Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II

Biochemistry ◽  
1986 ◽  
Vol 25 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Jon Minford ◽  
Yves Pommier ◽  
Jan Filipski ◽  
Kurt W. Kohn ◽  
Donna Kerrigan ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Cell Nuclei ◽  
Cleavage Activity ◽  
Dependent Protein ◽  
Dna Strand

Effects of DNA intercalating agents on topoisomerase II induced DNA strand cleavage in isolated mammalian cell nuclei

Biochemistry ◽  
1985 ◽  
Vol 24 (23) ◽  
pp. 6406-6410 ◽  
Author(s):  
Yves Pommier ◽  
Ronald E. Schwartz ◽  
Leonard A. Zwelling ◽  
Kurt W. Kohn
Keyword(s):  
Mammalian Cell ◽  
Topoisomerase Ii ◽  
Cell Nuclei ◽  
Dna Strand ◽  
Intercalating Agents

scholarly journals Characterization of an ATP-dependent DNA strand transferase from human cells.

1987 ◽  
Vol 7 (9) ◽  
pp. 3124-3130 ◽  
Author(s):  
D Ganea ◽  
P Moore ◽  
L Chekuri ◽  
R Kucherlapati
Keyword(s):  
Dna Sequences ◽  
Atp Hydrolysis ◽  
Reca Protein ◽  
Strand Exchange ◽  
Cell Nuclei ◽  
In Vitro Recombination ◽  
Dna Strand ◽  
Exchange Activity

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.

scholarly journals Histone carbonylation in vivo and in vitro

2000 ◽  
Vol 351 (3) ◽  
pp. 769-777 ◽  
Author(s):  
Georg T. WONDRAK ◽  
Daniel CERVANTES-LAUREAN ◽  
Elaine L. JACOBSON ◽  
Myron K. JACOBSON
Keyword(s):  
Histone H1 ◽  
Nuclear Proteins ◽  
Carbonyl Groups ◽  
Cell Nuclei ◽  
Strand Breaks ◽  
Core Histones ◽  
Dna Strand ◽  
And Function

Non-enzymic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity. Introduction of carbonyl groups into histones in vivo and in vitro was assessed by Western blot immunoassay and reductive incorporation of tritium from radiolabelled NaBH4 (sodium borohydride). Histone H1 extracted from bovine thymus, liver and spleen was found to contain significantly elevated amounts of protein-bound carbonyl groups as compared with core histones. The carbonyl content of nuclear proteins of rat pheochromocytoma cells (PC12 cells) was not greatly increased following oxidative stress induced by H2O2, but was significantly increased following alkylating stress induced by N-methyl-N´-nitro-N-nitrosoguanidine or by combined oxidative and alkylating stress. Free ADP-ribose, a reducing sugar generated in the nucleus in proportion to DNA strand breaks, was shown to be a potent histone H1 carbonylating agent in isolated PC12 cell nuclei. Studies of the mechanism of histone H1 modification by ADP-ribose indicate that carbonylation involves formation of a stable acyclic ketoamine. Our results demonstrate preferential histone H1 carbonylation in vivo, with potentially important consequences for chromatin structure and function.

Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks

1993 ◽  
Vol 105 (2) ◽  
pp. 563-569 ◽  
Author(s):  
D.J. Clarke ◽  
R.T. Johnson ◽  
C.S. Downes
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Topoisomerase Ii ◽  
Dna Strand Breaks ◽  
Temperature Sensitive ◽  
Dna Topoisomerase ◽  
Strand Breaks ◽  
Temperature Sensitive Mutants ◽  
Chromatid Segregation ◽  
Dna Strand

Yeast temperature-sensitive mutants of DNA topoisomerase II are incapable of chromosome condensation and anaphase chromatid segregation. In mammalian cells, topoisomerase II inhibitors such as etoposide (VP-16-123) have similar effects. Unfortunately, conclusions drawn from work with mammalian cells have been limited by the fact that the standard inhibitors of topoisomerase II also generate DNA strand breaks, which when produced by other agents (e.g. ionizing radiation) are known to affect progression into and through mitosis. Here we show that the anti-tumour agent ICRF-193, recently identified as a topoisomerase II inhibitor operating by a non-standard mechanism, generates neither covalent complexes between topoisomerase II and DNA, nor adjacent DNA strand breaks, in mitotic HeLa. However, the drug does prevent anaphase segregation in HeLa and PtK2 cells, with effects similar to those of etoposide. We therefore conclude that topoisomerase II function is required for anaphase chromosome segregation in mammalian cells, as it is in yeast.

