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T Lindahl

Publications and source records attributed to T Lindahl.

At least 37 records · Page 2Linked to original sources
PubMed1999

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage.

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G.C-->T.A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1(-/-) null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Animals↗
PubMed1999

Delayed DNA joining at 3' mismatches by human DNA ligases.

Repair synthesis catalysed by DNA polymerase beta at 1 nt gaps occurs in the main pathway of mammalian base excision repair. DNA polymerase beta has no exonucleolytic proof-reading ability, and exhibits high error frequency during DNA synthesis. Consequently, continuous correction of endogenous DNA damage by short-patch repair synthesis might lead to a high spontaneous mutation rate, unless subsequent steps in the repair pathway allow for selective removal of incorporation errors. We show here that both human DNA ligase I and III discriminate strongly between a correctly paired versus a mispaired residue at the 3' position of a nick in DNA, when assayed in the presence of physiological concentrations of KCl. The resulting delay in joining after misincorporation by DNA polymerase beta during gap filling could allow for removal of the mismatched terminal residue by a distinct 3' exonuclease.

Base Sequence↗
PubMed1999

A human DNA editing enzyme homologous to the Escherichia coli DnaQ/MutD protein.

Mammalian DNA polymerases alpha and beta lack 3' exonuclease activity and are unable to edit errors after DNA synthesis. However, editing exonucleases can be functions of separate polypeptides. We isolated a widely distributed DNA-specific 3' exonuclease from rabbit liver nuclei, sequenced tryptic peptides by mass spectrometry, and identified the corresponding human open reading frame. The protein expressed from the cloned human sequence exhibits 3' exonuclease activity. The human clone shares sequence homology with the editing function of the Escherichia coli DNA polymerase III holoenzyme, i.e., the DnaQ/MutD protein, and weakly with the editing 3' exonuclease domain of eukaryotic DNA polymerase epsilon. The gene maps to human chromosome 3p21.2-21.3. In a reconstituted human DNA repair system containing DNA polymerase beta and DNA ligase III-XRCC1, accurate rejoining of a 3' mismatched base residue at a single-strand break is dependent on addition of the exonuclease.

Amino Acid Sequence↗
PubMed1999

Base excision repair of oxidative DNA damage activated by XPG protein.

Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells. We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer. With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1. XPG protein promotes binding of hNth1 to damaged DNA. The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair. The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage.

Base Sequence↗
PubMed1998

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR).

Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody-antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (< 1.25 micrograms/ml) in a physiologically relevant range (1-40 micrograms/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.

Animals↗
PubMed1998

Structure of an XRCC1 BRCT domain: a new protein-protein interaction module.

The BRCT domain (BRCA1 C-terminus), first identified in the breast cancer suppressor protein BRCA1, is an evolutionarily conserved protein-protein interaction region of approximately 95 amino acids found in a large number of proteins involved in DNA repair, recombination and cell cycle control. Here we describe the first three-dimensional structure and fold of a BRCT domain determined by X-ray crystallography at 3.2 A resolution. The structure has been obtained from the C-terminal region of the human DNA repair protein XRCC1, and comprises a four-stranded parallel beta-sheet surrounded by three alpha-helices, which form an autonomously folded domain. The compact XRCC1 structure explains the observed sequence homology between different BRCT motifs and provides a framework for modelling other BRCT domains. Furthermore, the established structure of an XRCC1 BRCT homodimer suggests potential protein-protein interaction sites for the complementary BRCT domain in DNA ligase III, since these two domains form a stable heterodimeric complex. Based on the XRCC1 BRCT structure, we have constructed a model for the C-terminal BRCT domain of BRCA1, which frequently is mutated in familial breast and ovarian cancer. The model allows insights into the effects of such mutations on the fold of the BRCT domain.

Amino Acid Sequence↗
PubMed1998

Impaired platelet binding of fibrinogen due to a lower number of GPIIB/IIIA receptors in polycythemia vera.

