Magnar Bjørås

The base excision repair pathway

The base excision repair pathway has evolved to protect cells from the deleterious effects of endogenous DNA damage induced by hydrolysis, reactive oxygen species and other intracellular metabolites that modify DNA base structure. However, base excision repair is also important to resist lesions produced by ionizing radiation and strong alkylating agents, which are similar to those induced by endogenous factors.

Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA

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.

OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells

Although oxidative damage has long been associated with ageing and neurological disease, mechanistic connections of oxidation to these phenotypes have remained elusive. Here we show that the age-dependent somatic mutation associated with Huntington’s disease occurs in the process of removing oxidized base lesions, and is remarkably dependent on a single base excision repair enzyme, 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1). Both in vivo and in vitro results support a ‘toxic oxidation’ model in which OGG1 initiates an escalating oxidation–excision cycle that leads to progressive age-dependent expansion. Age-dependent CAG expansion provides a direct molecular link between oxidative damage and toxicity in post-mitotic neurons through a DNA damage response, and error-prone repair of single-strand breaks.

Opposite base‐dependent reactions of a human base excision repair enzyme on DNA containing 7,8‐dihydro‐8‐oxoguanine and abasic sites

The guanine modification 7,8‐dihydro‐8‐oxoguanine (8‐oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8‐oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8‐oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8‐oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8‐oxoG opposite C was the most efficient and was followed by strand cleavage via β‐elimination. However, significant removal of 8‐oxoG from mispairs (8‐oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8‐oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error‐free correction by a postreplicational repair mechanism.

Base Excision Repair

Base excision repair (BER) corrects DNA damage from oxidation, deamination and alkylation. Such base lesions cause little distortion to the DNA helix structure. BER is initiated by a DNA glycosylase that recognizes and removes the damaged base, leaving an abasic site that is further processed by short-patch repair or long-patch repair that largely uses different proteins to complete BER. At least 11 distinct mammalian DNA glycosylases are known, each recognizing a few related lesions, frequently with some overlap in specificities. Impressively, the damaged bases are rapidly identified in a vast excess of normal bases, without a supply of energy. BER protects against cancer, aging, and neurodegeneration and takes place both in nuclei and mitochondria. More recently, an important role of uracil-DNA glycosylase UNG2 in adaptive immunity was revealed. Furthermore, other DNA glycosylases may have important roles in epigenetics, thus expanding the repertoire of BER proteins.

New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA‐distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP‐C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA‐oxidizing agents and this effect is reverted by expression of wild‐type XPC. Upon oxidant exposure, XP‐C primary keratinocytes and fibroblasts accumulate 8,5′‐cyclopurine 2′‐deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8‐hydroxyguanine (8‐OH‐Gua) is also observed. We demonstrate that XPC–HR23B complex acts as cofactor in base excision repair of 8‐OH‐Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC‐HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP‐C patients.

Mitochondrial DNA Integrity Is Essential For Mitochondrial Maturation During Differentiation of Neural Stem Cells

Differentiation of neural stem cells (NSCs) involves the activation of aerobic metabolism, which is dependent on mitochondrial function. Here, we show that the differentiation of NSCs involves robust increases in mitochondrial mass, mitochondrial DNA (mtDNA) copy number, and respiration capacity. The increased respiration activity renders mtDNA vulnerable to oxidative damage, and NSCs defective for the mitochondrial 8‐oxoguanine DNA glycosylase (OGG1) function accumulate mtDNA damage during the differentiation. The accumulated mtDNA damages in ogg1−/− cells inhibit the normal maturation of mitochondria that is manifested by reduced cellular levels of mitochondrial encoded complex proteins (complex I [cI], cIII, and cIV) with normal levels of the nuclear encoded cII present. The specific cI activity and inner membrane organization of respiratory complexes are similar in wt and ogg1−/− cells, inferring that mtDNA damage manifests itself as diminished mitochondrial biogenesis rather than the generation of dysfunctional mitochondria. Aerobic metabolism increases during differentiation in wild‐type cells and to a lesser extent in ogg1−/− cells, whereas anaerobic rates of metabolism are constant and similar in both cell types. Our results demonstrate that mtDNA integrity is essential for effective mitochondrial maturation during NSC differentiation. STEM CELLS 2010;28:2195–2204

