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Biomedical subjects

Tomas Lindahl

Publications and source records attributed to Tomas Lindahl.

28 records · Page 2Linked to original sources
PubMed2004

Repair and genetic consequences of endogenous DNA base damage in mammalian cells.

Living organisms dependent on water and oxygen for their existence face the major challenge of faithfully maintaining their genetic material under a constant attack from spontaneous hydrolysis and active oxygen species and from other intracellular metabolites that can modify DNA bases. Repair of endogenous DNA base damage by the ubiquitous base-excision repair pathway largely accounts for the significant turnover of DNA even in nonreplicating cells, and must be sufficiently accurate and efficient to preserve genome stability compatible with long-term cellular viability. The size of the mammalian genome has necessitated an increased complexity of repair and diversification of key enzymes, as revealed by gene knock-out mouse models. The genetic instability characteristic of cancer cells may be due, in part, to mutations in genes whose products normally function to ensure DNA integrity.

Alkylation↗
PubMed2003

Minimal methylated substrate and extended substrate range of Escherichia coli AlkB protein, a 1-methyladenine-DNA dioxygenase.

The Escherichia coli AlkB protein, and two human homologs ABH2 and ABH3, directly demethylate 1-methyladenine and 3-methylcytosine in DNA. They couple Fe(II)-dependent oxidative demethylation of these damaged bases to decarboxylation of alpha-ketoglutarate. Here, we have determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA), short oligodeoxyribonucleotides, nucleotides, and nucleoside triphosphates. Methylated poly(dA) was the preferred AlkB substrate of those tested. The oligonucleotide trimer d(Tp1meApT) and even 5'-phosphorylated 1-me-dAMP were relatively efficiently demethylated, and competed with methylated poly(dA) for AlkB activity. A polynucleotide structure was clearly not essential for AlkB to repair 1-methyladenine effectively, but a nucleotide 5' phosphate group was required. Consequently, 1-me-dAMP(5') was identified as the minimal effective AlkB substrate. The nucleoside triphosphate, 1-me-dATP, was inefficiently but actively demethylated by AlkB; a reaction with 1-me-ATP was even slower. E. coli DNA polymerase I Klenow fragment could employ 1-me-dATP as a precursor for DNA synthesis in vitro, suggesting that demethylation of alkylated deoxynucleoside triphosphates by AlkB could have biological significance. Although the human enzymes, ABH2 and ABH3, demethylated 1-methyladenine residues in poly(dA), they were inefficient with shorter substrates. Thus, ABH3 had very low activity on the trimer, d(Tp1meApT), whereas no activity was detected with ABH2. AlkB is known to repair methyl and ethyl adducts in DNA; to extend this substrate range, AlkB was shown to reduce the toxic effects of DNA damaging agents that generate hydroxyethyl, propyl, and hydroxypropyl adducts.

Adenine↗
PubMed2003

Gene-targeted mice lacking the Ung uracil-DNA glycosylase develop B-cell lymphomas.

Mice deficient in the Ung uracil-DNA glycosylase have an increased level of uracil in their genome, consistent with a major role of Ung counteracting U:A base pairs arising by misincorporation of dUMP during DNA replication. A complementary uracil-excising activity apparently acts on premutagenic U:G lesions resulting from deamination of cytosine throughout the genome. However, Ung specifically processes U:G lesions targeted to immunoglobulin variable (V) genes during somatic hypermutation and class-switch recombination. Gene-targeted Ung(-/-) null mice remained tumour-free and showed no overt pathological phenotype up to approximately 12 months of age. We have monitored a large cohort of ageing Ung(-/-) mice and, beyond 18 months of age, they had a higher morbidity than Ung(+/+) controls. Post-mortem analyses revealed pathological changes in lymphoid organs, abnormal lymphoproliferation, and a greatly increased incidence of B-cell lymphomas in older Ung-deficient mice. These are the first data reporting the development of spontaneous malignancies in mice due to deficiency in a DNA glycosylase. Furthermore, they support a specific role for Ung in the immune system, with lymphomagenesis being related to perturbed processing of antibody genes in germinal centre B cells.

Animals↗
PubMed2003

Normal somatic hypermutation of Ig genes in the absence of 8-hydroxyguanine-DNA glycosylase.

The hypermutation cascade in Ig V genes can be initiated by deamination of cytosine in DNA to uracil by activation-induced cytosine deaminase and its removal by uracil-DNA glycosylase. To determine whether damage to guanine also contributes to hypermutation, we examined the glycosylase that removes oxidized guanine from DNA, 8-hydroxyguanine-DNA glycosylase (OGG1). OGG1 has been reported to be overexpressed in human B cells from germinal centers, where mutation occurs, and could be involved in initiating Ab diversity by removing modified guanines. In this study, mice deficient in Ogg1 were immunized, and V genes from the H and kappa L chain loci were sequenced. Both the frequency of mutation and the spectra of nucleotide substitutions were similar in ogg1(-/-) and Ogg1(+/+) clones. More importantly, there was no significant increase in G:C to T:A transversions in the ogg1(-/-) clones, which would be expected if 8-hydroxyguanine remained in the DNA. Furthermore, Ogg1 was not up-regulated in murine B cells from germinal centers. These findings show that hypermutation is unaffected in the absence of Ogg1 activity and indicate that 8-hydroxyguanine lesions most likely do not cause V gene mutations.

