Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene.

Chronic exposure of humans to benzene (BZ) causes acute myeloid leukemia (AML). Both BZ and therapy-related secondary AML are characterized by chromosomal translocations that may occur by inappropriate recombinational events. DNA topoisomerase II (topo II) is an essential sulfhydryl (SH)-dependent endonuclease required for replication, recombination, chromosome segregation, and chromosome structure. Topo II cleaves DNA at purine(R)/pyrimidine(Y) repeat sequences that have been shown to be highly recombinogenic in vivo. Certain antineoplastic drugs stabilize topo II-DNA cleavage complexes at RY repeat sequences, which leads to translocations of the type observed in leukemia. Hydroquinone (HQ) is metabolized to p-benzoquinone (BQ) in a peroxidase-mediated reaction in myeloid progenitor cells. BQ interacts wit SH groups of SH-dependent enzymes. Consequently, the aims of this research were to determine whether HQ and BQ are topo II inhibitors. The ability of the compounds to inhibit the activity of topo III was tested using an assay system that depends on the conversion, by homogeneous human topo II, of catenated kinetoplast DNA into open and/or nicked open circular DNA that can be separated from the catenated DNA by electrophoresis in a 1% agarose-ethidium bromide gel. We provide preliminary data that indicate that both HQ and BQ cause a time and concentration (microM)-dependent inhibition of topo II activity. These compounds, which potentially can form adducts with DNA, have no effect on the migration of the supercoiled and open circular forms in the electrophoretic gradient, and BQ-adducted KDNA can be decatenated by topo II. Using a pRYG plasmid DNA with a single RY repeat as a cleavage site, it was determined that BQ does not stimulate the production of linear DNA indicative of an inhibition of topo II religation of strand breaks by stabilization of the covalent topo III-DNA cleavage complex. Rather, BQ most probably inhibits the SH-dependent topo II by binding to an essential SH group. The inhibition of topo II by BQ has implications for the formation of deleterious translocations that may be involved in BZ-induced initiation of leukemogenesis

Investigation of the Substrate Recognition Characteristics and Kinetics of Mammalian Mitochondrial DNA Topoisomerase I

Topoisomerases are DNA-modifying enzymes found in prokaryotes, eukaryotes, viruses and organelles such as chloroplast and mitochondria. Information about these enzymes in eukaryotic systems is mostly limited to nuclear enzymes, although our laboratory has been characterizing the biochemical and biophysical properties of the mammalian mitochondrial topoisomerases. We have determined the polarity of the attachment of mitochondrial topoisomerase I to its substrate DNA. To study the substrate preference and kinetic parameters of mitochondrial topoisomerase I, selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors following a series of modification and optimization experiments of currently available methods for PCR-cloning. These mtDNA containing recombinant plasmids were used in a kinetic analysis of the highly purified enzyme. Recombinant plasmids containing the bovine mtDNA heavy and light strand origins of replication (pZT-Hori and pZT-Lori, respectively), a major transcription termination region (pZT-Term) and a portion of cytochrome b gene (pZT-Cytb) were prepared. Two other recombinant plasmids, containing non-mitochondrial DNA inserts (pZT-800 and pZT-400) served as control substrates. Southern hybridization using probes specific for either control or mtDNA-containing plasmids indicated a relative preference by the mitochondrial topoisomerase I to relax supercoils in pZT-Hori and pZT-Term. Quantitative determination of kinetic parameters derived from double-reciprocal Lineweaver-Burk plots showed that recombinant plasmids containing the heavy and light strand origins and the transcription termination region were preferentially relaxed by the mitochondrial enzyme with Km values 2.3 to 3.3-fold lower than controls. The Km values for pZT-Hori, pZT-Lori and pZT-Term were 21.0 +/- 0.9 μΜ, 25.2 +/- 1.0 μM and 17.0 +/- 0.8 μΜ respectively, while those for control plasmids were 57.5 +/- 2.1 μΜ and 56.3 +/- 2.3 μΜ. pZT-Cytb was not preferentially relaxed compared to the control plasmid (Km= 53.4 +/- 2.0 μΜ vs.56.3 +/- 2.3 μΜ respectively) indicating that mitochondrial topoisomerase I preferentially interacts with certain mtDNA sequences but not others. Identical experiments with the purified nuclear enzyme did not differentiate between control or mtDNA containing plasmids

