UBC9
functions to protect cells from DNA topoisomerase I poisons.
Herve R. Jacquiau (1), Robert C. A. M. van Waardenburg (1), Robert J. D. Reid
(2), Mary-Ann Bjornsti (1)
(1) Department of Molecular Pharmacology, St. Jude Children's Research
Hospital, 332 N. Lauderdale St., Memphis, TN 38105 USA
(herve.jacquiau@stjude.org); (2) Department of Biochemistry and Molecular
Pharmacology, Thomas Jefferson University, Philadelphia, PA 19107 USA
Camptothecin
(CPT) reversibly stabilizes a covalent DNA topoisomerase I (Top1p)-DNA
intermediate, which is converted into irreversible lethal lesions during S
phase. To define cellular processes that modulate cell sensitivity to
Top1p-induced DNA lesions, conditional mutants with enhanced sensitivity to
Top1p poisons were selected. Two self-poisoning top1 mutants were used.
Top1T722Ap mimics the action of CPT by inhibiting DNA religation, while
Top1N726Hp exhibits increased rates of DNA cleavage. Although these top1 mutants induce distinct
Top1p-DNA complexes, the same hypomorphic allele of UBC9, ubc9-10, was isolated. The
essential Ubc9 E2 enzyme conjugates the ubiquitin-like protein Smt3p to lysine
residues in specific proteins, which can alter protein function, sub-cellular
localization and/or complex formation. Mutation of the conserved Pro123 to Lys
in ubc9-10
enhances cell sensitivity to Top1p poisons, HU and other DNA damaging agents at
36°C. Immunoprecipitations revealed lower levels of Smt3p-protein conjugates,
although Ubc9p-Smt3p interactions were unaffected. While deletion of the Ulp2
protease or expression of human UBC9 restored ubc9Δ viability, neither
complemented ubc9-10 sensitivity to Top1T722Ap. However, a human UBC9-myc
construct partially restored ubc9-10 resistance to Top1p poisons. This suggests
alterations of C-terminal residues (such as Pro123 to Leu, or a myc tag) affect
Ubc9p substrate specificity, which induces CPT hypersensitivity.