Animals↗
PubMed2002

Reversal of DNA alkylation damage by two human dioxygenases.

The Escherichia coli AlkB protein protects against the cytotoxicity of methylating agents by repair of the DNA lesions 1-methyladenine and 3-methylcytosine, which are generated in single-stranded stretches of DNA. AlkB is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde. Here, we identify two human AlkB homologs, ABH2 and ABH3, by sequence and fold similarity, functional assays, and complementation of the E. coli alkB mutant phenotype. The levels of their mRNAs do not appear to correlate with cell proliferation but tissue distributions are different. Both enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an alpha-ketoglutarate-dependent reaction, and act by direct damage reversal with the regeneration of the unsubstituted bases. AlkB, ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde.

Adenine↗
PubMed2002

DNA base excision repair of uracil residues in reconstituted nucleosome core particles.

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.

Animals↗
PubMed2002

Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice.

BACKGROUND: We have previously proposed that deamination of cytosine to uracil at sites within the immunoglobulin loci by activation-induced deaminase (AID) triggers antibody diversification. The pattern of diversification (phase 1 or 2 hypermutation, gene conversion, or switch recombination) is viewed as depending on the mode of resolution of the dU/dG lesion. A major resolution mode involves excising the uracil, an activity that at least four different enzymes can accomplish in the mouse. RESULTS: Deficiency in UNG uracil-DNA glycosylase alone is sufficient to distort the pathway of hypermutation in mice. In ung(-/-) animals, mutations at dC/dG pairs are dramatically shifted toward transitions (95%), indicating that the generation of abasic sites (which can induce transversions) has been inhibited. The pattern of substitutions at dA/dT pairs is unaffected. Class-switch recombination is substantially, but not totally, inhibited. CONCLUSIONS: The results provide strong support for the DNA deamination model for antibody diversification with respect to class-switching as well as hypermutation and, in the context of this model, suggest that (i) UNG is the major mouse DNA glycosylase responsible for processing the programmed dU/dG lesions within the immunoglobulin locus; (ii) the second (dA/dT-biased) phase of mutation is probably triggered by recognition of the initiating dU/dG lesion; and (iii) switch recombination largely proceeds via formation of an abasic site, although (iv) an UNG-independent pathway of switch recombination exists, which could reflect action by another uracil-DNA glycosylase but might alternatively be explained by a distinct pathway of resolution, for example, one involving MSH2/MSH6 recognition of the dU/dG lesion.

Animals↗
PubMed2002

Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage.

Methylating agents generate cytotoxic and mutagenic DNA damage. Cells use 3-methyladenine-DNA glycosylases to excise some methylated bases from DNA, and suicidal O(6)-methylguanine-DNA methyltransferases to transfer alkyl groups from other lesions onto a cysteine residue. Here we report that the highly conserved AlkB protein repairs DNA alkylation damage by means of an unprecedented mechanism. AlkB has no detectable nuclease, DNA glycosylase or methyltransferase activity; however, Escherichia coli alkB mutants are defective in processing methylation damage generated in single-stranded DNA. Theoretical protein fold recognition had suggested that AlkB resembles the Fe(ii)- and alpha-ketoglutarate-dependent dioxygenases, which use iron-oxo intermediates to oxidize chemically inert compounds. We show here that purified AlkB repairs the cytotoxic lesions 1-methyladenine and 3-methylcytosine in single- and double-stranded DNA in a reaction that is dependent on oxygen, alpha-ketoglutarate and Fe(ii). The AlkB enzyme couples oxidative decarboxylation of alpha-ketoglutarate to the hydroxylation of these methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde.

Adenine↗
PubMed2002

Down-regulation of DNA repair synthesis at DNA single-strand interruptions in poly(ADP-ribose) polymerase-1 deficient murine cell extracts.

The functional involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in the repair of DNA single- and double-strand breaks, DNA base damage, and related repair substrate intermediates remains unclear. Using an in vitro DNA repair assay and cell extracts derived from PARP-1 deficient or wild-type murine embryonic fibroblasts, we investigated the DNA synthesis and ligation steps associated with the rejoining of DNA single-strand interruptions containing 3'-OH, and either 5'-OH or 5'-P termini. Complete repair leading to DNA rejoining was similar between PARP-1 deficient cells and wild-type controls and poly(ADP-ribose) synthesis was, as expected, greatly reduced in PARP-1 deficient cell extracts. The incorporation of [32P]dCMP into repaired DNA at the site of a lesion was reduced two-three-fold in PARP-1 deficient cell extracts, demonstrating a decrease in repair patch size. Addition of purified PARP-1 to levels approximating those present in wild-type extracts did not stimulate DNA repair synthesis. We conclude that PARP-1 is not required for the efficient processing and rejoining of single-strand interruptions with defined 3'-OH and 5'-OH or 5'-P termini. Decreased DNA repair synthesis observed in PARP-1 deficient cell extracts is associated with reduced cellular expression of several factors required for long-patch base excision repair (BER), including FEN-1 and DNA ligase I.

Animals↗