Contribution of topoisomerase I to conversion of single-strand into double-strand DNA breaks.

An in vitro system composed of nicked pBR322 DNA and purified topoisomerase I was employed to study the efficiency of the topoisomerase I-driven single-strand to double-strand DNA breaks conversion. At 1.4 x 10(5) topoisomerase I activity units per mg DNA about 20% single-strand nicks were converted into double-strand breaks during 30 min due to topoisomerase I action. Camptothecin inhibited the conversion. The conversion was also inhibited when the relaxing activity of the used topoisomerase I was increased by phosphorylation of the enzyme with casein kinase 2. The presented data suggest that topoisomerase I may be involved in production of double-stranded breaks in irradiated cells and that this process positively depends on the amount of topoisomerase I but not on its phosphorylation state.

Effect of protein kinase ck2 on topoisomerase I from plasmodia of the slime mold Physarum polycephalum.

Relaxing activity of Physarum topoisomerase I was increased by calf thymus protein kinase ck2, similarly as was the activity of mammalian topoisomerase I, despite a pronounced difference between amino-acid sequences of non-conserved domains of Physarum and mammalian enzymes. This feature of Physarum topoisomerase I was cancelled in nuclear extracts isolated from dibutyryl-cAMP treated plasmodia in which the activity of protein kinase ck2 was elevated.

Phosphorylation of topoisomerase I in L5178Y-S cells is associated with poly(ADP-ribose) metabolism.

The reason for different phosphorylation of topoisomerase I in two sublines of L5178Y murine lymphoma (LY cells) was investigated. Camptothecin-resistant LY-S cells show increased poly(ADP-ribose) level and lowered topoisomerase I phosphorylation compared to camptothecin-sensitive LY-R cells. In this study diminished phosphorylation of LY-S topoisomerase I was observed for sites recognized by casein kinase 2 but not for those phosphorylated by protein kinase C. Tryptic digests of LY-S topoisomerase I labeled in vitro by casein kinase 2 indicated that phosphorylation was similarly lowered at different sites. Activity of casein kinase 2 measured in nuclear extracts was about 1.7 times lower for LY-S than LY-R cells. This difference was diminished or eliminated by increasing casein concentration, diluting the extract or increasing the ionic strength. Activity of poly(ADP-ribose) polymerase was 5.3 times higher in LY-S than in LY-R nuclei. When the activity of the polymerase was inhibited by treatment of LY-S cells with benzamide, casein kinase 2-catalyzed phosphorylation of topoisomerase I increased. This was accompanied by an increase in sensitivity to camptothecin as reflected in the diminished viability of LY-S cells.

Topoisomerase I is differently phosphorylated in two sublines of L5178Y mouse lymphoma cells.

Two sublines of LY murine lymphoma, differing in sensitivity to CPT, served as source of topoisomerase I in order to compare the enzyme's properties. The activity of topoisomerase I isolated from LY-S cells of reduced sensitivity to CPT increased about 2-times more upon phosphorylation with casein kinase but was inhibited to a lesser extent upon dephosphorylation with alkaline phosphatase than the enzyme from the CPT-sensitive LY-R cells. The in vitro phosphorylation of LY-S enzyme restored its sensitivity to CPT. The in vitro incorporation of 32P into topoisomerase protein was about 1.7-times higher in LY-S than in LY-R enzyme. A reversed incorporation ratio was observed upon metabolic labelling. The level of topoisomerase I protein, determined by Western blot analysis using scleroderma anti-topoisomerase I antibodies, was about 1.5-times higher in LY-S than in LY-R cells. The level of topoisomerase I mRNA was similar in both sublines. These results indicate that the reduced sensitivity of LY-S cells to CPT is based on the lowered phosphorylation of topoisomerase I protein but does not depend on the expression of topoisomerase I gene.

The sensitivity to camptothecin of DNA topoisomerase I in L5178Y-S lymphoma cells.

Sensitivity to camptothecin (CPT) of type I DNA topoisomerases isolated from two L5178Y (LY) sublines was examined in reaction media containing either aspartate or chloride. The enzyme from LY-S cells was sensitive to the drug in the presence of 120 mM K-aspartate, but the sensitivity was markedly reduced in the presence of 120 mM KCl. The enzyme from LY-R cells was similarly sensitive to camptothecin in the presence of either aspartate or chloride. The optimum ionic strength for the relaxation reaction catalyzed by both LY-R and LY-S type I DNA topoisomerases was similar. We suggest that sensitivity of the LY-S enzyme to CPT depends on the amount of cleavable complex formed, which in turn depends on the ionic conditions of the assay.

Sensitivity to inhibitors of type II topoisomerases from mouse L5178Y lymphoma strains that are resistant or sensitive to X-radiation.

Sensitivity to topoisomerase II inhibitors was tested at the cellular and enzyme level for two strains of mouse L5178Y lymphoma cells: resistant (LY-R) and sensitive (LY-S) to X-radiation. Differences in the susceptibility to inhibitors between LY-R and LY-S cells depended on the inhibitor used and were observed for adriamycin and VP-16, but not for mitoxantrone. On the other hand, isolated enzymes displayed the same sensitivity to all inhibitors tested regardless of the cell line. These results exclude the presence of altered topoisomerase II in LY-S cells as a possible reason for the collateral sensitivity of LY-S cells to X-radiation and topoisomerase II inhibitors.

