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T Lindahl

Publications and source records attributed to T Lindahl.

At least 73 records · Page 4Linked to original sources
PubMed1994

Dual function for poly(ADP-ribose) synthesis in response to DNA strand breakage.

Soluble extracts of human cells repair gamma-ray-induced single-strand breaks in DNA. Accompanying NAD-dependent automodification of poly(ADP-ribose) polymerase is required for effective DNA rejoining. The kinetics of poly(ADP-ribose) synthesis by this polymerase, and subsequent polymer degradation by poly(ADP-ribose) glycohydrolase, have been compared with the rate of DNA repair. The results agree with previous in vivo data. In response to addition of gamma-irradiated plasmid DNA, rapid and heavy automodification of poly(ADP-ribose) polymerase occurred in NAD-containing human cell extracts. After 2 min at 30 degrees C, when very little DNA rejoining had yet occurred, synthesis of long polymers essentially ceased, although only a minor fraction of the NAD had been consumed. Poly(ADP-ribose) chains were then reduced to oligomer size by poly(ADP-ribose) glycohydrolase. These short chains were present for longer times and were sufficient to permit DNA repair. Thus, most but not all poly(ADP-ribose) synthesis could be suppressed without marked inhibition of DNA repair, and prolonged occurrence of long poly(ADP-ribose) chains in consequence to glycohydrolase inhibition did not improve DNA repair. The temporary presence of short poly(ADP-ribose) chains on poly(ADP-ribose) polymerase avoids inhibition of excision-repair by that protein, but the initial very transient formation of long and branched chains of poly(ADP-ribose) in response to DNA damage apparently serves an entirely different purpose. Local poly(ADP-ribose) synthesis in the vicinity of a DNA strand interruption causes negative charge repulsion, and this may function to prevent accidental homologous recombination events within tandem repeat DNA sequences.

Cell Line↗
PubMed1994

Enzymatic repair of oxidative DNA damage.

Oxidative DNA damage, including both mutagenic and cytotoxic lesions, is implicated in aging and cancer. Studies of the processes which correct such damage in mammalian cells are, however, still in their early stages. Here we have summarized our recent work which demonstrates new features of mammalian oxidative DNA damage repair, such as (a) a functional role for poly(ADP-ribosyl)ation in the rejoining of DNA strand breaks and (b) the defective repair of oxidative DNA damage in xeroderma pigmentosum cells.

Animals↗
PubMed1994

Release of 5'-terminal deoxyribose-phosphate residues from incised abasic sites in DNA by the Escherichia coli RecJ protein.

Excision of deoxyribose-phosphate residues from enzymatically incised abasic sites in double-stranded DNA is required prior to gap-filling and ligation during DNA base excision-repair, and a candidate deoxyribophosphodiesterase (dRpase) activity has been identified in E. coli. This activity is shown here to be a function of the E. coli RecJ protein, previously described as a 5'-->3' single-strand specific DNA exonuclease involved in a recombination pathway and in mismatch repair. Highly purified preparations of dRpase contained 5'-->3' exonuclease activity for single-stranded DNA, and homogeneous RecJ protein purified from an overproducer strain had both 5'-->3' exonuclease and dRpase activity. Moreover, E. coli recJ strains were deficient in dRpase activity. The hydrolytic dRpase function of the RecJ protein requires Mg2+; in contrast, the activity of E. coli Fpg protein, that promotes the liberation of 5'-->3'Rp residues from DNA by beta-elimination, is suppressed by Mg2+. Several other E. coli nucleases, including exonucleases I, III, V, and VII, endonucleases I, III and IV and the 5'-->3' exonuclease function of DNA polymerase I, are unable to act as a dRpase. Nevertheless, E. coli fpg recJ double mutants retain capacity to repair abasic sites in DNA, indicating the presence of a back-up excision function.

Apurinic Acid↗
PubMed1994

DNA repair. DNA surveillance defect in cancer cells.

A defect in a protein involved in DNA-mismatch correction accounts for a common type of hereditary colon cancer, adding to the evidence that DNA repair is central to counteracting the transformation of human cells.

Adenosine Triphosphatases↗
PubMed1994

Different active sites of mammalian DNA ligases I and II.

