DNA repair in mammalian cells: Base excision repair: the long and short of it.

Base excision repair (BER) is the primary DNA repair pathway that corrects base lesions that arise due to oxidative, alkylation, deamination, and depurinatiation/depyrimidination damage. BER facilitates the repair of damaged DNA via two general pathways - short-patch and long-patch. The shortpatch BER pathway leads to a repair tract of a single nucleotide. Alternatively, the long-patch BER pathway produces a repair tract of at least two nucleotides. The BER pathway is initiated by one of many DNA glycosylases, which recognize and catalyze the removal of damaged bases. The completion of the BER pathway is accomplished by the coordinated action of at least three additional enzymes. These downstream enzymes carry out strand incision, gap-filling and ligation. The high degree of BER conservation between E. coli and mammals has lead to advances in our understanding of mammalian BER. This review will provide a general overview of the mammalian BER pathway. (Part of a Multi-author Review).

Deficiency of the Cockayne syndrome B (CSB) gene aggravates the genomic instability caused by endogenous oxidative DNA base damage in mice.

The Cockayne syndrome B protein (CSB) has long been known to be involved in the repair of DNA modifications that block the RNA polymerase in transcribed DNA sequences (transcription-coupled repair). Recent evidence suggests that it also has a more general role in the repair of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG). In mammalian cells, 8-oxoG is a substrate of the repair glycosylase OGG1. Mice without this enzyme accumulate 8-oxoG in the genome and have elevated spontaneous mutation rates. To elucidate the role of CSB in the prevention of mutations by oxidative DNA base damage, we have generated mice that are deficient in Csb or Ogg1 or both genes and carry a non-transcribed bacterial lacI gene for mutation analysis (Big Blue mice). Our results indicate that the overall spontaneous mutation frequencies in the livers of Csb(m/m)/Ogg1-/- -mice are elevated not only compared with heterozygous control mice (factor 3.3), but also with Ogg1-/- -animals (factor 1.6). Sequence analysis revealed that the additional mutations caused by CSB deficiency in an Ogg1-/- background are mostly G:C to T:A transversions and small deletions. For all mouse strains, the background levels of oxidative purine modifications in the livers correlate linearly with the numbers of G:C to T:A transversions observed. The data indicate that CSB is involved in the inhibition of mutations caused by spontaneous oxidative DNA base damage in a non-transcribed gene.

Genome instability and DNA damage accumulation in gene-targeted mice.

Six major pathways for DNA repair have been identified. These include (1) DNA repair by direct reversal, (2) base excision repair, (3) mismatch repair, (4) nucleotide excision repair, (5) homologous recombination, and (6) non-homologous end-joining. In addition, several other cellular processes influence the response to DNA damage. The generation of gene-targeted organisms is crucial for assessing the relative contribution of single DNA repair proteins and DNA repair pathways in maintaining genome stability. In particular, the accumulation of DNA damage, mutations and cancer in unexposed gene-targeted animals illuminates the spontaneous load of a particular lesion and the relative significance of a single gene in a specific pathway. Strategies for the generation of gene-targeted mice have been available for 15 years and more than 100 different genes relevant to DNA repair have been targeted. This review describes some important progress made toward understanding spontaneous DNA damage and its repair, exemplified through one, or a few, gene-targeted mice from each major DNA repair pathway.

A novel splice site mutation in the Cockayne syndrome group A gene in two siblings with Cockayne syndrome.

Cockayne syndrome (CS) is mainly caused by mutations in the Cockayne syndrome group A or B (CSA or CSB) genes which are required for a sub-pathway of nucleotide excision repair entitled transcription coupled repair. Approximately 20% of the CS patients have mutations in CSA, which encodes a 44 kDa tryptophane (Trp, W) and aspartic acid (Asp, D) amino acids (WD) repeat protein. Up to now, nine different CSA mutations have been identified. We examined two Somali siblings 9 and 12 years old with clinical features typical of CS including skin photosensitivity, progressive ataxia, spasticity, hearing loss, central and peripheral demyelination and intracranial calcifications. Molecular analysis showed a novel splice acceptor site mutation, a G to A transition in the -1 position of intervening sequence 6 (g.IVS6-1G>A), in the CSA (excision repair cross-complementing 8 (ERCC8)) gene. IVS6-1G>A results in a new 28 amino acid C-terminus and premature termination of the CSA protein (G184DFs28X). A review of the CSA protein and the 10 known CSA mutations is also presented.

Repair of methyl lesions in DNA and RNA by oxidative demethylation.

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.

Repair and mutagenesis at oxidized DNA lesions in the developing brain of wild-type and Ogg1-/- mice.

OGG1 (8-oxoguanine DNA glycosylase-1) is one of the main DNA glycosylases present in mammalian cells. The enzyme removes 7,8-dihydro-8-oxoguanine (8-oxoG) lesions, believed to be the most important oxidized lesions due to their relatively high incidence and their miscoding properties. This study shows that in prenatal mice brains the repair capacity for 8-oxoG is 5-10-fold higher than in adult mice brains. Western blot analysis and repair activity in extracts from Ogg1(-/-) mice revealed that OGG1 was responsible for the efficient 8-oxoG removal from prenatal mice. To investigate how OGG1 protects against oxidative stress-induced mutagenesis, pregnant Big Blue/wild-type and Big Blue/Ogg1(-/-) mice were exposed to nontoxic doses of gamma radiation. A 2.5-fold increase in the mutation frequency in Ogg1(-/-) mouse brains was obtained by exposure to 3.5 Gy at day 19 postfertilization. This was largely due to GC to TA transversions, believed to originate from 8-oxoG mispairing with A during replication. Furthermore, rapid cell divisions seemed to be required for fixation of mutations, as a similar dose of radiation did not increase the mutation frequency, or the frequency of GC to TA transversion, in the adult brain.