Unusual Structure of a Human Middle Repetitive DNA

The L2Hs sequences are a polymorphic, interspersed, middle repetitive DNA family unique to human genomes. Genomic fingerprinting indicates that these DNAs vary from one individual to another and between tissues of the same individual. Sequence analysis reveals that they are AT-rich (76%) and contain many unusual sequence arrangements (palindromes, inverted and direct repeats). These sequence properties confer on the L2Hs elements the potential to fold into non-B-form structures, a characteristic of recombination hot spots. To test this hypothesis carbodiimide, osmium tetroxide and S\sb1 nuclease were used as single-strand specific probes to study a recombinant plasmid, pN6.4.39, containing a single L2Hs segment. Different forms of the plasmid substrate were analyzed, including linear molecules and circular forms of low, intermediate and high superhelical densities. In addition, plasmid DNA in growing E. coli cells were analyzed. Modified plasmid DNA was analyzed by primer extension in a sequencing-type reaction format. These studies demonstrate that the L2Hs sequences: (1) assume non-B-form structures both in vitro and in vivo, (2) map to predicted cruciform structures, (3) behave as C-type extrusion sequences, and (4) that these unusual DNA structures are dependent on plasmid superhelicity

Elucidation of non-B DNA-induced mutagenesis mechanisms : DNA repair proteins are required for the processing of H-DNA and Z-DNA in eukaryotes

The vast majority of all cancers result from some form of genetic instability, thus it is important to study the mechanisms involved. The integrity of DNA can be influenced by secondary structure, and DNA can adopt alternative structures that do not conform to the Watson-Crick B-DNA helix (i.e. non-B DNA). To date, >12 different types of non-B DNA structures have been described including H-DNA and Z-DNA, and these structure-forming sequences are abundant in the human genome, occurring 1/3,000 and 1/50,000 base-pairs for Z-DNA and H-DNA, respectively. Non-B DNA can alter DNA metabolism and contribute to the development of many human diseases. Specific to this project, translocations occurring in the c-MYC and BCL-2 genes, which are shown to contain non-B DNA-forming sequences at translocation breakpoint “hotspots”, are characteristic of certain leukemias and lymphomas. However, the mechanism(s) involved in this process remains undefined. Previously, we found these structures to be mutagenic in bacteria, human cells, and mice, largely by stimulating the formation of DNA double strand breaks (DSBs). We speculated that the helical distortions produced by non-B DNA may be recognized as “damage” by the cell, eliciting an error-prone repair response, resulting in genomic instability. Using genetic-based mutation-reporter assays, we have shown for the first time that these structures are mutagenic in yeast. Furthermore, we have identified DNA repair proteins from both the nucleotide excision repair (NER) and mismatch repair (MMR) pathways to be involved in H-DNA and Z-DNA-induced mutagenesis via distinct mechanisms in both yeast and human cells. We further characterized the functions of these proteins using biochemical and molecular biology assays and found that they are enriched at sites of H-DNA and/or Z-DNA, and have cleavage activity at or near the structure. Taken together, these results suggest that non-B structures are processed in an error-prone fashion via various novel structure-specific repair pathways in which repair proteins from multiple pathways cooperate. The results obtained have enhanced our knowledge of DNA structure-induced genetic instability in disease etiology, and will guide future studies in the development of novel strategies to treat and/or prevent genetic diseases.Pharmac

Analysis of the repair of topoisomerase II DNA damage

A large number of anti-cancer chemotherapeutics target DNA topoisomerases. Etoposide is a specific topoisomerase II poison which causes reversible double strand DNA breaks. The focus of this project is to analyze the repair of DNA damage induced by etoposide.. Double strand DNA break repair is mediated by through either non-homologous end joining (NHEJ) or homologous recombination. NHEJ repairs through direct ligation of a double stranded break while homologous recombination utilizes a homologous template to recover the wild type sequence. A reporter cassette, RYDR-GFP, has been stably integrated into HeLa cells. This reporter contains an ultra-high affinity topoisomerase II cleavage site (RY) placed in the middle of a mutant GFP sequence. Flanking this sequence is a corresponding stretch of wild type GFP that is used as template to repair the break and restore gene function yielding GFP positive cells. Titrations with etoposide have shown that a logarithmic increase in drug concentration yields a corresponding increase in repair through homologous recombination (HR). This result demonstrates that topoisomerase II mediated damage is efficiently repaired by the process of HR. To examine NHEJ repair, a doxycycline inducible, stably integrated NHEJ HeLa cell reporter cassette was also evaluated. The data indicates that repair of topoisomerase II mediated DNA damage occurs more efficiently through the HR pathway. Collectively, the data suggests that tumor cells proficient in HR repair may effectively elude treatment by topoisomerase II targeting drugs