Differential Expression of Two Glial Glutamate Transporters in the Rat Brain: an In Situ Hybridization Study

The distributions of the mRNAs encoding the l‐glutamate transporters GLT1 and GLAST were examined in the rat brain by in situ hybridization using 35S‐labelled oligonucleotide probes. Probes directed to GLT1 produced dense labelling in the hippocampus, neocortex and neostriatum, and weak labelling in the cerebellum. In contrast, GLAST mRNA appeared to be greatly enriched in the cerebellum compared to other brain regions. While the intensity of the labelling for GLAST and GLT1 varied among different regions, their cellular distributions appeared to coincide inasmuch as both mRNAs were mainly expressed by glial cells. Labelling occurred, inter alia, in glial cells throughout the hippocampus, and in Golgi epithelial cells in the Purkinje cell layer of the cerebellum.

Autophagy contributes to therapy-induced degradation of the PML/RARA oncoprotein

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid and/or arsenic trioxide represents a paradigm in targeted cancer therapy because these drugs cause clinical remission by affecting the stability of the fusion oncoprotein promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARA). The authors of previous studies have implicated the ubiquitin-proteasome pathway as the main mechanism involved in therapy-induced PML/RARA degradation. Here we have investigated a role of autophagy, a protein degradation pathway that involves proteolysis of intracellular material within lysosomes. We found that both all-trans retinoic acid and arsenic trioxide induce autophagy via the mammalian target of rapamycin pathway in APL cells and that autophagic degradation contributes significantly both to the basal turnover as well as the therapy-induced proteolysis of PML/RARA. In addition, we observed a correlation between autophagy and therapy-induced differentiation of APL cells. Given the central role of the PML/RARA oncoprotein in APL pathogenesis, this study highlights an important role of autophagy in the development and treatment of this disease.

The Saccharomyces cerevisiae Homologues of Endonuclease III from Escherichia coli, Ntg1 and Ntg2, Are Both Required for Efficient Repair of Spontaneous and Induced Oxidative DNA Damage in Yeast

ABSTRACT Endonuclease III from Escherichia coli is the prototype of a ubiquitous DNA repair enzyme essential for the removal of oxidized pyrimidine base damage. The yeast genome project has revealed the presence of two genes in Saccharomyces cerevisiae,NTG1 and NTG2, encoding proteins with similarity to endonuclease III. Both contain the highly conserved helix-hairpin-helix motif, whereas only one (Ntg2) harbors the characteristic iron-sulfur cluster of the endonuclease III family. We have characterized these gene functions by mutant and enzyme analysis as well as by gene expression and intracellular localization studies. Targeted gene disruption of NTG1 and NTG2produced mutants with greatly increased spontaneous and hydrogen peroxide-induced mutation frequency relative to the wild type, and the mutation response was further increased in the double mutant. Both enzymes were found to remove thymine glycol and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (faPy) residues from DNA with high efficiency. However, on UV-irradiated DNA, saturating concentrations of Ntg2 removed only half of the cytosine photoproducts released by Ntg1. Conversely, 5-hydroxycytosine was removed efficiently only by Ntg2. The enzymes appear to have different reaction modes, as judged from much higher affinity of Ntg2 for damaged DNA and more efficient borhydride trapping of Ntg1 to abasic sites in DNA despite limited DNA binding. Northern blot and promoter fusion analysis showed that NTG1 is inducible by cell exposure to DNA-damaging agents, whereas NTG2 is constitutively expressed. Ntg2 appears to be a nuclear enzyme, whereas Ntg1 was sorted both to the nucleus and to the mitochondria. We conclude that functions of both NTG1 and NTG2 are important for removal of oxidative DNA damage in yeast.