Hans Krokan

Accumulation of small fragments of DNA in isolated HeLa cell nuclei due to transient incorporation of dUMP

[3H]dUMP was incorporated into DNA of isolated S-phase HeLa S3 cell nuclei during DNA synthesis. The incorporated radioactivity was made acid soluble during a chase with excess TTP. A partially purified DNA polymerase alpha incorporated [3H]dUMP into activated salmon sperm DNA. The incorporation rate was equal to the incorporation of [3H]TMP, and the radioactivity incorporated was not made acid soluble during a chase. The nuclei thus have the ability to remove misincorporated uracil. From cytosol we have partially purified an enzyme (80 times purification) that splits the N-glycosidic bond between uracil and deoxyribose in dUMP-containing DNA. This uracil-N-glycosidase has a molecular weight of about 50 000. It does not accept dUTP or RNA as substrates. Pulse labelling of isolated nuclei with radioactive deoxyribonucleoside triphosphates in the presence of dUTP lead to a large accumulation of label in small DNA fragments. The size of these fragments was about 80 nucleotides in a 60 s pulse and no increase in size was observed with increasing pulse length. The corresponding value for control experiments with no dUTP, was 200 nucleotides and the fragments increased in size with increasing pulse length. About 90% of the radioactivity was found in the small fragments after a 3 min pulse when the concentration of dUTP in the test mixture was 100 micrometer and no exogenous TTP was present. In control experiments with no dUTP present, only 14% of the radioactivity was found in small DNA pieces. When test mixture containing dUTP was preincubated with cytosol for 60 s before adding the isolated nuclei, the small fragments increased in size to that of DNA fragments found in control incubations; also the relative amount of label bound to the fragments returned to the levels found in the controls. Increasing the TTP concentration from 5 micrometer to 1.88 mM in the absence of exogenous dUTP had no effect on the size of the DNA fragments.

Uracil-DNA glycosylase in HeLa S3 cells: interconvertibility of 50 and 20 kDa forms and similarity of the nuclear and mitochondrial form of the enzyme.

Uracil-DNA glycosylase activity in HeLa S3 cells was found in nuclei (70%), mitochondria (15%) and cytosol (15%) after fractionation in hypotonic buffers. After fractionation in isotonic buffers the activity in cytosol was increased, apparently as a consequence of leakage from the nuclei. Both in the nuclear and the mitochondrial fraction, a major 50 and a minor 18 kDa form were found after gel filtration in the presence of 0.5 M NaCl. However, after glycerol, gradient sedimentation or gel filtration in the presence of 2 M NaCl or 20% glycerol most of the 50 kDa form dissociated into a 22 kDa form, which was also the smallest catalytically active form found after partial trypsin digestion. The dissociation of the 50 kDa form was reversible. Biochemical properties of the nuclear and mitochondrial forms were very similar. Thus, they had similar apparent Km values, pH optima, heat sensitivities and activation energies, and were stimulated 2-5-fold by 40-60 mM monovalent salt.

CHARACTERIZATION OF ANTIBODIES SPECIFIC FOR UV‐DAMAGED DNA BY ELISA

Abstract— The specificity of affinity purified antibodies raised against UV‐irradiated DNA was examined using an enzyme‐linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV‐irradiated DNA was given a second dose of 340‐nm UV light, the binding of antibodies was abrogated. Taken together, this indicates a major specificity for (6‐4)‐photoproducts, which are photochemically reversed by UV light in the 340‐nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with OsO4. Previously, we have used these antibodies for the detection of UV‐induced DNA damage and its repair in human skin in vivo. These findings indicate that (6‐4)‐photoproducts, considered highly mutagenic, are repaired in human skin.

Preferential association of uracil-DNA glycosylase activity with replicating SV40 minichromosomes.

Uracil-DNA glycosylase re_leases free uracil from dUMP-residues in DNA ([l-S], reviewed [6]). Uracil in DNA may arise from misincorporation of dUMP during DNA replication [7-91 or deamination of cytosine in DNA [lo]. Much information is available on the biophysical and biochemical properties [l-6] of uracil-DNA glycosylase, but little is known about how this enzyme is organized in chromatin. Isolated SV40- or polyoma minichromosomes [ 1 l-131 serve as useful models for studies on chromatin proteins. Thus, DNA polymerases (Y and y [13,14-171 as well as T-antigen [ 18,191 are associated with replicating SV40 and/or polyoma minichromosomes, while DNA polymerase 0 and topoisomerase I cosedimented with mature minichromosomes [ 171. Here, we show that uracil-DNA glycosylase, a major DNA repair enzyme, is preferentially Pssociated with replicating SV40 minichromosomes. somes, 0.5 ml extract was layered on a linear 5-30% sucrose gradient in 10 mM Hepes (pH 7.8), 5 mM KC1 and 0.5 mM dithiothreitol [ 1 l] and centrifuged at 37 000 rev./min in a Beckman SW 40 rotor for 150 min at 4°C. Fractions of 0.34 ml were collected from the bottom of the tube. Uracil-DNA glycosylase was tested by incubating aliquots of 30 ~1 from the fractions with 10 ~1 assay buffer (200 mM NaCl, 8 mM EDTA, 160 mM Tris- HCl (pH 7.5) and 12 yM d [3H] UMP-containing DNA, spec. act. 500 /.Li/pmol) for 45 min at 30°C. The release of acid- or ethanol-soluble radioactivity was monitored [5]. The amount of radioactivity released did not exceed 30% of the added radioactivity. The release of [3H]uracil was linear with time (up to 45 min) and with the amount of extract added (up to 30 ~1). Cytochrome c oxidase [22] was determined in aliquots of 100 ~1 from extracts or gradient frac- tions. The detection limit was 0.025 nmol cytochrome c oxidized/min at 25°C. 2.

