期刊
BIOCHEMISTRY
卷 44, 期 34, 页码 11546-11554出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi050811l
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资金
- NICHD NIH HHS [5 T32 HD07043, T32 HD007043] Funding Source: Medline
- NIGMS NIH HHS [GM33944, R01 GM033944-21, R01 GM033944] Funding Source: Medline
The enzymatic function of the C-terminal domain of eukaryotic topoisomerase 11 is not well defined. This region of the enzyme is highly variable and hydrophilic and contains nuclear localization signals and phosphorylation sites. In contrast to eukaryotic topoisomerase 11, type 11 enzymes from chlorella virus completely lack the C-terminal domain. These viral enzymes are characterized by a robust DNA cleavage activity, high coordination between their two active site tyrosyl residues, and reduced sensitivity to anticancer drugs. As a first step toward characterizing the contribution of the C-terminal domain of human topoisomerase II alpha to enzyme function, the protein was truncated at amino acid 1175, which corresponds to the C-terminal residue of Paramecium bursaria chlorella virus-1 topoisomerase 11 as determined by BLAST sequence alignment. Although the overall catalytic activity of the resulting enzyme, hTop2 alpha Delta 1175, was lower than that of full-length topoisomerase II alpha, the mutant protein displayed a double-stranded DNA cleavage activity that was similar to 2-3-fold higher. While the DNA breaks created by hTop2 alpha Delta 1175 were primarily double stranded, cuts generated by topoisomerase II alpha were primarily single stranded. Thus, the enhanced cleavage observed for hTop2 alpha Delta 1175 appears to be due, at least in part, to an increase in active site coordination. Finally, hTop2 alpha Delta 1175 displayed a distinctly lower susceptibility to anticancer agents than did topoisomerase II alpha, despite the fact that it showed a similar binding affinity for etoposide. Therefore, the C-terminal domain of human topoisomerase II alpha appears to play significant roles in modulating the DNA cleavage/ligation reaction of the enzyme and its response to anticancer agents.
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