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T Lindahl

Publications and source records attributed to T Lindahl.

At least 19 recordsLinked to original sources
PubMed2001

Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.

DNA Damage↗
PubMed2001

Excision of deaminated cytosine from the vertebrate genome: role of the SMUG1 uracil-DNA glycosylase.

Gene-targeted mice deficient in the evolutionarily conserved uracil-DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung(-) mutants. A complementary uracil-DNA glycosylase activity detected in ung(-/-) murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil-DNA glycosylase in UNG-deficient mice. SMUG1 is present at similar levels in cell nuclei of non-proliferating and proliferating tissues, indicating a replication- independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti-mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA.

Animals↗
PubMed2001

BRCT domain interactions in the heterodimeric DNA repair protein XRCC1-DNA ligase III.

Proteins involved in DNA repair, or its coordination with DNA replication and mitosis through cell cycle checkpoints, are vital in the concerted cellular response to DNA damage that maintains the integrity of the genome. The "BRCT" domain (BRCA1 carboxy terminal) was noted as a putative protein-protein interaction motif in the breast cancer suppressor gene, BRCA1, and subsequently identified in over 50 proteins involved in DNA repair, recombination, or cell cycle control. The heterodimer of the DNA repair proteins, XRCC1 and DNA ligase III, was the first example of a functional interaction via BRCT modules. The only three-dimensional crystal structure of a BRCT domain was solved for this region of XRCC1. Key amino acid residues mediating the interaction with DNA ligase III were identified here by targeted mutagenesis of the XRCC1 BRCT domain. The consequences of these mutations on protein folding were assessed. A structural model of the DNA ligase III BRCT domain was constructed and similarly tested by mutation of corresponding residues required for the interaction with XRCC1. These data identify the XRCC1-DNA ligase III heterodimer interface and provide the first demonstration of the surface contacts coordinating a functional BRCT-BRCT protein interaction.

Amino Acid Substitution↗
PubMed2001

The expression of vascular endothelial growth factor correlates with mutant p53 and poor prognosis in human breast cancer.

Wild-type p53 protein has been shown to inhibit angiogenesis through thrombospondin in the preclinical setting. Here, we determined the associations between the expression of the angiogenic factor vascular endothelial growth factor (VEGF) and the p53 status, including different mutation sites and types, in primary breast cancer. Cytosols from 224 primary breast cancer patients were analyzed with an enzyme immunoassay for determination of human VEGF165 protein content. p53 status was determined by cDNA-based sequencing of the entire coding region, by immunohistochemistry (IHC), and by a p53 luminometric immunoassay (LIA) method. Statistically significant associations was found between higher VEGF content and non-wild-type p53 status for all methods; sequence-based data (P = 0.0019), IHC data (P = 0.0068), and the LIA method (r = 0.427; P > 0.001). Highest VEGF values were detected in tumors with p53 insertions, deletions, and stop codon mutations (P = 0.0043). Combining p53 status and VEGF content resulted in additional prognostic information, relapse-free survival (RFS; P = 0.0377), overall survival (OS; P = 0.0319), and breast cancer corrected survival (BCCS; P = 0.0292). In multivariate analysis, the relative hazard increased when the VEGF data were added to the p53 status, with a relative hazard of 1.7 for RFS and 3.0 for BCCS, compared with 1.1 for RFS and 1.4 for BCCS among the patients with either high VEGF content or p53 mutation. Higher VEGF content was statistically significantly correlated with a worse outcome for patients with estrogen receptor-positive tumors receiving adjuvant tamoxifen: RFS (P = 0.0471), OS (P = 0.0134), BCCS (P = 0.0064), as well as in multivariate analysis with point estimates of 3.4 and 2.1 for BCCS and RFS, respectively. VEGF expression is related to p53 status in human breast cancer patients. Combining VEGF with p53 status resulted in better prognostic prediction.

Adult↗
PubMed2001

Human DNA repair genes.

Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process.

DNA↗
PubMed2001

Keynote: past, present, and future aspects of base excision repair.