Bovine DNA ligases I and II were adenylylated in the presence of [alpha-32P]ATP and digested with limiting amounts of trypsin or V8 protease. The generation of radioactive peptides of decreasing size was monitored by polyacrylamide gel electrophoresis and autoradiography. Active site peptides obtained by complete proteolytic digestions with trypsin, V8, or Lys-C protease were also compared. The partial digestion products of DNA ligases I and II were entirely different, with no indication of extensive sequence homology. Furthermore, the sequence of the active site region of DNA ligase I is clearly different from that of DNA ligase II. Similar analysis of a third chromatographically distinct mammalian DNA ligase indicated that it is different from DNA ligase I but related to DNA ligase II.

Adenosine Monophosphate↗
PubMed1994

Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I.

Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A subline of identical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially active enzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% of normal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nicked DNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion of nicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNA replication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation of Okazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragments remain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extracts shows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purified normal DNA ligase I.

Adenosine Monophosphate↗
PubMed1993

DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions.

Plasmid DNA was gamma-irradiated or treated with H2O2 in the presence of Cu2+ to generate oxygen free radical-induced lesions. Open circular DNA molecules were removed by ethidium bromide/CsCl density gradient centrifugation. The closed circular DNA fraction was treated with the Escherichia coli reagent enzymes endonuclease III (Nth protein) and Fpg protein. This treatment converted DNA molecules containing the major base lesions pyrimidine hydrates and 8-hydroxyguanine to a nicked form. Remaining closed circular DNA containing other oxygen radical-induced base lesions was used as a substrate for nucleotide excision-repair in a cell-free system. Extracts from normal human cells, but not extracts from xeroderma pigmentosum cells, catalyzed repair synthesis in this DNA. The repair defect in the latter extracts could be specifically corrected by in vitro complementation. The data suggest that accumulation of endogenous oxidative damage in cellular DNA from xeroderma pigmentosum patients contributes to the increased frequency of internal cancers and the neural degeneration occurring in serious cases of the syndrome.

Cell Line↗
PubMed1993

Instability and decay of the primary structure of DNA.

Although DNA is the carrier of genetic information, it has limited chemical stability. Hydrolysis, oxidation and nonenzymatic methylation of DNA occur at significant rates in vivo, and are counteracted by specific DNA repair processes. The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and ageing, and also sets limits for the recovery of DNA fragments from fossils.

Aging↗
PubMed1993

NAD(+)-dependent repair of damaged DNA by human cell extracts.

Rejoining of DNA single-strand breaks generated by treatment of plasmids with gamma-rays, neocarzinostatin, or bleomycin was catalyzed inefficiently by human cell extracts. The reaction was strongly promoted by the addition of NAD+, which was employed for rapid and transient synthesis of poly(ADP-ribose). The DNA rejoining reaction was accompanied by DNA repair replication, apparently due to replacement of damaged residues at termini. Selective depletion of poly(ADP-ribose) polymerase from cell extracts improved the repair of DNA exposed to a variety of DNA-damaging agents by removing the NAD+ dependence of the repair reaction. NAD(+)-promoted DNA repair by soluble cell extracts also occurred with alkylated DNA as substrate and was suppressed by 3-aminobenzamide. A similar stimulatory effect by NAD+ was observed for repair of ultraviolet-irradiated DNA, and this could be ascribed to the presence of pyrimidine hydrates as minor radiation-induced DNA lesions. No effect was observed on the sealing of gamma-irradiated DNA by supplementation of cell extracts with purified mammalian DNA ligase I or DNA ligase II. The results indicate that poly(ADP-ribose) polymerase interferes with base excision-repair processes because bound enzyme molecules block DNA strand interruptions. Release of bound poly-(ADP-ribose) polymerase following automodification, or physical removal of the protein from reaction mixtures, facilitates DNA repair.

Adenine Phosphoribosyltransferase↗
PubMed1993

Induction of the adaptive response of Escherichia coli to alkylation damage by the environmental mutagen, methyl chloride.

Methyl chloride (MeCl) is an abundant environmental mutagen and carcinogen and may be one of several environmental alkylating agents against which the protection of an adaptive response is required in microorganisms. Both MeCl and methyl iodide (MeI), at micromolar concentrations, induced the adaptive response to alkylation damage in Escherichia coli. This response is regulated by the Ada protein which is converted into a transcriptional activator by self-methylation on repair of methylphosphotriesters in methylated DNA. However, using high amounts of Ada protein, activation of Ada occurred in vitro following direct protein methylation by both MeI (in agreement with previously published data) and MeCl. Activation was enhanced when methyl halide treatments were performed in the presence of DNA. An unadapted E. coli cell contains only 2 to 4 molecules of Ada protein, and presents an extremely small target of 2 to 4 specific cysteine residues per cell for activation of Ada by direct protein methylation in vivo. Thus, it is proposed that induction of the adaptive response in vivo initially occurs via efficient repair by the Ada protein of a low number of methylphosphotriesters in DNA. When the cellular Ada protein level has substantially increased, a greater probability of direct methylation and activation of Ada at cysteine-69 by MeCl may sustain and further increase induction of the adaptive response.

