Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase IIalpha.
Topoisomerase II (topo II) is a dyadic enzyme found in all eukaryotic cells. Topo II is involved in a number of cellular processes related to DNA metabolism, including DNA replication, recombination and the maintenance of genomic stability. We discovered a correlation between the development of postnatal testis and increased binding of topo IIalpha to the chromatin fraction. We used this observation to characterize DNA-binding specificity and catalytic properties of purified testis topo IIalpha. The results indicate that topo IIalpha binds a substrate containing the preferred site with greater affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Interestingly, topo IIalpha displayed high-affinity and cooperativity in binding to the scaffold associated region. In contrast to the preferred site, however, high-affinity binding of topo IIalpha to the scaffold-associated region failed to result in enhanced catalytic activity. Intriguingly, competition assays involving scaffold-associated region revealed an additional DNA-binding site within the dyadic topo IIalpha. These results implicate a dual role for topo IIalpha in vivo consistent with the notion that its sequestration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.
Bakshi
R
R
Department of Biochemistry, Indian Institute of Science, Bangalore.
Galande
S
S
Muniyappa
K
K
D000818:Animals; D000951:Antigens, Neoplasm; D001665:Binding Sites; D002384:Catalysis; D002843:Chromatin / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D011068:Poly G; D051381:Rats; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology* / Q000254:growth & development; D059005:Topoisomerase II Inhibitors
D000951:Antigens, Neoplasm; D002843:Chromatin; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D059005:Topoisomerase II Inhibitors; D011068:Poly G; C026628:poly(dG); D004250:DNA Topoisomerases, Type II