Footprinting of yeast DNA topoisomerase II lysyl side chains involved in substrate binding and interdomainal interactions.
Footprinting of yeast DNA topoisomerase II and its NH2- and COOH-terminal truncation derivatives was carried out to map the locations of lysyl side chains that are involved in enzyme-DNA interaction, in the binding of ATP, or in interaction between domains of the same enzyme molecule. Several conclusions were drawn based on these measurements and the crystal structures of a 92-kDa fragment of the yeast enzyme and a 43-kDa fragment of Escherichia coli gyrase B-subunit. First, the footprinting results support the model previously inferred from the 92-kDa fragment crystal structure that the main site of DNA binding is comprised of a pair of semicircular grooves. Second, the binding of a nonhydrolyzable ATP analog to the yeast enzyme appears to affect citraconylation at a minimum of six lysines in the ATPase domain of each polypeptide. Two of these lysines are probably involved in contacting the nucleotide directly, and one probably becomes buried when the two ATPase domains of a dimeric enzyme come into contact upon ATP binding; for the others, changes in lysine reactivity appear to reflect allosteric changes following ATP binding. Third, from a comparison of the footprint of the intact enzyme and those of the truncated polypeptides comprised of either the NH2- or the COOH-terminal half of the intact polypeptide, it appears that there are few contacts between the NH2- and COOH-terminal half of yeast DNA topoisomerase II.
Li
W
W
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Wang
J C
JC
D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D001665:Binding Sites; D004250:DNA Topoisomerases, Type II / Q000737:chemistry*; D004271:DNA, Fungal / Q000378:metabolism; D005650:Fumarates / Q000378:metabolism; D008239:Lysine / Q000737:chemistry*; D008298:Maleates / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011485:Protein Binding; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
D004271:DNA, Fungal; D005650:Fumarates; D008298:Maleates; D010446:Peptide Fragments; C073341:citraconic acid; D000266:Adenylyl Imidodiphosphate; D004250:DNA Topoisomerases, Type II; D008239:Lysine