We have previously described a stimulus-specific defect in platelet aggregation in polycythaemia vera (PV) after stimulation with surface receptor dependent agonists such as platelet activating factor (PAF). In contrast, responses to phorbol myristate acetate (PMA) were normal. We now report that after PAF stimulation, using flow cytometry, the amount of fibrinogen bound to its receptor was significantly lower in PV platelets with a median MFI of 6.0 (range 4.1-17.3) compared to controls, 12.8 (range 8-21.3; n=11; p<0.01). We found no evidence of preactivation of PV platelets. Quantitative analysis of GPIIIa gave a significantly lower number of GPIIIa on resting PV platelets, 14300 subunits of GPIIIa (range 8500-15500) vs. 19800 for controls (range 13400-26800; n=12; p<0.01). Both patients and controls increased their number of receptors on the cell surface after stimulation with PAF and PMA, but the significant difference in the number of receptors per cell remained. Indirect evaluation of PAF receptor function showed that activation of CD 62 did not differ in PV and controls after PAF stimulation. Additionally, although the basal level of serotonin in platelet-rich plasma was significantly lower in PV, there was a threefold increase of the basal level after stimulation with PAF for both PV and control platelets, also indicating a normal interaction of PAF with its receptor. Although our results indicate both an impaired PAF induced aggregation in PV and a lower number of GPIIb/IIIa complexes on single platelets, whether these phenomena are related remains uncertain.

Adult↗
PubMed1998

Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4.

Saccharomyces cerevisiae DNA ligase IV (LIG4) has been shown previously to be involved in non-homologous DNA end joining and meiosis. The homologous mammalian DNA ligase IV interacts with XRCC4, a protein implicated in V(D)J recombination and double-strand break repair. Here, we report the discovery of LIF1, a S.cerevisiae protein that strongly interacts with the C-terminal BRCT domain of yeast LIG4. LIG4 and LIF1 apparently occur as a heterodimer in vivo. LIF1 shares limited sequence homology with mammalian XRCC4. Disruption of the LIF1 gene abolishes the capacity of cells to recircularize transformed linearized plasmids correctly by non-homologous DNA end joining. Loss of LIF1 is also associated with conditional hypersensitivity of cells to ionizing irradiation and with reduced sporulation efficiency. Thus, with respect to their phenotype, lif1 strains are similar to the previously described lig4 mutants. One function of LIF1 is the stabilization of the LIG4 enzyme. The finding of a XRCC4 homologue in S.cerevisiae now allows for mutational analyses of structure-function relationships in XRCC4-like proteins to define their role in DNA double-strand break repair.

Amino Acid Sequence↗
PubMed1998

Enhanced onset of platelet inhibition with a loading dose of ticlopidine in ASA-treated stable coronary patients.

Antiplatelet therapy reduces the rate of complications of coronary angioplasty. Ticlopidine, in addition to acetyl salicytic acid (ASA), improves the results after coronary stenting, but early complications still occur. Ticlopidine given in the standard way has a slow onset of action. Eighteen ASA-treated stable coronary patients were randomized to standard ticlopidine treatment (250 mg twice daily) or to a loading dose of 1500 mg followed by 250 mg twice daily. After one day, standard treatment had a very modest antiplatelet effect, as assessed by ADP-induced platelet fibrinogen binding, whereas the loading dose resulted in a considerably better platelet inhibition; relative inhibition, 5.6-10.6 vs. 24.9-35.3% (p<0.005) with final ADP concentrations 3 and 0.6 microM, respectively. It is possible that a loading dose of ticlopidine, given the day before the procedure, might reduce the complications related to angioplasty and stenting.

Angina Pectoris↗
PubMed1998

Association between deficiency of free protein S and anticardiolipin antibodies in patients </= 65 years of age with acute ischemic stroke and TIA.

A possible association between anticardiolipin antibodies (ACA), which are a marker for increased risk of cerebral ischemia, and deficiency of free Protein S, a naturally occurring anticoagulant, has been suspected in some studies of ischemic stroke, particularly in young adults. In order to investigate this further, we prospectively studied all stroke patients </= 65 years of age admitted to our stroke unit during 1991-1992. A total of 66 patients with acute ischemic stroke or transient ischemic attacks (TIA) (embolic/thrombotic infarction n = 30, embolic infarction n = 13, thrombotic infarction n = 10, and TIA n = 13) were analysed for ACA, protein C and S, free protein S and antithrombin III (AT III). Traditional risk factors were scrutinized in each patient. Eight patients had some previously undetected derangement of the coagulation process; five had elevated ACA levels, four had low, free Protein S levels, and three had low AT III levels. None of the patients showed any decrease in total protein C or S levels. A striking association between the presence of ACA and free protein S deficiency was noted. All patients with free protein S deficiency had concomitant elevated ACA levels. Sixteen patients had had a previous episode of ischemic stroke/TIA or mycocardial infarction, two of them had lowered AT III levels. Thirty-four patients had one or more elevated infectious parameters but with no clear correlation to derangement of the coagulation factors. We conclude that a probable association between ACA and free protein S deficiency exist in ischemic stroke patients, and that it may have a pathogenetic importance. Copyright 1998 Lippincott Williams & Wilkins

Journal Article↗
PubMed1998

Impaired platelet function in endotoxemic pigs analyzed by flow cytometry.

