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T Lindahl

Publications and source records attributed to T Lindahl.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cloning and characterization of a functional human homolog of Escherichia coli endonuclease III.

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic/apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron/sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron/sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic/apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells.

Adenocarcinoma↗
PubMed1997

Repair and processing events at DNA ends.

Cell nuclei contain several abundant enzymes that bind rapidly and avidly to exposed termini of DNA. The properties and physiological roles of such factors are described; they include poly (ADP-ribose) polymerase, DNA-dependent protein kinase, several DNA ligases and excision-repair enzymes. Telomeres normally seem shielded from these activities by telomere-binding proteins. If incomplete protection of telomeres occurred, the functions of the DNA end-specific enzymes would be relevant for processing of telomeres. This could include alternative pathways for telomere propagation in telomerase-negative cells.

DNA↗
PubMed1997

DNA excision repair pathways.

The major DNA excision repair pathways of base excision repair for endogenous DNA lesions and nucleotide excision repair for DNA damage inflicted by ultraviolet light have been reconstructed with purified mammalian proteins and details of these repair mechanisms are emerging. Similar data are becoming available with regard to mismatch repair for correction of replication errors. Deletion of individual DNA repair proteins in knockout mice provides information on the roles of such factors in vivo and recent three-dimensional structures of several repair enzymes explain their detailed modes of action.

Animals↗
PubMed1997

Age-specific reference values for serum prostate-specific antigen in a community-based population of healthy Swedish men.

To establish normal reference values for prostate-specific antigen (PSA) in a Swedish population we investigated 878 healthy men, 56-75 years of age. They were randomly selected from a population of 9171 males in this group. Cancer of the prostate was excluded by digital rectal examination. When digital rectal examination was suspicious for carcinoma of the prostate and/or serum PSA > 4 micrograms l-1, fine-needle aspiration biopsy was performed. Central values, values of variance and reference limits were defined by a non-parametric method in four age groups. A strong positive correlation between PSA values and age was found and the variance increased with age. The relationship between PSA value and age was non-linear. For the age group 56-60 the upper reference limit (95th percentile) was 4.6 micrograms l-1 (confidence interval, CI: 3.9-5.5). For the age groups 61-65, 66-70 and 71-75 the corresponding values were 4.4 (3.8-5.2), 7.6 (6.5-8.9) and 8.4 micrograms l-1 (7.2-9.8) respectively. For the age groups studied the increment over time of the PSA value was 2-8% per year depending on age, with an average increment per year over 15 years of 4.3%. Overall, 11% of our reference sample had a serum PSA level > 4 micrograms l-1. We consider our study population to be representative for a normal Swedish male population in these age groups.

Aged↗
PubMed1996

Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.

Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.

Base Composition↗
PubMed1996

The Croonian Lecture, 1996: endogenous damage to DNA.

Although DNA is the carrier of stable genetic information, this giant molecule exhibits slow turnover in cells as a consequence of endogenous damage. DNA lesions result from hydrolysis, and from exposure to active oxygen and reactive metabolites. These major forms of damage to the heterocyclic bases and to the DNA backbone structure are now well characterized. Most DNA repair enzymes have apparently evolved to prevent genomic instability caused by endogenous lesions, the only exception being those that counteract ultraviolet light damage inflicted by the sun. Despite the efficiency of DNA repair pathways, some forms of endogenous DNA damage still cause mutagenic alterations and may result in human disease.

DNA Damage↗
PubMed1996

DNA ligase IV from HeLa cell nuclei.

A human cDNA encoding a previously unrecognized DNA ligase IV has been identified (Wei, Y.-F., Robins, P., Carter, K., Caldecott, K., Pappin, D. J. C., Yu, G.-L., Wang, R.-P., Shell, B. K., Nash, R. A., Schär, P., Barnes, D. E., Haseltine, W. A., and Lindahl, T. (1995) Mol. Cell. Biol. 15, 3206-3216). Antibodies have been raised against predicted peptide sequences of DNA ligase IV and used to identify the enzyme during purification from HeLa cell nuclei. The 96-kDa DNA ligase IV and the 103-kDa DNA ligase III co-migrate during SDS-polyacrylamide gel electrophoresis and have similar column fractionation properties, which complicates the distinction between the two enzymes, but they have been separated by Mono S liquid chromatography. During initial size fractionation by gel chromatography in 1 M NaCl, DNA ligase IV elutes in the same position as the DNA ligase III-XRCC1 protein complex, indicating that DNA ligase IV is also bound to another protein or occurs as a dimer. DNA ligase IV has been purified free from other DNA ligases, and its enzymatic properties have been examined. The purified protein effectively joins single-strand breaks in a double-stranded polydeoxynucleotide in an ATP-dependent reaction. The substrate specificity of DNA ligase IV differs from those of the other two cloned human DNA ligases, I and III, with regard to their ability to join the hybrid substrates oligo(dT).poly(rA) and oligo(rA).poly(dT). DNA ligase IV occurs in part as an enzyme-adenylate complex in HeLa cell nuclear extracts.

Adenosine Triphosphate↗
PubMed1996

Molecular cloning and functional analysis of a Schizosaccharomyces pombe homologue of Escherichia coli endonuclease III.

The Escherichia coli endonuclease III (Nth-Eco) protein is involved in the removal of damaged pyrimidine residues from DNA by base excision repair. It is an iron-sulphur enzyme possessing both DNA glycosylase and apurinic/apyrimidinic lyase activities. A database homology search identified an open reading frame in genomic sequences of Schizosaccharomyces pombe which encodes a protein highly similar to Nth-Eco. The gene has been subcloned in an expression vector and the protein purified to apparent homogeneity. The S.pombe Nth homologue (Nth-Spo) is a 40.2 kDa protein of 355 amino acids. Nth-Spo possesses glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polymers. The eukaryotic protein removes urea more efficiently than the prokaryotic enzyme, whereas its efficiency in excising thymine glycol is lower. A nicking assay was used to show that the enzyme also exhibits an AP lyase activity on UV- and gamma-irradiated DNA substrates. These findings show that Nth protein is structurally and functionally conserved from bacteria to fission yeast.