Reduced sensitivity to camptothecin of topoisomerase I from a L5178Y mouse lymphoma subline sensitive to X-radiation.

Murine L517BY (LY) lymphoma sublines, LY-R (X-radiation resistant) and LY-S (X-radiation sensitive) displayed a difference in susceptibility to camptothecin: susceptibility of LY-S cells to the alkaloid was shifted towards higher concentrations as compared to LY-R cells. A similar difference was observed at the level of genomic DNA when a number of DNA-protein cross-links was determined or single-strand breaks were revealed by the fluorescent nucleoid halo assay. Activities of topoisomerases I and II were the same in both sublines. In turn, a higher resistance to camptothecin was found for the isolated LY-S topoisomerase I in the DNA cleavage test, suggesting that an altered enzyme was responsible for the susceptibility difference observed at the cellular level. In the relaxation test the enzymes from the two sublines showed a different sensitivity to beta-lapachone, an activator of topoisomerase I, but were similarly sensitive to all inhibitors, except camptothecin.

Effects of fluorodeoxyuridine and nalidixic acid on the activity of topoisomerase I in plasmodia of Physarum polycephalum.

1. A regulatory coupling between the rate of cellular transcription and the activity of topoisomerase I was investigated in plasmodia of Physarum polycephalum treated with fluorodeoxyuridine or nalidixic acid. 2. Fluorodeoxyuridine at concentrations above 40 micrograms/ml lowered both the incorporation of [3H]uridine and the activity of topoisomerase I to 10% of corresponding control values. 3. Nalidixic acid, in the range of concentrations between 20-50 micrograms/ml did not inhibit the incorporation of [3H]uridine but lowered the activity of topoisomerase I by about half. 4. It is suggested that a coupling between the level of transcription and the activity of topoisomerase I in Physarum plasmodia involves only about a half of the topoisomerase I activity and is limited to transcription occurring on ribosomal genes.

Topoisomerase I in actively growing plasmodia and during differentiation of the slime mold Physarum polycephalum.

A type I topoisomerase has been purified from nuclei of a slime mold Physarum polycephalum and its activity was tested during spherulation. The final preparation contained a single polypeptide of about 100 kDa. Basic properties of Physarum topoisomerase I (substrate specificity, ionic requirement, sensitivity to inhibitors) were similar to those of topoisomerases from higher eukaryotes. Specific features of Physarum enzyme were that it was rapidly inactivated at 45 degrees C and did not react with antibodies against human topoisomerase I. The activity of topoisomerase I in developed dormant spherules decreased approx. 2-fold, as compared with a 4-fold decrease of RNA and a 10-fold decrease of DNA synthesis. Basic properties of the enzyme remained unchanged during spherulation.

Nuclear matrix proteins of Physarum polycephalum.

The proteins of nuclear matrix preparations from Physarum polycephalum were compared with analogous mammalian fractions by gel electrophoresis, DNA-binding studies and immunological tests. Polypeptides of 28 and 36 K dalton, which dominate in Physarum preparations, differed from calf thymus matrix proteins in that they were basic and showed low affinity to DNA. These polypeptides were present at about 1.2 mg per mg of nuclear DNA. Polypeptides of higher molecular weight occurred in the preparation at about 0.5 mg per mg of nuclear DNA. At least some of the latter proteins showed high affinity to DNA and cross-reacted with the antiserum against calf thymus matrix proteins.

Similarity in active site arrangement of neutral protease from calf thymus chromatin and trypsin.

1. Susceptibility to inhibitors of neutral protease from calf thymus chromatin has been compared with that of trypsin. The chromatin protease reacts stoichiometrically with the inhibitors specific for trypsin (diisopropylfluorophosphate, tosyl-lysyl chloromethane, soybean trypsin inhibitor and Kunitz basic inhibitor from pancreas), but not with the inhibitor specific for chymotrypsin (tosyl-phenylalanyl chloromethane). 2. Chromatin protease, similarly as trypsin, cleaves Lys-X and Arg-X peptide bonds. 3. It is concluded that the structure of active site region of both enzymes is very similar.

The presence of serine protease in pea embryo chromatin.

1. It has been shown that chromatin from pea seedlings contains a proteolytic enzyme, similar to that of mammalian chromatin. 2. The protease was isolated from chromatin by acid extraction and partly characterized. It is a serine-type enzyme, sensitive to DFP, of low mol. wt. (about 18 000 - 20 000), with optimum pH at about 8 with [3H]N-acetylated histone as a substrate. 3. In chromatin complex, histones fl and f3 are preferentially degraded.

The activation by staphylokinase of human plasminogen.

The activation of human plasminogen by a highly purified staphylokinase was investigated using casein or an active site titrant (p-nitrophenyl-p-guanidinobenzoate, NPGB) as a substrate. The reaction rate was time dependent, showing a pronounced lag period with either substrate. Saturation curve estimated from the caseinolytic assay was sigmoid, but changed to quasi-hyperbolic in the presence of pre-formed human plasmin. With NPGB, the extent of plasminogen conversion into esterolytic plasmin was directly proportional to staphylokinase concentration, and the saturation point was reached when the molar concentration of staphylokinase equaled that of plasminogen. It is concluded that staphylokinase acts stoichiometrically, forms an equimolar complex with plasminogen, and thus is not an enzyme but a modifier. Staphylokinase-activated plasminogen exhibits properties of a hysteretic enzyme.