Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA

Abstract

For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study, we provide evidence that the Schizosaccharomyces pombe Rad32(Mre11) nuclease activity is involved in the removal of both Top2 from 5' DNA ends as well as Top1 from 3' ends in vivo. A ctp1(CtIP) deletion is defective for Top2 removal but overproficient for Top1 removal, suggesting that Ctp1(CtIP) plays distinct roles in removing topoisomerases from 5' and 3' DNA ends. Analysis of separation of function mutants suggests that MRN-dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anticancer drugs.

Figure 1

Temperature sensitivity of rad50S to CPT and TOP-53 suggests that the MRN complex is involved in the removal of covalently bound Top1 and Top2 in mitotic cells. a) The rad50S mutant is only slightly sensitive to MMS and γ-irradiation, but is temperature sensitive to CPT (Top1 poison) and TOP-53 (Top2 poison). b) Sensitivity of a rad32^mre11-D65N putative nuclease dead mutant to various DNA damaging agents is identical to that of rad50S at 34 °C.

Figure 2

Representative examples showing increased levels of covalently bound Top1 and Top2 in rad50∆ and rad32^mre11-D65N compared to WT. The arrow indicates where the top and bottom fractions of the CsCl gradient have been loaded.

Figure 3

a) sensitivity of ctp1∆ against various DNA damaging agents is identical to that of rad50∆ and the rad50∆ ctp1∆ double mutant. ctp1∆ is epistatic to rad32^mre11-D65N. b) ctp1∆ is defective for the removal of Top2, but over-proficient for Top1 removal in mitotic cells. c) In line with results from the DLPD assay, deletion of pku80 rescues the CPT sensitivity of ctp1∆, but not that of rad50∆.

Figure 4

a) Autoradiograph showing the TOP-53 dependent Top2-oligo release product in WT cells, which is absent in rad32^mre11-D65N and ctp1∆. The control lane C shows the drug-independent Top2-oligo product in S. cerevisiae. This experiment was repeated 3 times. b) Density trace of the autoradiograph presented in panel a. In WT, the TOP-53 dependent release product is visible as a small peak left of the spontaneous drug-independent release product.