Amino Acid Sequence↗
PubMed1995

Repair and recombination. How to make ends meet.

The repair of double-stranded breaks in DNA and the recombination of antibody gene V(D)J segments share a common pathway involving the Ku protein, which binds DNA ends, and its associated protein kinase.

Animals↗
PubMed1995

Enzymes acting at strand interruptions in DNA.

Endogenous and environmental DNA-damaging agents often generate single-strand interruptions in DNA. The lesions trigger a complex set of cellular reactions. In most eukaryotic cells, cellular poly(ADP-ribose) formation is the most acute response to such damage. Recently, such events have been amenable to study with soluble cell-free extracts of human cells. These investigations clarify the modulating role on DNA repair by poly (ADP-ribose), and suggest that the primary function of this unusual polymer is to act as an antirecombinant agent. Similar biochemical studies of subsequent repair events have revealed a branched pathway for the ubiquitous DNA base excision-repair process. The alternative pathway provides the cell with back-up functions for individual steps in this essential form of DNA repair.

Animals↗
PubMed1995

Post-translational modification of poly(ADP-ribose) polymerase induced by DNA strand breaks.

There are one million molecules of poly(ADP-ribose) polymerase (PARP) in mammalian cell nuclei and the enzyme is found in most eukaryotes, with the notable exception of yeasts. In response to DNA damage caused by ionizing radiation or alkylating agents, PARP binds to strand interruptions in DNA and undergoes rapid automodification with synthesis of long branched polymers of highly negatively charged poly(ADP-ribose). DNA repair occurs after dissociation of modified PARP from DNA strand breaks. Biochemical data with enzyme-depleted extracts and studies of enzyme-deficient mice show that PARP does not participate directly in DNA repair. Possible roles for poly(ADP-ribose) synthesis are discussed.

Animals↗
PubMed1995

Recognition and processing of damaged DNA.

Base excision-repair, which is required for correction of spontaneous hydrolytic and oxidative damage to DNA as well as lesions inflicted by alkylating agents, is a relatively well understood repair pathway. Mammalian factors involved in this pathway are reviewed, with emphasis on current uncertainties. Most DNA replication and repair enzymes in mammalian cell nuclei, e.g. DNA polymerases alpha, beta, delta, and epsilon, have direct counterparts in yeast. In contrast, the abundant enzymes in mammalian cell nuclei that bind and are activated specifically by DNA strand interruptions, poly(ADP-ribose) polymerase and DNA-dependent protein kinase, have not been detected in yeast; nor has p53, which is elevated in response to DNA strand breaks. We have found a family of four distinct DNA ligases in human cell nuclei, whereas only a single DNA ligase has been detected in yeast. It would appear that the cellular responses to DNA strand breaks may differ markedly between higher and lower eukaryotes.

Amino Acid Sequence↗
PubMed1994

Partial expression of GP Ib measured by flow cytometry in two patients with Bernard-Soulier syndrome.

Bernard-Soulier syndrome is a rare, congenital bleeding disorder caused by absent or defective GP Ib platelet membrane receptor for the von Willebrand factor (vWF). We studied two brothers with moderate bleeding symptoms. Bleeding time was prolonged and ristocetin-induced platelet aggregation was absent. Flow cytometric analysis showed that both boys had a subnormal expression of GP Ib. One antibody used (AN51) was bound only to 30% of the platelets and at a subnormal density. A second antibody (SZ2) also bound at a subnormal density but a normal fraction of the platelets were immunoreactive. Ristocetin stimulation of the patients' platelets in the presence of plasma resulted in a low binding of vWF, about 30% of healthy controls. On the other hand the expression of GP IIb/IIIa on the platelet membrane appeared to be supernormal even when the increased platelet size was taken into account as shown by the ratio between the density of GP IIIa and CD 9 structures. We conclude that these brothers have a variant of the Bernard-Soulier syndrome with a low expression of a GP Ib receptor.

Bernard-Soulier Syndrome↗
PubMed1994

Reconstitution of the DNA base excision-repair pathway.

BACKGROUND: The base excision-repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision-repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision-repair, we have attempted to reconstitute the pathway in vitro using purified enzymes. RESULTS: We report here the successful reconstitution of the base excision-repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5' terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5' nuclease function of DNA polymerase I. CONCLUSIONS: Repair of uracil in DNA is achieved by a very short-patch excision-repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.

Bacterial Proteins↗
PubMed1994

Structural and functional homology between mammalian DNase IV and the 5'-nuclease domain of Escherichia coli DNA polymerase I.

A nuclear 42-kDa 5'-->3'-exonuclease, DNase IV, was found previously in animal tissues. The enzyme has been purified from HeLa cells and shown to possess two catalytic properties characteristic of the 5'-nuclease function of Escherichia coli DNA polymerase I,-DNase IV removes single-stranded 5' regions from splayed-arm DNA structures by endonucleolytic incision at the bifurcation point and possesses RNase H activity. Determination of the molecular masses of tryptic and V8 peptides of DNase IV by mass spectrometry identified the enzyme as the human homolog of the Schizosaccharomyces pombe Rad2 protein. The protein sequence retains conserved residues and shows significant homology to the sequences of the 5'-nuclease domain of E. coli DNA polymerase I and related microbial enzymes.

Amino Acid Sequence↗