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Endogenous factors constitute a substantial source of damage to the genomic DNA. The type of damage includes a number of different base lesions and single- and double-strand breaks. Unrepaired DNA damage can give rise to mutations and may cause cell death. A number of studies have demonstrated an association between aging and the accumulation of DNA damage. This may be attributed to reduced DNA repair with age, although this is apparently not a general feature for all types of damage and repair mechanisms. Therefore, detailed studies that improve our knowledge of DNA repair systems as well as mutagenic and toxic effects of DNA lesions will help us to gain a better insight into the mechanisms of aging. The aim of this review is to provide a brief description of cytotoxic and mutagenic endogenous DNA lesions that are mainly repaired by base excision repair and single-strand break repair pathways and to discuss the potential role of DNA lesions and DNA repair dysfunction in the onset of human aging.

Elaborate repair pathways counteract the deleterious effects of DNA damage by mechanisms that are understood in reasonable detail. In contrast, repair of damaged RNA has not been widely explored. This may be because aberrant RNAs are generally assumed to be degraded rather than repaired. The reason for this view is well founded, since conserved surveillance mechanisms that degrade abnormal RNAs are thoroughly documented. Numerous proteins and protein-RNA complexes are involved in the metabolism of different RNA species, assuring correct transcription, splicing, posttranscriptional modifications, transport, translation and timely degradation of the molecule. However, like DNA, RNA is under constant attack of various environmental and endogenous agents that damage the molecule, such as alkylating agents, radiation and free radicals. Importantly, many DNA damaging drugs used in cancer therapy also modify RNA, presumably causing delayed or faulty translation. This may result in generation of inactive proteins, dominant negative proteins or toxic protein aggregates. Several lines of evidence indicate RNA repair as a possible cellular defence mechanism to cope with RNA damage. Thus, there are convincing examples of tRNA repair by elongation of truncated forms, and repair of cleaved tRNA by T4 phage proteins. In addition, in vitro repair of aberrant tRNA methylation by a methyl transferase has been reported. Finally, recent reports on repair of chemically methylated RNA by AlkB and a human homologue (hABH3) in vitro and in vivo strengthen the idea of RNA base repair as a cellular defence mechanism.

Deamination of cytosine in DNA results in mutagenic U:G mispairs, whereas incorporation of dUMP leads to U:A pairs that may be genotoxic directly or indirectly. In both cases, uracil is mainly removed by a uracil-DNA glycosylase (UDG) that initiates the base excision repair pathway. The major UDGs are mitochondrial UNG1 and nuclear UNG2 encoded by the UNG-gene, and nuclear SMUG1. TDG and MBD4 remove uracil from special sequence contexts, but their roles remain poorly understood. UNG2 is cell cycle regulated and has a major role in post-replicative removal of incorporated uracils. UNG2 and SMUG1 are both important for prevention of mutations caused by cytosine deamination, and their functions are non-redundant. In addition, SMUG1 has a major role in removal of hydroxymethyl uracil from oxidized thymines. Furthermore, UNG-proteins and SMUG1 may have important functions in removal of oxidized cytosines, e.g. isodialuric acid, alloxan and 5-hydroxyuracil after exposure to ionizing radiation. UNG2 is also essential in the acquired immune response, including somatic hypermutation (SHM) required for antibody affinity maturation and class switch recombination (CSR) mediating new effector functions, e.g. from IgM to IgG. Upon antigen exposure B-lymphocytes express activation induced cytosine deaminase that generates U:G mispairs at the Ig locus. These result in GC to AT transition mutations upon DNA replication and apparently other mutations as well. Some of these may result from the generation of abasic sites and translesion bypass synthesis across such sites. SMUG1 can not complement UNG2 deficiency, probably because it works very inefficiently on single-stranded DNA and is down-regulated in B cells. In humans, UNG-deficiency results in the hyper IgM syndrome characterized by recurrent infections, lymphoid hyperplasia, extremely low IgG, IgA and IgE and elevated IgM. Ung(-/-) mice have a similar phenotype, but in addition display dysregulated cytokine production and develop B cell lymphomas late in life.

Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 A resolution and analyse key site-directed mutants. The hABH3 structure reveals the beta-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His1-X-Asp/Glu-X(n)-His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.

Uracil may arise in DNA as a result of deamination of cytosine or through incorporation of dUMP instead of dTMP during replication. We have studied the steady-state levels of uracil in the DNA of primary cells and mouse embryonic fibroblast (MEF) cell lines from mice deficient in the Ung uracil-DNA glycosylase. The results show that the levels of uracil in the DNA of Ung(-/-) cells strongly depend on proliferation, indicating that the uracil residues originate predominantly from misincorporation during replication. Treatment with 5-fluoro-2'-deoxyuridine (5-FdUrd) or 5-fluorouracil (5-FU) gives rise to a dose-dependent increase of uracil in Ung(-/-) MEFs (up to 1.5-fold) but not in wild-type cells. Interestingly, Ung(-/-) MEFs accumulate AP-sites as well as uracil in response to 5-FdUrd but not to 5-FU. This accumulation of repair intermediates suggests a loss of tightly co-ordinated repair in the absence of Ung, and correlates with stronger inhibition of cell proliferation in response to 5-FdUrd, but not to 5-FU, in Ung(-/-) MEFs compared with wild-type cells. However, other cytotoxic effects of these fluoropyrimidines are comparable in both wild-type and Ung-deficient cells, demonstrating that excision of uracil from DNA by the Ung uracil-DNA glycosylase is not a prerequisite for obtaining cytotoxicity.