Covalent alterations of DNA bases, which may have promutagenic or cytotoxic effects, are major consequences of endogenous DNA damage caused by hydrolysis, reactive oxygen species, and several metabolites and coenzymes. A common strategy for initiation of DNA base excision repair (BER) involves a DNA glycosylase that binds the altered deoxynucleoside in an extrahelical position and catalyzes cleavage of the base-sugar bond. Subsequently, an AP endonuclease or AP lyase activity incises the abasic site, followed by short-patch gap-filling, excision of the base-free sugar-phosphate residue, and ligation. The initial work that resulted in the discovery of DNA glycosylases and AP endonucleases is briefly reviewed. In recent years, it has been shown that the latter steps of the BER pathway differ greatly between mammalian cells and microorganisms such as yeast and bacteria. Three distinct subpathways of BER occur in mammalian cells, and these have been individually reconstituted with purified enzymes. Gene knockout mice are now revealing specific roles and backup mechanisms for repair functions in murine cells, and the results in general are also applicable to human cells. Future developments in the field of base excision repair include definition by proteomics of all factors involved in handling many different types of DNA lesions, clarification of mechanisms of repair of chromatin at a high level of accuracy, manifestation of repair proteins as drug targets for cellular sensitization to ionizing radiation and anticancer medicines, and elucidation of cross-talk between the base excision repair factors and other cellular proteins involved in a variety of stress responses.

Aging↗
PubMed2001

Age-related and tissue-specific accumulation of oxidative DNA base damage in 7,8-dihydro-8-oxoguanine-DNA glycosylase (Ogg1) deficient mice.

Mutations that influence the repair of oxidative DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation. We have analysed the steady-state levels and repair kinetics of oxidative DNA modifications in cells of homozygous ogg1(-/-) null mice, which are deficient in Ogg1 protein, a DNA repair glycosylase that removes the miscoding base 8-hydroxyguanine (8-oxoG) from the genome. Oxidative purine modifications including 8-oxoG were quantified by means of an alkaline elution assay in combination with Fpg protein, the bacterial functional analogue of Ogg1 protein. In primary hepatocytes of adult ogg1(-/-) mice aged 9-12 months, the steady-state level of the lesions was 2.8-fold higher than in wild-type control mice. In contrast, no difference between ogg1(-/-) and wild-type mice was observed in splenocytes, spermatocytes and kidney cells. In hepatocytes of ogg1(-/-) mice, but not in wild-type controls, the steady-state levels increased continuously over the whole lifespan. No significant accumulation of the oxidative base modifications was observed in ogg1(-/-) fibroblasts in culture when they were kept confluent for 8 days. Both in confluent and proliferating ogg1(-/-) fibroblasts, the global repair of additional oxidative base modifications induced by photosensitization was 4-fold slower than in wild-type cells. The results suggest that the consequences of an Ogg1 defect are restricted to slowly proliferating tissues with high oxygen metabolism such as liver, because of a back-up mechanism for the repair of 8-oxoG residues that is independent of transcription and replication.

Age Factors↗
PubMed2001

Enzymatic mutation detection method evaluated for detection of p53 mutations in cDNA from breast cancers.

BACKGROUND: Rapid, reproducible, and easily run methods with high sensitivity and specificity are required for mutation screening of clinical samples. We evaluated the Enzymatic Mutation Detection (EMD(TM)) method by analysis of archival cDNA from 203 breast cancer patients and comparison with results of cDNA-based sequencing of the tumor suppressor gene p53. METHODS: The EMD technology uses the T4 endonuclease VII, which cleaves double-stranded DNA at sites where a DNA mismatch is present because of mispairing or an insertion/deletion of nucleotides. The EMD analyses were carried out by dividing the p53 gene into two overlapping fragments that were analyzed separately. After PCR amplification, the fragments were hybridized with wild-type p53 and subsequently exposed to the EMD enzyme. Cleavage products were analyzed and scored using an ALF(TM) automated DNA sequencer and ALFwin Fragment Analyzer software (VER: 1.02). RESULTS: The EMD technique had sensitivities of 45% and 64% and specificities of 83% and 84% for the two fragments, respectively. Patients with EMD-positive, wild-type p53 tumors had a survival similar to that of patients with EMD-negative, wild-type p53 tumors. Node-positive patients with p53 mutated tumors according to sequencing had a statistically significantly worse overall survival than those with p53 wild-type tumors (P = 0.016), whereas this difference in survival was not detected when p53 status was determined with EMD (P = 0.47). CONCLUSIONS: EMD had insufficient sensitivity for consideration in screening for the p53 gene in this archival material. Sequencing must still be considered as the standard procedure.

Breast Neoplasms↗
PubMed2000

Defective neurogenesis resulting from DNA ligase IV deficiency requires Atm.

Ataxia telangiectasia results from mutations of ATM and is characterized by severe neurodegeneration and defective responses to DNA damage. Inactivation of certain DNA repair genes such as DNA ligase IV results in massive neuronal apoptosis and embryonic lethality in the mouse, indicating the occurrence of endogenously formed DNA double-strand breaks during nervous system development. Here we report that Atm is required for apoptosis in all areas of the DNA ligase IV-deficient developing nervous system. However, Atm deficiency failed to rescue deficits in immune differentiation in DNA ligase IV-null mice. These data indicate that ATM responds to endogenous DNA lesions and functions during development to eliminate neural cells that have incurred genomic damage. Therefore, ATM could be important for preventing accumulation of DNA-damaged cells in the nervous system that might eventually lead to the neurodegeneration observed in ataxia telangiectasia.