scholarly journals Purification and characterization of a DNA-binding heterodimer of 52 and 100 kDa from HeLa cells

1993 ◽  
Vol 290 (1) ◽  
pp. 267-272 ◽  
Author(s):  
W W Zhang ◽  
L X Zhang ◽  
R K Busch ◽  
J Farrés ◽  
H Busch
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Topoisomerase Ii ◽  
Sequence Similarity ◽  
Peptide Sequence ◽  
Upstream Region ◽  
Cell Nuclei ◽  
Sperm Dna ◽  
Multiple Species

In studies of protein binding to the upstream region of the human proliferation-associated antigen p120 gene, a heterodimer of 52 and 100 kDa proteins was purified from HeLa cells. A 1:1 ratio of p52 and p100 was constant throughout the purification. The heterodimer was localized to cell nuclei, as shown by immunofluorescence. The pI values of the p52 and p100 were 7.8 and 8.6 respectively. The peptide sequences obtained for p52 (QSNKTFNLEKQNHTPRKKHQ and PLRGKQLRVRFAAHSASLTVR) and for p100 (PGGPKPGGGPGLSTPGGHPKPPHRGGGEPPRGRQ and GPGPGQSGPKPPIPPPPPHQQ) were not found in the computer databanks. One p52 peptide sequence, PLRGKQLRVRFA, shows considerable sequence similarity to a conserved motif in topoisomerase II of multiple species. The p52/100 heterodimer bound to different DNA probes. The binding was competed by poly(dI-dC), sonicated salmon sperm DNA, and circular or linearized plasmid DNA. The optimal DNA binding for the heterodimer was at pH 7-9 with low salt. The DNA-binding subunit of the heterodimer was the p100 polypeptide, as shown by u.v.-cross-linking assays and Southwestern blots.

scholarly journals DNA Repair Functions That Control Sensitivity to Topoisomerase-Targeting Drugs

2004 ◽  
Vol 3 (1) ◽  
pp. 82-90 ◽  
Author(s):  
Mobeen Malik ◽  
John L. Nitiss
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Excision Repair ◽  
Dna Strand Breaks ◽  
Dna Topology ◽  
Strand Breaks ◽  
Wide Range ◽  
Dna Strand

ABSTRACT DNA topoisomerases play critical roles in a wide range of cellular processes by altering DNA topology to facilitate replication, transcription, and chromosome segregation. Topoisomerases alter DNA topology by introducing transient DNA strand breaks that involve a covalent protein DNA intermediate. Many agents have been found to prevent the religation of DNA strand breaks induced by the enzymes, thereby converting the enzymes into DNA-damaging agents. Repair of the DNA damage induced by topoisomerases is significant in understanding drug resistance arising following treatment with topoisomerase-targeting drugs. We have used the fission yeast Schizosaccharomyces pombe to identify DNA repair pathways that are important for cell survival following drug treatment. S. pombe strains carrying mutations in genes required for homologous recombination such as rad22A or rad32 (homologues of RAD52 and MRE11) are hypersensitive to drugs targeting either topoisomerase I or topoisomerase II. In contrast to results observed with Saccharomyces cerevisiae, S. pombe strains defective in nucleotide excision repair are also hypersensitive to topoisomerase-targeting agents. The loss of DNA replication or DNA damage checkpoints also sensitizes cells to both topoisomerase I and topoisomerase II inhibitors. Finally, repair genes (such as the S. pombe rad8+ gene) with no obvious homologs in other systems also play important roles in causing sensitivity to topoisomerase drugs. Since the pattern of sensitivity is distinct from that seen with other systems (such as the S. cerevisiae system), our results highlight the usefulness of S. pombe in understanding how cells deal with the unique DNA damage induced by topoisomerases.

Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage

Biochemistry ◽  
1985 ◽  
Vol 24 (23) ◽  
pp. 6410-6416 ◽  
Author(s):  
Yves Pommier ◽  
Jon K. Minford ◽  
Ronald E. Schwartz ◽  
Leonard A. Zwelling ◽  
Kurt W. Kohn
Keyword(s):  
Topoisomerase Ii ◽  
Dna Intercalators ◽  
Dna Strand ◽  
Strand Passage
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