Ingrun Alseth

Crystal structure and mutational analysis of human uracil-DNA glycosylase: Structural basis for specificity and catalysis

Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.

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.

Structures of Endonuclease V with DNA Reveal Initiation of Deaminated Adenine Repair

Endonuclease V (EndoV) initiates a major base-repair pathway for nitrosative deamination resulting from endogenous processes and increased by oxidative stress from mitochondrial dysfunction or inflammatory responses. We solved the crystal structures of Thermotoga maritima EndoV in complex with a hypoxanthine lesion substrate and with product DNA. The PYIP wedge motif acts as a minor groove–damage sensor for helical distortions and base mismatches and separates DNA strands at the lesion. EndoV incises DNA with an unusual offset nick 1 nucleotide 3′ of the lesion, as the deaminated adenine is rotated ∼90° into a recognition pocket ∼8 Å from the catalytic site. Tight binding by the lesion-recognition pocket in addition to Mg2+ and hydrogen-bonding interactions to the DNA ends stabilize the product complex, suggesting an orderly recruitment of downstream proteins in this base-repair pathway.

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 Alzheimers disease and Parkinsons 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.

A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD

Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3‐methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E.u2003coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E.u2003coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N‐alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B.u2003cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.

Base excision repair activities required for yeast to attain a full chronological life span

The chronological life span of yeast, the survival of stationary (G0) cells over time, provides a model for investigating certain of the factors that may influence the aging of non‐dividing cells and tissues in higher organisms. This study measured the effects of defined defects in the base excision repair (BER) system for DNA repair on this life span. Stationary yeast survives longer when it is pregrown on respiratory, as compared to fermentative (glucose), media. It is also less susceptible to viability loss as the result of defects in DNA glycosylase/AP lyases (Ogg1p, Ntg1p, Ntg2p), apurinic/apyrimidinic (AP) endonucleases (Apn1p, Apn2p) and monofunctional DNA glycosylase (Mag1p). Whereas single BER glycosylase/AP lyase defects exerted little influence over such optimized G0 survival, this survival was severely shortened with the loss of two or more such enzymes. Equally, the apn1Δ and apn2Δ single gene deletes survived as well as the wild type, whereas a apn1Δapn2Δ double mutant totally lacking in any AP endonuclease activity survived poorly. Both this shortened G0 survival and the enhanced mutagenicity of apn1Δapn2Δ cells were however rescued by the overexpression of either Apn1p or Apn2p. The results highlight the vital importance of BER in the prevention of mutation accumulation and the attainment of the full yeast chronological life span. They also reveal an appreciable overlap in the G0 maintenance functions of the different BER DNA glycosylases and AP endonucleases.

Separation-of-Function Mutants Unravel the Dual-Reaction Mode of Human 8-Oxoguanine DNA Glycosylase

7,8-Dihydro-8-oxoguanine (8oxoG) is a major mutagenic base lesion formed when reactive oxygen species react with guanine in DNA. The human 8oxoG DNA glycosylase (hOgg1) recognizes and initiates repair of 8oxoG. hOgg1 is acknowledged as a bifunctional DNA glycosylase catalyzing removal of the damaged base followed by cleavage of the backbone of the intermediate abasic DNA (AP lyase/β-elimination). When acting on 8oxoG-containing DNA, these two steps in the hOgg1 catalysis are considered coupled, with Lys249 implicated as a key residue. However, several lines of evidence point to a concurrent and independent monofunctional hydrolysis of the N-glycosylic bond being the inxa0vivo relevant reaction mode of hOgg1. Here, we present biochemical and structural evidence for the monofunctional mode of hOgg1 by design of separation-of-function mutants. Asp268 is identified as the catalytic residue, while Lys249 appears critical for the specific recognition and final alignment of 8oxoG during the hydrolysis reaction.

Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma

The human hMYH and NEIL1 genes encode DNA glycosylases involved in repair of oxidative base damage and mutations in these genes are associated with certain cancers. Primary sclerosing cholangitis (PSC), a chronic cholestatic liver disease characterized by inflammatory destruction of the biliary tree, is often complicated by the development of cholangiocarcinoma (CCA). Here, we aimed to investigate the influence of genetic variations in the hMYH and NEIL1 genes on risk of CCA in PSC patients. The hMYH and NEIL1 gene loci in addition to the DNA repair genes hOGG1, NTHL1 and NUDT1 were analyzed in 66 PSC patients (37 with CCA and 29 without cancer) by complete genomic sequencing of exons and adjacent intronic regions. Several single-nucleotide polymorphisms and mutations were identified and severe impairment of protein function was observed for three non-synonymous variants. The NEIL1 G83D mutant was dysfunctional for the major oxidation products 7,8-dihydro-8-oxoguanine (8oxoG), thymine glycol and dihydrothymine in duplex DNA, and the ability to perform δ-elimination at abasic sites was significantly reduced. The hMYH R260Q mutant had severe defect in adenine DNA glycosylase activity, whereas hMYH H434D could excise adenines from A:8oxoG pairs but not from A:G mispairs. We found no overall associations between the 18 identified variants and susceptibility to CCA in PSC patients; however, the impaired variants may be of significance for carcinogenesis in general. Our findings demonstrate the importance of complete resequencing of selected candidate genes in order to identify rare genetic variants and their possible contribution to individual susceptibility to cancer development.

Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe mag1 gene encodes a DNA repair enzyme with sequence similarity to the AlkA family of DNA glycosylases, which are essential for the removal of cytotoxic alkylation products, the premutagenic deamination product hypoxanthine and certain cyclic ethenoadducts such as ethenoadenine. In this paper, we have purified the Mag1 protein and characterized its substrate specificity. It appears that the substrate range of Mag1 is limited to the major alkylation products, such as 3-mA, 3-mG and 7-mG, whereas no significant activity was found towards deamination products, ethenoadducts or oxidation products. The efficiency of 3-mA and 3-mG removal was 5–10 times slower for Mag1 than for Escherichia coli AlkA whereas the rate of 7-mG removal was similar to the two enzymes. The relatively low efficiency for the removal of cytotoxic 3-methylpurines is consistent with the moderate sensitivity of the mag1 mutant to methylating agents. Furthermore, we studied the initial steps of Mag1-dependent base excision repair (BER) and genetic interactions with other repair pathways by mutant analysis. The double mutants mag1 nth1, mag1 apn2 and mag1 rad2 displayed increased resistance to methyl methanesulfonate (MMS) compared with the single mutants nth1, apn2 and rad2, respectively, indicating that Mag1 initiates both short-patch (Nth1-dependent) and long-patch (Rad2-dependent) BER of MMS-induced damage. Spontaneous intrachromosomal recombination frequencies increased 3-fold in the mag1 mutant suggesting that Mag1 and recombinational repair (RR) are both involved in repair of alkylated bases. Finally, we show that the deletion of mag1 in the background of rad16, nth1 and rad2 single mutants reduced the total recombination frequencies of all three double mutants, indicating that abasic sites formed as a result of Mag1 removal of spontaneous base lesions are substrates for nucleotide excision repair, long- and short-patch BER and RR.

Inosine in DNA and RNA.

Deamination of the nucleobases in DNA and RNA is a result of spontaneous hydrolysis, endogenous or environmental factors as well as deaminase enzymes. Adenosine is deaminated to inosine which is miscoding and preferentially base pairs with cytosine. In the case of DNA, this is a premutagenic event that is counteracted by DNA repair enzymes specifically engaged in recognition and removal of inosine. However, in RNA, inosine is an essential modification introduced by specialized enzymes in a highly regulated manner to generate transcriptome diversity. Defect editing is seen in various human disease including cancer, viral infections and neurological and psychiatric disorders. Enzymes catalyzing the deaminase reaction are well characterized and recently an unexpected function of Endonuclease V in RNA processing was revealed. Whereas bacterial Endonuclease V enzymes are classified as DNA repair enzymes, it appears that the mammalian enzymes are involved in processing of inosine in RNA. This yields an interesting yet unexplored, link between DNA and RNA processing. Further work is needed to gain understanding of the impact of inosine in DNA and RNA under normal physiology and disease progression.