Alkylation lesions in DNA and RNA result from endogenous compounds, environmental agents and alkylating drugs. Simple methylating agents, e.g. methylnitrosourea, tobacco-specific nitrosamines and drugs like temozolomide or streptozotocin, form adducts at N- and O-atoms in DNA bases. These lesions are mainly repaired by direct base repair, base excision repair, and to some extent by nucleotide excision repair (NER). The identified carcinogenicity of O(6)-methylguanine (O(6)-meG) is largely caused by its miscoding properties. Mutations from this lesion are prevented by O(6)-alkylG-DNA alkyltransferase (MGMT or AGT) that repairs the base in one step. However, the genotoxicity and cytotoxicity of O(6)-meG is mainly due to recognition of O(6)-meG/T (or C) mispairs by the mismatch repair system (MMR) and induction of futile repair cycles, eventually resulting in cytotoxic double-strand breaks. Therefore, inactivation of the MMR system in an AGT-defective background causes resistance to the killing effects of O(6)-alkylating agents, but not to the mutagenic effect. Bifunctional alkylating agents, such as chlorambucil or carmustine (BCNU), are commonly used anti-cancer drugs. DNA lesions caused by these agents are complex and require complex repair mechanisms. Thus, primary chloroethyl adducts at O(6)-G are repaired by AGT, while the secondary highly cytotoxic interstrand cross-links (ICLs) require nucleotide excision repair factors (e.g. XPF-ERCC1) for incision and homologous recombination to complete repair. Recently, Escherichia coli protein AlkB and human homologues were shown to be oxidative demethylases that repair cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues. Numerous AlkB homologues are found in viruses, bacteria and eukaryotes, including eight human homologues (hABH1-8). These have distinct locations in subcellular compartments and their functions are only starting to become understood. Surprisingly, AlkB and hABH3 also repair RNA. An evaluation of the biological effects of environmental mutagens, as well as understanding the mechanism of action and resistance to alkylating drugs require a detailed understanding of DNA repair processes.

Cellular and humoral defence mechanisms are essential for the survival of individuals and species. Thus, DNA repair prevents mutations and cytotoxicity from DNA damage, thereby reducing the risks of inappropriate cell death, developmental defects, premature ageing and cancer. Similarly, antigen-dependent acquired immune responses prevent infections and also have a role in cancer prevention. DNA repair is highly complex and functions in an intricate network that also involves transcription, replication, cell cycle regulation, and the immune system. DNA damage is repaired by at least four major mechanisms, each requiring many different proteins. In addition there are "subpathways", and back-up mechanisms both within and between pathways. Various defects in DNA repair result in different forms of cancer, e.g. the rare syndrome Xeroderma pigmentosum and the more common diseases early-onset breast cancer and hereditary non-polyposis colon cancer. Surprisingly, recent research has revealed molecular interactions between the ancient DNA repair mechanisms and the much younger acquired immune system. Thus, the classical base excision enzyme uracil-DNA glycosylase encoded by the UNG gene is also involved in somatic hypermutation and class switch recombination, e.g. from IgM antibodies to IgG, yielding secreted high affinity antibodies. Mutations in both alleles of UNG result in a hyper-IgM syndrome with life-threatening infections. Furthermore, it has recently become clear that not only DNA, but also RNA and proteins are repaired. Thus, certain aberrant methylations in RNA are repaired by oxidative demethylation in one step restoring the normal base, and at least in a bacterial model system this increases survival several-fold after exposure to methylating agents. Proteins are repaired both at the peptide amino acid level and at the structural level. RNA and protein repair are likely to be important to prevent the formation of cytotoxic protein aggregates of the types known to cause neurodegenerative diseases e.g. Alzheimer's, Parkinson's and Huntington's diseases, and other diseases as well. In conclusion, recent research has demonstrated an unexpected complexity of cellular defence mechanisms that function in intricate networks, rather than as independent mechanisms. The new knowledge opens for interventions that are based on a deeper understanding of the mechanisms of defence.

