Svein Bjelland

AlkB Homologue 2–Mediated Repair of Ethenoadenine Lesions in Mammalian DNA

Endogenous formation of the mutagenic DNA adduct 1,N(6)-ethenoadenine (epsilon A) originates from lipid peroxidation. Elevated levels of epsilon A 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 epsilon A 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 epsilon A lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2(-/-) mice indicates that mABH2 is the principal dioxygenase for epsilon A 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 epsilon A lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of epsilon A, comprise the cellular defense against epsilon A lesions.

Cloning and expression in Escherichia coli of a gene for an alkylbase DNA glycosylase from Saccharomyces cerevisiae ; a homologue to the bacterial alkA gene

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, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E. coli.

Structural basis for enzymatic excision of N1-methyladenine and N3-methylcytosine from DNA.

N1‐methyladenine (m1A) and N3‐methylcytosine (m3C) are major toxic and mutagenic lesions induced by alkylation in single‐stranded DNA. In bacteria and mammals, m1A and m3C were recently shown to be repaired by AlkB‐mediated oxidative demethylation, a direct DNA damage reversal mechanism. No AlkB gene homologues have been identified in Archaea. We report that m1A and m3C 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 effect a robust excision of 1,N6‐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.

Cellular effects of 5-formyluracil in DNA

5-Formyluracil is a major oxidation product of thymine, formed in DNA in yields comparable to that of 8-oxo-7,8-dihydroguanine by exposure to gamma-irradiation. Whereas the repair pathways for removal and the biological effects of persisting 8-oxo-7,8-dihydroguanine are much elucidated, much less attention has been paid to the cellular implications of 5-formyluracil in DNA. Here we review the present state of knowledge in this important area within research on oxidative DNA damage.

Different efficiencies of the Tag and AlkA DNA glycosylases from Escherichia coli in the removal of 3-methyladenine from single-stranded DNA.

Escherichia coli possesses two different DNA repair glycoselases, Tag and AlkA, which have similar ability to remove the alkylation product 3‐methyladenine from double‐stranded DNA. In this study we show that these enzymes have quite different activities for the excision of 3‐methyladenine from single‐stranded DNA, AlkA being 10–20 times more efficient than Tag. We propose that AlkA and perhaps other glycosylases as well may have an important role in the excision of base damage from single‐stranded regions transiently formed in DNA during transcription and replication.

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.

Opposite-base dependent excision of 5-formyluracil from DNA by hSMUG1

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 [32P]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 non-cognate 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.

Excision of the doubly methylated base N 4 ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Cytosine (C) in DNA is often modified to 5-methylcytosine (m5C) to execute important cellular functions. Despite the significance of m5C for epigenetic regulation in mammals, damage to m5C has received little attention. For instance, almost no studies exist on erroneous methylation of m5C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m5C into N4,5-dimethylcytosine (mN4,5C) in DNA, mN4,5C is probably present in vivo. We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at mN4,5C residues in vitro. The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m5C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that mN4,5C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.