Animals↗
PubMed2000

Fossil DNA.

Explore the source record for details and available documents.

Animals↗
PubMed2000

Defective processing of methylated single-stranded DNA by E. coli AlkB mutants.

Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated lambda phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by gamma irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells.

Bacterial Proteins↗
PubMed2000

Removal of oxygen free-radical-induced 5',8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells.

Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base excision-repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5' position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5'R or 5'S form of 5',8-cyclo-2-deoxyadenosine was a substrate for the human nucleotide excision-repair enzyme complex. The R diastereoisomer was more efficiently repaired than the S isomer. No correction of the lesion by direct damage reversal or base excision repair was detected. Dual incision around the lesion depended on the core nucleotide excision-repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals.

Base Sequence↗
PubMed2000

Distinct repair activities of human 7,8-dihydro-8-oxoguanine DNA glycosylase and formamidopyrimidine DNA glycosylase for formamidopyrimidine and 7,8-dihydro-8-oxoguanine.

7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and are involved in mutagenic and/or lethal events in cells. Both lesions are repaired by human 7, 8-dihydro-8-oxoguanine DNA glycosylase (hOGG1) and formamidopyrimidine DNA glycosylase (Fpg) in human and Escherichia coli cells, respectively. In the present study, the repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The k(cat)/K(m) values of hOGG1 for 8-oxoG and me-Fapy were comparable, and this was also the case for Fpg. However, the k(cat)/K(m) values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg. Analysis of the Schiff base intermediate by NaBH(4) trapping implied that lower substrate affinity and slower hydrolysis of the intermediate for hOGG1 than Fpg accounted for the difference. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8- (hOGG1) and 12-fold (Fpg) between the most and least preferred bases. Surprisingly, such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair. The repair efficiency of me-Fapy randomly incorporated in M13 DNA varied at the sequence level, but orders of preferred and unpreferred repair sites were quite different for hOGG1 and Fpg. The distinctive activities of hOGG1 and Fpg including enzymatic parameters (k(cat)/K(m)), paired base, and sequence context effects may originate from the differences in the inherent architecture of the DNA binding domain and catalytic mechanism of the enzymes.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication.

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.

Animals↗
PubMed2000

Suppression of spontaneous mutagenesis in human cells by DNA base excision-repair.

The chemical instability of the covalent structure of DNA, and in vivo exposure of DNA to reactive oxygen species and endogenously produced alkylating agents, has triggered the evolution of several specific DNA repair pathways. A major strategy of repair involves the initial removal of an altered base from DNA by a member of the enzyme family of DNA glycosylases. The currently known enzymes of this type in mammalian cells are reviewed, and the subsequent base excision-repair (BER) steps that achieve restoration of the intact DNA structure are also described. The specific problem of retaining high accuracy in this essentially error-free repair process is discussed.

Animals↗
PubMed2000

Can axillary dissection be avoided by improved molecular biological diagnosis?

Axillary dissection is presently a routine staging procedure in the management of breast cancer. The use of adjuvant systemic treatment is largely based on the diagnosis of axillary metastases. Routine axillary dissection leads to acute and chronic side-effects in a large proportion of patients. The sentinel node technique is presently explored with the aim of decreasing the need for standard axillary dissection. A complementary way forward is to analyse the primary breast cancer for molecular markers with prognostic significance with reference to the risk for metastatic capacity and thereby obtain a 'biological staging' and identify those patients in need of systemic adjuvant therapy. A large number of molecular biological factors have been shown to have prognostic significance in breast cancer e.g. c-erbB-2, p53, uPA, PAI-I and VEGF. This article reviews the expression of these and other factors in the primary breast cancers in relation to the risk for axillary and systemic metastatic disease, with the long-term aim of excluding routine axillary dissection.

Axilla↗
PubMed1999

Quality control by DNA repair.

Faithful maintenance of the genome is crucial to the individual and to species. DNA damage arises from both endogenous sources such as water and oxygen and exogenous sources such as sunlight and tobacco smoke. In human cells, base alterations are generally removed by excision repair pathways that counteract the mutagenic effects of DNA lesions. This serves to maintain the integrity of the genetic information, although not all of the pathways are absolutely error-free. In some cases, DNA damage is not repaired but is instead bypassed by specialized DNA polymerases.

Animals↗