Excision of uracil from DNA by the hyperthermophilic Afung protein is dependent on the opposite base and stimulated by heat-induced transition to a more open structure.

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.

Repair of 8-oxodeoxyguanosine lesions in mitochondrial dna depends on the oxoguanine dna glycosylase (OGG1) gene and 8-oxoguanine accumulates in the mitochondrial dna of OGG1-defective mice.

Mitochondria are not only the major site for generation of reactive oxygen species, but also one of the main targets of oxidative damage. One of the major products of DNA oxidation, 8-oxodeoxyguanosine (8-oxodG), accumulates in mitochondrial DNA (mtDNA) at levels three times higher than in nuclear DNA. The main pathway for the repair of 8-oxodG is the base excision repair pathway initiated by oxoguanine DNA glycosylase (OGG1). We previously demonstrated that mammalian mitochondria from mice efficiently remove 8-oxodG from their genomes and isolated a protein from rat liver mitochondria with 8-oxoguanine (8-oxodG) DNA glycosylase/apurinic DNA lyase activity. In the present study, we demonstrated that the mitochondrial 8-oxodG DNA glycosylase/apurinic DNA lyase activity is the mitochondrial isoform of OGG1. Using mouse liver mitochondria isolated from ogg1(-/-) mice, we showed that the OGG1 gene encodes for the mitochondrial 8-oxodG glycosylase because these extracts have no incision activity toward an oligonucleotide containing a single 8-oxodG DNA base lesion. Consistent with an important role for the OGG1 protein in the removal of 8-oxodG from the mitochondrial genome, we found that mtDNA isolated from liver from OGG1-null mutant animals contained 20-fold more 8-oxodG than mtDNA from wild-type animals.

5-Formyluracil and its nucleoside derivatives confer toxicity and mutagenicity to mammalian cells by interfering with normal RNA and DNA metabolism.

Oxidation of the methyl group of thymine yields 5-(hydroxymethyl)uracil (5-hmU) and 5-formyluracil (5-foU) as major products. Whereas 5-hmU appears to have normal base pairing properties, the biological effects of 5-foU are rather poorly characterised. Here, we show that the colony forming ability of Chinese hamster fibroblast (CHF) cells is greatly reduced by addition of 5-foU, 5-formyluridine (5-foUrd) and 5-formyl-2'-deoxyuridine (5-fodUrd) to the growth medium. There are no toxic effects of 5-fodUrd on cells defective in thymidine kinase or thymidylate synthetase, suggesting that the toxicity may be caused by 5-fodUrd phosphorylation and subsequent inhibition of thymidylate synthetase. Whereas 5-fodUrd was the most effective 5-foU derivative causing cell growth inhibition, the corresponding ribonucleoside 5-foUrd was more effective in inhibiting [3H]uridine incorporation in non-dividing rat nerve cells in culture, suggesting that 5-foUrd exerts its toxicity through interference with RNA rather than DNA synthesis. Addition of 5-foU and 5-fodUrd was also found to promote mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus of CHF cells; 5-fodUrd being three orders of magnitude more potent than 5-foU. In contrast, neither 5-hmU nor 5-(hydroxymethyl)-2'-deoxyuridine induced HPRT mutations. The mutation induction indicates that 5-foU will be incorporated into DNA and has base pairing properties different from that of thymine. These results suggest that 5-foU residues, originating from incorporation of oxidised bases, nucleosides or nucleotides or by oxidation of DNA, may contribute significantly to the damaging effects of oxygen radical species in mammalian cells.

[Life without DNA repair]. / Et liv uten DNA-reparasjon.

BACKGROUND: 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. MATERIAL AND METHODS: 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. RESULTS: 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. INTERPRETATION: 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.

Transcription coupled repair of 8-oxoguanine in murine cells: the ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences.

To assess the role of the Ogg1 DNA glycosylase in the transcription-coupled repair (TCR) of the mutagenic lesion, 7, 8-dihydro-8oxoguanine (8-OxoG), we have investigated the removal of this lesion in wild-type and ogg1(-/-) null mouse embryo fibroblast (MEF) cell lines. We used nonreplicating plasmids containing a single 8-OxoG.C base pair in a different assay that allowed us to study the removal of 8-OxoG located in a transcribed sequence (TS) or in a nontranscribed sequence (NTS). The results show that the removal of 8-OxoG in a wild-type MEF cell line is faster in the TS than in the NTS, indicating TCR of 8-OxoG in murine cells. In the homozygous ogg1(-/-) MEF cell line, 8-OxoG was not removed from the NTS whereas there was still efficient 8-OxoG repair in the TS. Expression of the mouse Ogg1 protein in the homozygous ogg1(-/-) cell line restored the ability to remove 8-OxoG in the NTS. Therefore, we have demonstrated that Ogg1 is essential for the repair of 8-OxoG in the NTS but is not required in the TS. These results indicate the existence of an Ogg1-independent pathway for the TCR of 8-OxoG in vivo.

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage.

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.

Base excision repair of oxidative DNA damage activated by XPG protein.

Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells. We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer. With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1. XPG protein promotes binding of hNth1 to damaged DNA. The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair. The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage.

Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase.

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7, 8-dihydroguanine). In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen.

Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).

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 and processing events at DNA ends.

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.

Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.

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.

Post-translational modification of poly(ADP-ribose) polymerase induced by DNA strand breaks.

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.

Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts.

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.