Drosophila melanogaster topoisomerase IIIalpha preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development.
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.
Plank
Jody L
JL
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Chu
Shin Hai
SH
Pohlhaus
Jennifer Reineke
JR
Wilson-Sali
Tina
T
Hsieh
Tao-Shih
TS
D000818:Animals; D002021:Buffers; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D018507:Gene Expression Regulation, Developmental; D005804:Genes, Lethal; D008274:Magnesium; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011679:Pupa / Q000502:physiology; D012492:Salts; D013379:Substrate Specificity; D013696:Temperature
D002021:Buffers; D004278:DNA, Superhelical; D012492:Salts; D004264:DNA Topoisomerases, Type I; D008274:Magnesium