Svein Bjelland | University of Stavanger (original) (raw)

Papers by Svein Bjelland

The EMBO Journal

An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase acti... more An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate. Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant. Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA. DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E.coli. One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA. DNA containing the open reading frame was subcloned in E.coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity. It appeared that such activity was expressed at levels sufficiently high for enzyme purification. The molecular weight of the purified protein was determined by SDS-PAGE to be 35 000 daltons, in good agreement with the 34 340 value calculated from the sequence. The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confi'rming the related nature of this enzyme to the AlkA DNA glycosylase from E.coli.

The methyl group of thymine can be oxidised by reactive oxygen species resulting in the mutagenic... more The methyl group of thymine can be oxidised by reactive oxygen species resulting in the mutagenic 5-formyluracil (fU) residue. fU is removed from DNA in vitro by several DNA glycosylases of Escherichia coli including the AlkA (3-methyladenine DNA glycosylase II), Fpg, Nth and Nei proteins, initiating the base excision repair pathway. The possible involvement of the E. coli nucleotide excision repair complex, UvrABC, has hitherto not been investigated. In a recent study we found that alkA influences the distribution of mutations induced by addition of 5-formyl-2’-deoxyuridine (fdU) to the growth culture of E. coli, proposing that the AlkA glycosylase is able to alleviate as well as promote induction of certain base substitutions dependent on the fdU concentration. Here we present in vivo evidence to indicate that the UvrA protein/UvrABC complex exhibits similar effects. The aim of the project is to understand this at a molecular level by analysing, in addition to uvrA, uvrB and uvrC ...

Mutation research, Jan 9, 2001

5-Formyluracil (5-foU) is a major oxidation product of thymine formed in yields comparable to tha... more 5-Formyluracil (5-foU) is a major oxidation product of thymine formed in yields comparable to that of 8-oxoguanine in DNA by ionizing radiation. Whereas the mutagenic effects of 8-oxoguanine are well understood, the investigation of the biological implications of 5-foU has so far been limited. Here we demonstrate that 5-formyl-2'-deoxyuridine (5-fodUrd) supplied to the growth medium of Escherichia coli induces several base substitutions at different frequencies at position 461 in the lacZ gene in the following order: A.T-->G.C>G.C-->A.T>G.C-->T.A>A.T-->T.A>A.T-->C.G. No induction of G.C-->C.G transversions was observed. It is inferred that 5-fodUrd will be incorporated into the DNA during cell growth, forming mispairs with guanine, cytosine and thymine during replication. It, thus, appears that cell growth in the presence of 5-fodUrd may represent a good model for elucidating the cellular effects of 5-foU residues in DNA.

The Journal of biological chemistry, Jan 2, 1994

The alkA gene of Escherichia coli encodes a DNA glycosylase involved in base excision repair of D... more The alkA gene of Escherichia coli encodes a DNA glycosylase involved in base excision repair of DNA alkylation damage. In an attempt to define the reactions of the AlkA enzyme with methylated DNA, we discovered that the enzyme released substantial amounts of radioactivity from [methyl-3H]thymidine-labeled DNA even without any exposure of the DNA to methylating agents. The excised material was identified by chromatography as two different oxidized derivatives of thymine, 5-hydroxymethyluracil and 5-formyluracil. These products are formed in such DNA by one and two consecutive decays, respectively, of the tritiums of the labeled methyl group. Kinetic analysis showed that both the apparent Km and Vmax values for 5-formyluracil removal are within the same range as found for 3-methyladenine removal, suggesting that this catalytic property of AlkA is also significant under in vivo conditions. Removal of 5-hydroxymethyluracil proceeds at a rate that is 1-3 orders of magnitude slower. Since...

Toxicology Letters, 2001

Oxidation of the methyl group of thymine yields 5-(hydroxymethyl)uracil (5-hmU) and 5-formyluraci... more 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 [ 3 H]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. : S 0 3 7 8 -4 2 7 4 ( 0 0 ) 0 0 3 0 8 -8 A. Klungland et al. / Toxicology Letters 119 (2001) 71-78 72

The EMBO Journal, 2007

N 1 -methyladenine (m 1 A) and N 3 -methylcytosine (m 3 C) are major toxic and mutagenic lesions ... more 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 AlkBmediated 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 possess 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.