Effect of cytosol on DNA synthesis in isolated HeLa cell nuclei

Abstract Cytosol obtained by centrifugation of cytoplasm from synchronized S-phase HeLa cells at 200 000 × g for 30 min had a stimulatory effect on the rate and extent of DNA synthesis in isolated nuclei. The cytosol preserved the ability of isolated nuclei to initiate early nascent intermediates (primary DNA pieces). The stimulatory activity was partially separated from the DNA polymerase activity present in the cytosol.

Enzymatic repair of premutagenic DNA lesions in human epidermis Quantitation of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities

Extracts of human epidermis prepared by the suction blister method were used to measure O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activities. Although both activities were detected in all extracts examined, a 4-5-fold interindividual variation in activity was found. No obvious correlation of the two enzyme activities with the age of the patient was observed. Neither was there any correlation between the level of uracil-DNA glycosylase activity and O6-methylguanine-DNA methyltransferase activity.

DNA replication intermediates in whole HeLa cells and isolated nuclei

Replicative intermediates have been studied in intact HeLa cells and in nuclei isolated from such cells. In whole cells the smallest DNA (primary DNA pieces) observed after pulse labelling with [3H]thymidine were 90-160 nucleotides long, and the size of the molecules in this class of DNA did not increase with increasing pulse length. Some increase in size was, however, observed when cells were pulse labelled at 25 degrees C instead of 37 degrees C. Chase experiments using nuclei from pulse-labelled cells suggested that the primary DNA pieces could be chased rapidly into DNA of high molecular weight (30-70 S, corresponding to a molecular weight of 0.7 - 10(7)-6.4-10(7)). Longer chases showed that the label eventually accumulated in DNA with s values greater than 150 S. In isolated nuclei the primary DNA pieces after a 1 min pulse at 37 degrees C were approximately 200 nucleotides long. Primary pieces of this size were also rapidly chased into the 30-70 S region. However, during longer pulses in vitro a fraction of the primary DNA pieces grew beyond their normal size to reach a size of up to 2000-3000 nucleotides before being attached to the 30-70 S molecules.

The effect of phospholipase C on DNA synthesis, morphology and phospholipid content of isolated HeLa cell nuclei

Isolated HeLa cell nuclei have been treated with purified phospholipase C (Bacillus cereus) and sphingomyelinase (Staphylococcus aureus). The phospholipids of untreated nuclei consisted of about 67% phosphatidylcholine, 23% phosphatidylethanolamine, 7% sphingomyelin, 2% phosphatidylserine and 1% phosphatidylinositol. Phospholipase C degraded 80-90% of the total phospholipids of the nuclei. Such nuclei seemed ultrastructurally intact, and had an average diameter and a protein loss during incubation which were not significantly different from those of controls. Their rate of DNA synthesis was only slightly reduced after treatment with phospholipase C alone and slightly more reduced when phospholipase C was used in combination with sphingomyelinase. This suggests that the polar head-groups of the nuclear phospholipids are of very limited importance in DNA synthesis. Since it has been reported that phospholipase C treatment releases nascent DNA from a membrane complex, the absence of a concommitant reduction in DNA synthesis may suggest that this complex is not necessary for the replication of DNA. Phospholipase C did not significantly influence the stability of the DNA product and gave only a slight inhibition of cytosol and nuclear DNA polymerases when tested with exogenous template.

UV-Irradiated SV40 minichromosomes as substrates for DNA repair endonucleases

UV-irradiated SV40 minichromosomes have been shown to be a substrate for a purified DNA repair endonuclease. A UV-repair endonuclease activity was also found to be associated with the isolated SV40 minichromosomes themselves. It appeared to have similar properties to the enzymes described from other mammalian sources.

Preservation by glycerol of DNA synthetic capacity of isolated HeLa S3 cell nuclei during long term storage at low temperature

Abstract A method for storage of nuclei which preserves the DNA synthetic activity is presented. Freezing of nuclei in a hypotonic buffer containing 70% glycerol preserves fully the DNA synthetic capacity. DNA synthesis continues at sites that were active in vivo and proceeds through synthesis of early nascent intermediates (primary DNA pieces) which are subsequently joined into DNA of larger molecular weight.