Abstract
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.
Highlights
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
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
Tagging of Yeast DNA Topoisomerase II Fragments for Radiolabeling at Their NH2 or COOH Termini—Because of its large size (1428 amino acids), footprinting of the singlepolypeptide yeast DNA topoisomerase II was carried out with both NH2-terminal and COOH-terminal-tagged derivatives of it to improve the resolution of the footprinting products by SDS-polyacrylamide gel electrophoresis
Summary
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. The passage of the T-segment through the G-segment involves transesterification between a pair of active-site tyrosines, Tyr782 [10], and a pair of DNA phosphoryl groups that are 5Ј-staggered and separated by 4 base pairs This reaction breaks the pair of phosphodiester bonds in the G-segment and at the same time forms a pair of phosphotyrosine links between the enzyme and the DNA; an opening or gate in the G-segment is created. It was found that if a duplex DNA possesses a 4-nucleotide 5Ј-overhang, the singlestranded overhang could be joined to the active site tyrosine 782 through a narrow channel at one end of the semicircular groove [6] These observations led to the hypothesis that the conformation of the enzyme observed in the particular crystal closely resembles that of the enzyme after it has cleaved the DNA G-segment and widened the DNA gate [6]. We applied the two-step lysine footprinting method [15] to map the lysyl residues of yeast DNA topoisomerase II that became protected against citraconylation
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