Journal of Bacteriology, 2010

Hydrolytic deamination of cytosine to uracil in cellular DNA is a major source of C-to-T transiti... more Hydrolytic deamination of cytosine to uracil in cellular DNA is a major source of C-to-T transition mutations if uracil is not repaired by the DNA base excision repair (BER) pathway. Since deamination increases rapidly with temperature, hyperthermophiles, in particular, are expected to succumb to such damage. There has been only one report of crenarchaeotic BER showing strong similarities to that in most eukaryotes and bacteria for hyperthermophilic Archaea. Here we report a different type of BER performed by extract prepared from cells of the euryarchaeon Archaeoglobus fulgidus. Although immunodepletion showed that the monofunctional family 4 type of uracil-DNA glycosylase (UDG) is the principal and probably only UDG in this organism, a ␤-elimination mechanism rather than a hydrolytic mechanism is employed for incision of the abasic site following uracil removal. The resulting 3 remnant is removed by efficient 3-phosphodiesterase activity followed by single-nucleotide insertion and ligation. The finding that repair product formation is stimulated similarly by ATP and ADP in vitro raises the question of whether ADP is more important in vivo because of its higher heat stability.

International Journal of Radiation Biology, 2009

Purpose: The aim of this study was to determine the excision efficiency of hSMUG1 (human single-s... more Purpose: The aim of this study was to determine the excision efficiency of hSMUG1 (human single-strand-selective monofunctional uracil-DNA glycosylase) for 5-formyluracil (fU), a major thymine lesion formed by ionizing radiation, opposite all normal bases in DNA, to possibly explain mutation induction by fU in the DNA of mammalian cells. Materials and methods: An enzymatically [ 32 P]labelled fU-containing 36 nucleotide DNA sequence plus its complementary sequence (with an A, C, G or T residue inserted opposite fU) was subjected to hSMUG1 in a pH 7.5-buffer, followed by NaOH-mediated cleavage of the resultant abasic sites. Cleaved and uncleaved DNA were separated by denaturing electrophoresis and quantified by autoradiography. Results: The hSMUG1 excised fU from DNA opposite all normal bases with the highest activity when opposite noncognate C or T followed by G and cognate A. Conclusions: The predominant T!G and T!A transversions induced by fU in mammalian cells may be explained by replicative incorporation of C and T, respectively, opposite the lesion and subsequent SMUG1-initiated repair of fU.

Cancer Research, 2008

Endogenous formation of the mutagenic DNA adduct 1,N 6ethenoadenine (EA) originates from lipid pe... more Endogenous formation of the mutagenic DNA adduct 1,N 6ethenoadenine (EA) originates from lipid peroxidation. Elevated levels of EA in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for EA lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair EA lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2 À/À mice indicates that mABH2 is the principal dioxygenase for EA repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of EA lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of EA, comprise the cellular defense against EA lesions. [Cancer Res 2008;68(11):4142-9] Requests for reprints:

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1983

Five preparations of bovine thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydr... more Five preparations of bovine thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase (EC 1.8.4.2) and one preparation of bovine liver protein-disulphide isomerase (EC 5.3.4.1) from four different laboratories showed immunological identity in double immunodiffusion and rocket-line immunoelectrophoresis. Consequently, thiol:protein'disulphide oxidoreductase/glutathione-insulin transhydrogenase and protein-disulphide isomerase, formerly classified as two separate enzymes, should be considered as alternative activities of the same enzyme.

Analytical Biochemistry, 1984

A rapid purification procedure of thiol:protein-disulfide oxidoreductase (EC 1.8.4.2) from bovine... more A rapid purification procedure of thiol:protein-disulfide oxidoreductase (EC 1.8.4.2) from bovine liver has been developed. The procedure is based on that of D. F. Carmichael, J. E. Morin, and J. E. Dixon (1977, J. Biol. Chem. 252, 7163-7167), and contains the following steps: homogenization in Triton X-100, selective heat denaturation, chromatography on CM-Sephadex C-50, and chromatography on DEAE-Sephadex A-50. The final preparation has a high specific activity and a high level of purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Biochemistry Usa, Dec 1, 1995

Oxidative agents produce several different types of base modifications in DNA, and only a few of ... more Oxidative agents produce several different types of base modifications in DNA, and only a few of these have been properly characterized with respect to mechanisms of formation and biological implications. We have established a procedure using neutral thermal hydrolysis and reverse phase high-