Genomes are damaged by spontaneous decay, chemicals, radiation and replication errors. DNA damage may cause mutations resulting in inheritable disease, cancer and ageing. Oxidative stress from ionising radiation and oxidative metabolism causes base damage, as well as strand breaks in DNA. Base damage is mostly indirect and caused by reactive oxygen species (ROS) generated, e.g. O2(.-) (superoxide radical), OH. (hydroxyl radical) and H2O2 (hydrogen peroxide). ROS also oxidise RNA, lipids, proteins and nucleotides. The first line of defence against ROS is enzymatic inactivation of superoxide by superoxide dismutase and inactivation of the less toxic hydrogen peroxide by catalase. As a second line of defence, incorporation of damaged bases into DNA is prevented by enzymes that hydrolyse oxidised dNTPs (e.g. 8-oxodGTP) to the corresponding dNMP. The third line of defence is repair of oxidative damage in DNA by an intricate network of DNA repair mechanisms. Base excision repair (BER), transcription-coupled repair (TCR), global genome repair (GGR), mismatch repair (MMR), translesion synthesis (TLS), homologous recombination (HR) and non-homologous end-joining (NHEJ) all contribute to repair of oxidative DNA damage. These mechanisms are also integrated with other cellular processes such as cell cycle regulation, transcription and replication and even use some common proteins. BER is the major pathway for repair of oxidative base damage, with TCR and MMR being important backup pathways for repair of transcribed strands and newly replicated strands, respectively. In recent years, several new DNA glycosylases that initiate BER of oxidative damage have been identified. These have specificities overlapping with previously known DNA glycosylases and serve as backups, and may have distinct roles as well. Thus, there is both inter- and intra-pathway complementation in repair of oxidative base damage, explaining the limited effects of absence of single DNA glycosylases in animal model systems.

Repair of DNA damage is essential for maintaining genome integrity, and repair deficiencies in mammals are associated with cancer, neurological disease and developmental defects. Alkylation damage in DNA is repaired by at least three different mechanisms, including damage reversal by oxidative demethylation of 1-methyladenine and 3-methylcytosine by Escherichia coli AlkB. By contrast, little is known about consequences and cellular handling of alkylation damage to RNA. Here we show that two human AlkB homologues, hABH2 and hABH3, also are oxidative DNA demethylases and that AlkB and hABH3, but not hABH2, also repair RNA. Whereas AlkB and hABH3 prefer single-stranded nucleic acids, hABH2 acts more efficiently on double-stranded DNA. In addition, AlkB and hABH3 expressed in E. coli reactivate methylated RNA bacteriophage MS2 in vivo, illustrating the biological relevance of this repair activity and establishing RNA repair as a potentially important defence mechanism in living cells. The different catalytic properties and the different subnuclear localization patterns shown by the human homologues indicate that hABH2 and hABH3 have distinct roles in the cellular response to alkylation damage.

XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLβ and PNK colocalize with XRCC1 in replication foci and that POLβ and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLβ and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLβ to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] We analyzed error sources in genomic uracil assays. [•] We present a method that ameliorates possible technical shortcomings. [•] Nucleotides co-eluted in DNA isolation step, causing overestimation. [•] In vitro cytosine deamination to uracil caused overestimation. [•] We report 400–600 uracils per genome in repair-proficient cells. [ABSTRACT FROM AUTHOR]

Abstract: Cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions induced in DNA and RNA in vitro and in pre-damaged DNA and RNA bacteriophages in vivo are repaired by the Escherichia coli (E. coli) protein AlkB and a human homolog, ALKBH3. However, it is not known whether endogenous RNA is repaired in vivo by repair proteins present at physiological concentrations. The concept of RNA repair as a biologically relevant process has therefore remained elusive. Here, we demonstrate AlkB-mediated repair of endogenous RNA in vivo by measuring differences in lesion-accumulation in two independent AlkB-proficient and deficient E. coli strains during exposure to methyl methanesulfonate (MMS). Repair was observed both in AlkB-overproducing strains and in the wild-type strains after AlkB induction. RNA repair appeared to be highest in RNA species below 200 nucleotides in size, mainly comprising tRNAs. Strikingly, at least 10-fold more lesions were repaired in RNA than in DNA. This may be a consequence of some 30-fold higher levels of aberrant methylation in RNA than in DNA after exposure to MMS. A high primary kinetic isotope effect (>10) was measured using a deuterated methylated RNA substrate, D3-1me(rA), demonstrating that it is the catalytic step, and not the search step that is rate-limiting. Our results demonstrate that RNA repair by AlkB takes place in endogenous RNA as part of an adaptive response in wild-type E. coli cells. [Copyright &y& Elsevier]

Abstract: Aberrant methylations in DNA are repaired by base excision repair (BER) and direct repair by a methyltransferase or by an oxidative demethylase of the AlkB type. Yang et al. [Nature 452 (2008) 961–966] have now solved the crystal structure of AlkB and human AlkB homolog 2 (hABH2) in complex with DNA using an ingenious crosslinking strategy to stabilize the DNA–protein complex. AlkB proteins have similar catalytic domains, but different DNA recognition motifs. Whereas AlkB mainly makes contact with the damaged strand, hABH2 makes numerous contacts with both strands. hABH2 flips out the damaged base and fills the vacant space by a hydrophobic amino acid residue similar to DNA glycosylases, essentially without distorting the double helix structure. In contrast, AlkB squeezes together the bases flanking the flipped-out base to maintain the base stack. This unprecedented flipping mechanism and the differences between AlkB and hABH2 in contacting the DNA strands explain their preferences for single stranded- and double stranded DNA, respectively. [Copyright &y& Elsevier]