Endotoxin or bacterial lipopolysaccharide (LPS) is a structural component of Gram-negative bacteria. It is believed to be the major pathogenic factor of Gram-negative sepsis, and may result in intravascular coagulation and in a shock syndrome that is characterized by thrombocytopenia, leucopenia, hypotension, fever, reduced delivery of oxygen, metabolic acidosis and ultimately death. We have previously shown that both endotoxemic pigs and patients with Gram-negative sepsis have elevated levels of platelet microvesicles in their blood, which indicates platelet activation. In this study, we have used flow cytometry and fluorescein-labeled chicken anti-human fibrinogen to evaluate the in vivo effect of endotoxin on platelet function in a porcine model. Endotoxin infusion in pigs caused impaired platelet function when platelets were stimulated with adenosine-diphosphate in vitro ( P < 0.001). We also found a similarly decreased platelet function in patients with Gram-negative sepsis. Since flow cytometry is a rapid method for determination of platelet function, this method may turn out to be a useful tool in clinical situations. Our results may contribute to our understanding of the bleeding problems that may occur in septic shock and in disseminated intravascular coagulation.

Journal Article↗
PubMed1997

A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks.

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase.

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7, 8-dihydroguanine). In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen.

Amino Acid Sequence↗
PubMed1997

Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.

Cell-Free System↗
PubMed1997

XRCC1 protein interacts with one of two distinct forms of DNA ligase III.

Human DNA ligase III (103 kDa) has been shown to interact directly with the 70 kDa DNA repair protein, XRCC1. Here, the binding sites have been defined. Subcloned fragments of XRCC1 have been expressed and assayed for their ability to associate with DNA ligase III by far Western and affinity precipitation analyses. The C-terminal 96 amino acids of XRCC1 are necessary and sufficient for the specific interaction with DNA ligase III. A similar approach with the 103 kDa DNA ligase III has identified the C-terminal 148 amino acids of this enzyme as containing the binding site for XRCC1. An alternative 96 kDa form of DNA ligase III, abundant in testes, has been described [Chen, J., et al. (1995) Mol. Cell. Biol. 15, 5412-5422]. These two forms of DNA ligase III have identical N-terminal regions but differ toward their C termini and may be alternatively spliced products of the same gene. Antipeptide antibodies directed against the different C termini of the two forms of the enzyme indicate that both of them occur in vivo. The C-terminal region of the 96 kDa derivative of DNA ligase III is not able to interact with XRCC1. These findings indicate that only the larger form of DNA ligase III acts together with XRCC1, suggesting a role for this isoform of the enzyme in base excision repair.

Amino Acid Sequence↗
PubMed1997

Specific function of DNA ligase I in simian virus 40 DNA replication by human cell-free extracts is mediated by the amino-terminal non-catalytic domain.

The joining of Okazaki fragments during lagging strand DNA replication in mammalian cells is believed to be due to DNA ligase I. This enzyme is composed of a 78-kDa carboxyl-terminal catalytic domain and a 24-kDa amino-terminal region that is not required for ligation activity in vitro. Extracts of the human cell line 46BR.1G1, in which DNA ligase I is mutationally altered, supported aberrant in vitro SV40 DNA replication; the joining of Okazaki fragments was defective, and unligated intermediates were unstable. Human DNA ligase I, but not DNA ligase III or bacteriophage T4 DNA ligase, complemented both defects in 46BR.1G1 extracts. The catalytic domain of DNA ligase I was 10-fold less effective in complementation experiments than the full-length protein, indicating that the amino-terminal region of the enzyme is required for efficient lagging strand DNA replication. Moreover, in vitro SV40 DNA replication in normal human cell extracts was inhibited by an excess of either full-length DNA ligase I or the amino-terminal region of the protein, but not by the catalytic domain. This inhibition may be mediated by the interaction of the amino-terminal region of DNA ligase I with other replication proteins.

Cell Line↗