherichia coli.

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G·C→T·A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1 −/− null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells . These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.

Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.

There are one million molecules of poly(ADP-ribose) polymerase (PARP) in mammalian cell nuclei and the enzyme is found in most eukaryotes, with the notable exception of yeasts. In response to DNA damage caused by ionizing radiation or alkylating agents, PARP binds to strand interruptions in DNA and undergoes rapid automodification with synthesis of long branched polymers of highly negatively charged poly(ADP-ribose). DNA repair occurs after dissociation of modified PARP from DNA strand breaks. Biochemical data with enzyme-depleted extracts and studies of enzyme-deficient mice show that PARP does not participate directly in DNA repair. Possible roles for poly(ADP-ribose) synthesis are discussed.

This work describes the isolation and characterization of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants in normal and Escherichia coli tag gene expressing Chinese hamster fibroblast, RJKO, cells. It was previously shown that increased removal of 3-alkylated adenine, effected by 3-methyladenine DNA glycosylase I (Tag), reduces the frequencies of hprt mutations induced by alkylating agents which produce mostly N-alkylation (MMS and EMS) to half the normal rate. In order to identify which type of mutation is suppressed by increased 3-alkyladenine repair we have determined the DNA base sequence changes of the hprt cDNA in 61 independent MMS- and EMS-induced mutant clones. For both cell types and irrespective of the agent used, the majority of mutations were GC to AT transitions originating in the non-transcribed strand. Only 6/55 base substitutions occurred at AT base pairs: five AT to GC transitions and one AT to CG transversion. Six mutations were found to be deletions. These results indicate that 3-alkylated adenines in DNA are not directly premutagenic. The fact that the mutation frequency is reduced by increased 3-alkyladenine removal might be explained by postulating the existence in mammalian cells of an SOS-like response turned on by cytotoxic lesions like 3-alkyladenine, or, alternatively, that increased removal of 3-alkyladenine increases the number of single-strand breaks in DNA, which stalls DNA replication and allows a prolonged time for DNA repair by the alkyltransferase.

Transcription does not always stall at base damage in DNA and can create mutated transcripts from miscoding lesions. In this issue of Molecular Cell, present genetic analysis of E. coli to indicate that the highly mutagenic purine modification, 8-oxoguanine, is subject to transcription-coupled repair despite transcriptional bypass and generation of mutant transcripts.

Recently, 5-hydroxymethylcytosine (5hmC) was identified in mammalian genomic DNA. The biological role of this modification remains unclear; however, identifying the genomic location of this modified base will assist in elucidating its function. We describe a method for the rapid and inexpensive identification of genomic regions containing 5hmC. This method involves the selective glucosylation of 5hmC residues by the b-glucosyltransferase from T4 bacteriophage creating b-glucosyl-5-hydroxymethylcytosine (b-glu-5hmC). The b-glu-5hmC modification provides a target that can be efficiently and selectively pulled down by J-binding protein 1 coupled to magnetic beads. DNA that is precipitated is suitable for analysis by quantitative PCR, microarray or sequencing. Furthermore, we demonstrate that the J-binding protein 1 pull down assay identifies 5hmC at the promoters of developmentally regulated genes in human embryonic stem cells. The method described here will allow for a greater understanding of the temporal and spatial effects that 5hmC may have on epigenetic regulation at the single gene level. The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

First lineage specification in the mammalian embryo leads to formation of the inner cell mass (ICM) and trophectoderm (TE), which respectively give rise to embryonic and extraembryonic tissues. We show here that this first differentiation event is accompanied by asymmetric distribution of trimethylated histone H3 lysine 27 (H3K27me3) on promoters of signaling and developmentally-regulated genes in the mouse ICM and TE. A genome-wide survey of promoter occupancy by H3K4me3 and H3K27me3 indicates that both compartments harbor promoters enriched in either modification, and promoters co-enriched in trimethylated H3K4 and H3K27 linked to developmental and signaling functions. The majority of H3K4/K27me3 co-enriched promoters are distinct between the two lineages, primarily due to differences in the distribution of H3K27me3. Derivation of embryonic stem cells leads to significant losses and gains of H3K4/K27me3 co-enriched promoters relative to the ICM, with distinct contributions of (de)methylation events on K4 and K27. Our results show histone trimethylation asymmetry on promoters in the first two developmental lineages, and highlight an epigenetic skewing associated with embryonic stem cell derivation.

In order to investigate the importance of 3-methyladenine in cellular sensitivity to chemical methylating agents we have constructed retroviral vectors for the integration and expression of the Escherichia coli tag gene in mammalian cells. The tag gene encodes 3-methyladenine DNA glycosylase-1 which specifically removes 3-alkyladenines from DNA. The constructs were introduced into Chinese hamster V79 cells by liposome mediated transfection or into murine haemopoietic stem cells by cocultivation with a lipofected, virus-packaging cell line. In both cases, stable transfectants were selected for resistance to the antibiotic, G418, conferred by expression of the neo gene carried by the vector. Measurements of 3-methyladenine DNA glycosylase activity in cell extracts showed an up to 10-fold increase in cell lines with stably integrated tag gene sequences. These cell lines were significantly more resistant to the cytotoxic effects of methylmethanesulfonate and N-methyl-N-nitrosourea than their parent cell lines, indicating that 3-methyladenine repair is a limiting factor in cellular resistance to these methylating agents. Furthermore, the mutation frequency induced by methylmethanesulfonate was reduced to 50% of normal by expression of 3-methyladenine I activity in the Chinese hamster cells, indicating that m3A is not only a cytotoxic but also a premutagenic lesion in mammalian cells. It is concluded that an alkylation repair gene function of a type only thought to be present in bacteria can yield a hyperresistant phenotype when transferred to mammalian cells.

5-Hydroxymethylcytosine (5hmC) was recently described as a stable modification in mammalian DNA. 5hmC is formed by the enzymatic oxidation of 5-methylcytosine (5meC). Overwhelming evidence supports the notion that 5meC has a negative effect on transcription; however, only recently has the effect that 5hmC has on transcription begun to be studied. Using model substrates including the CMVIE promoter and a generic gene body we have directly assessed the effect that 5hmC, both at the promoter and in the gene body, has on in vitro gene transcription. We show that the presence of the 5hmC modifications strongly represses transcription. We also demonstrate that the inhibition of transcriptional activity is primarily due to the presence of 5hmC in the promoter and that 5hmC in the gene body has a minimal effect on transcription. Thus, we propose that the presence of 5hmC in promoter prevents the binding of essential transcription factors or recruits factors that repress transcription.

Cell nuclei contain several abundant enzymes that bind rapidly and avidly to exposed termini of DNA. The properties and physiological roles of such factors are described; they include poly (ADP-ribose) polymerase, DNA-dependent protein kinase, several DNA ligases and excision-repair enzymes. Telomeres normally seem shielded from these activities by telomere-binding proteins. If incomplete protection of telomeres occurred, the functions of the DNA end-specific enzymes would be relevant for processing of telomeres. This could include alternative pathways for telomere propagation in telomerase-negative cells.