Abstract
Eukaryotic type IA topoisomerases are important for the normal function of the cell, and in some cases essential for the organism, although their role in DNA metabolism remains to be elucidated. In this study, we cloned Drosophila melanogaster topoisomerase (topo) IIIalpha from an embryonic cDNA library and expressed and purified the protein to >95% homogeneity. This enzyme partially relaxes a hypernegatively supercoiled plasmid substrate consistent with other purified topo IIIs. A novel, covalently closed bubble substrate was prepared for this study, which topo IIIalpha fully relaxed, regardless of the handedness of the supercoils. Experiments with the bubble substrate demonstrate that topo IIIalpha has much different reaction preferences from those obtained by plasmid substrate-based assays. This is presumably due to the fact that solution conditions can affect the structure of plasmid based substrates and therefore their suitability as a substrate. A mutant allele of the Top3alpha gene, Top3alpha191, was isolated through imprecise excision mutagenesis of an existing P-element inserted in the first intron of the gene. Top3alpha191 is recessive lethal, with most of the homozygous individuals surviving to pupation but never emerging to adulthood. Whereas this mutation can be rescued by a Top3alpha transgene, ubiquitous overexpression of D. melanogaster topo IIIbeta cannot rescue this allele.
Highlights
DNA topoisomerases are ubiquitous enzymes found in all cells and some viruses that regulate the topology of DNA in such cellular processes as replication, transcription, and recombination [1, 2]
Cloning of D. melanogaster Topoisomerase III␣—To isolate and study the Top3␣ gene from D. melanogaster, primers were designed from the genomic sequence to amplify a region from the highly conserved type IA core region of the gene, and this PCR product was used to screen a D. melanogaster embryonic cDNA library
This result was repeated, with the same outcome, using Positively Supercoiled (PSC) bubble substrate created with reverse gyrase. These results show that the handedness of the supercoils in the substrate is inconsequential to topo III␣ activity; only the presence of single-stranded DNA matters
Summary
DNA topoisomerases are ubiquitous enzymes found in all cells and some viruses that regulate the topology of DNA in such cellular processes as replication, transcription, and recombination [1, 2]. Reaction of the annealed circles with reverse gyrase results in relaxed, covalently closed bubble substrate (Fig. 3B, lane 3), which is negatively supercoiled with the same technique used to create the HNSC plasmid substrate. It is apparent that there is no detectable increase in the amount of nicked material when the NSC bubble substrate is reacted with topo III␣, whereas the intensity of the relaxed product band is comparable with the NSC starting material (Fig. 4B).
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