Adaptation, Physiological↗
PubMed1993

DNA repair.

Multiple DNA repair processes are required to maintain the integrity of the cellular genome. Recent advances, including elucidation of three-dimensional structures of DNA repair enzymes, and the cloning and characterization of DNA repair genes implicated in human inherited disease, have given new insights into the surprising complexity of cellular responses to DNA damage.

Animals↗
PubMed1993

Chicken anti-protein G for the detection of small amounts of protein G.

Streptococcal protein G, similar to staphylococcal protein A, is the Fc-binding proteins that seems best suited for affinity-purification of mouse, rat or human monoclonal antibodies. We have developed a sandwich-ELISA for monitoring of protein G leakage from such columns. It utilizes microtitre plates coated with chicken anti-protein G to capture protein G, and biotinylated chicken anti-protein G to detect bound protein G. Chicken IgG is one of a very few IgG that does not show any Fc reactivity with protein G. It is thus possible to make an antigen-specific assay for protein G. This assay can be used to detect protein G in IgG containing solutions, such as monoclonal antibody preparations. The method can be used to detect 1 x 10(-7) g protein G/1 in the presence of human serum or human IgG.

Animals↗
PubMed1992

Chicken antibodies: a tool to avoid interference by complement activation in ELISA.

MicroELISA plates coated with mammalian IgG will activate the human complement system. It has been shown that this activation of the complement system may interfere in solid-phase immunometric assays, and that there is a difference between IgG from different species and between different IgG subclasses in their ability to activate the human complement system. We have studied the ability of mammalian IgG and avian IgG to activate the human complement system. We show that chicken IgG do not activate the human complement system, and chicken IgG can thus be used in solid-phase immunometric assays to reduce interference by complement activation.

Animals↗
PubMed1992

Structure of the human DNA ligase I gene.

The gene encoding DNA ligase I, the major DNA ligase activity in proliferating mammalian cells, maps to human chromosome 19q13.2-13.3. We have determined the complete structure of the gene, which is composed of 28 exons spanning 53kb on this chromosome. The first exon is untranslated, and utilises a GC dinucleotide instead of the canonical GT splice donor. The 5' flanking region lacks a TATA box and is highly GC-rich, as is characteristic of a 'housekeeping' gene. In common with the promoters of genes encoding other DNA replication enzymes, such as DNA polymerase alpha, the 5' flanking region of the DNA ligase I gene contains recognition elements for several transcription factors which may mediate increased expression in quiescent cells in response to growth factors.

Base Sequence↗
PubMed1992

Growth retardation and immunodeficiency in a patient with mutations in the DNA ligase I gene.

DNA ligases are required for the replication, repair, and recombination of DNA. A cell line derived from a young woman with growth retardation, sun sensitivity, and immunodeficiencies, who died aged 19 with lymphoma, showed two different miscoding mutations at the DNA ligase I locus on chromosome 19, one in each allele. One of the mutations was inherited from the mother and has also been detected in two healthy brothers. The patient had features similar to those of Bloom's syndrome (BS), but cell lines from BS patients do not have mutations in the DNA ligase I gene. This patient seems to represent a distinct genetic entity.

Cell Line↗
PubMed1992

Mutations in the DNA ligase I gene of an individual with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents.

Two missense mutations occurring in different alleles of the DNA ligase I gene, encoding the major DNA ligase in proliferating mammalian cells, were detected in a human fibroblast strain (46BR). These cells exhibit retarded joining of Okazaki fragments during DNA replication and hypersensitivity to a variety of DNA-damaging agents. 46BR was derived from a patient who displayed symptoms of immunodeficiency, stunted growth, and sun sensitivity. A strongly reduced ability of DNA ligase I to form a labeled enzyme-adenylate intermediate correlated with the genetic defect in 46BR cells. The data indicate that human DNA ligase I is required for joining of Okazaki fragments during lagging-strand DNA synthesis and the completion of DNA excision repair.

Alleles↗