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. 2000 Apr 11;97(8):4046-51.
doi: 10.1073/pnas.080536597.

SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase-mediated DNA damage

Y Mao  1 , M Sun, S D Desai, L F Liu
Affiliations

SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase-mediated DNA damage

Y Mao et al. Proc Natl Acad Sci U S A. .

Abstract

Ubiquitin/26S proteasome-dependent degradation of topoisomerase I (TOP1) has been suggested to be a unique repair response to TOP1-mediated DNA damage. In the current study, we show that treatment of mammalian cells or yeast cells expressing human DNA TOP1 with camptothecin (CPT) induces covalent modification of the TOP1 by SUMO-1/Smt3p, a ubiquitin-like protein. This conclusion is based on the following observations: (i) Mammalian DNA TOP1 conjugates induced by CPT were cross-reactive with SUMO-1/Smt3p-specific antibodies both in yeast expressing human DNA TOP1 as well as mammalian cells. (ii) The formation of TOP1 conjugates was shown to be dependent on UBC9, the E2 enzyme for SUMO-1/Smt3p. (iii) TOP1 physically interacts with UBC9. (iv) Ubc9 mutant yeast cells expressing human DNA TOP1 was hypersensitive to CPT, suggesting that UBC9/SUMO-1 may be involved in the repair of TOP1-mediated DNA damage.

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Figures

Figure 1
Figure 1
CPT induces the formation of hTOP1 conjugates in human cells and yeast cells expressing hTOP1. (A) Human lung fibroblast WI38 cells were treated with 25 μM CPT in 1% DMSO (+) or 1% DMSO (−) for 15 min and then lysed using the alkali lysis procedure described in Materials and Methods. Samples were analyzed on 6% SDS-PAGE and immunoblotted with anti-hTOP1 antibodies. hTOP1 conjugates were marked by a bracket. (B) Yeast JN362a harboring YCpGAL1-hTOP1 plasmid was induced by 2% galactose for 12 h and then treated with 100 μM CPT in 1% DMSO (+) or 1% DMSO (−) for 30 min. Cells were lysed using the alkali/glass bead methods. The SDS-PAGE and immunoblotting using anti-hTOP1 antibodies were performed as described in A.
Figure 2
Figure 2
CPT induces Smt3p modification of hTOP1 in yeast. (A) Modification of hTOP1 by HF-Smt3p in yeast. JN362a was cotransformed with YCpGAL1-hTOP1 and HF-Smt3 plasmids. After 2% galactose induction, cells were treated with either 100 μM CPT in 1% DMSO (+) or 1% DMSO (−) for 30 min. Cells were lysed using the alkali lysis procedure described in Materials and Methods. After SDS-PAGE, immunoblotting was performed using antibodies against hTOP1. Lanes 1 and 2: vector control, no HF-Smt3p expression. The high molecular weight bands induced by CPT were marked with *. Lanes 3 and 4: overexpression of HF-Smt3p from HF-Smt3p expressing plasmid. The new series of high molecular weight bands were indicated by arrow heads. Lanes 5 and 6: overexpression of HF-Smt3p (G97) from plasmid expressing mutant HF-Smt3p(Smt3p (G97)). (B) The same six samples shown in A were also analyzed in parallel by SDS-PAGE and immunoblotted with anti-FLAG antibodies instead of anti-hTOP1 antibodies. (C) Yeast cell lysates from JN362a cells harboring HF-Smt3p (lanes 1 and 2) or cells harboring HF-Smt3p(G97) (lanes 3 and 4) were prepared as described in A. Lysates were immunoblotted with anti-hTOP1 antibodies. (D) The lysates from C were immunoprecipated with anti-hTOP1 antibodies as described in Materials and Methods. The beads fractions were analyzed on 5% SDS-PAGE and immunoblotted with anti-FLAG antibodies. Lanes 1 and 2: IP beads fractions from JN362a expressing hTOP1 and HF-Smt3p with (+) (lane 1) or without (−) (lane 2) CPT treatment. Lanes 3 and 4: beads fractions from JN362a expressing hTOP1 and HF-Smt3p(G97) with (+) (lane 3) or without (−) (lane 4) CPT treatment. The 100-kDa bands indicated in B and D are protein(s) in yeast lysates which are cross-reactive to anti-FLAG antibodies.
Figure 3
Figure 3
CPT -induced formation of hTOP1-Smt3p conjugates is UBC9 dependent in yeast. (A) Yeast ubc9 ts mutant strain Y0174 was transformed with YCpGAL-hTOP1 plasmid. Cells were treated with CPT (+) (lanes 1 and 3) or DMSO (−) (lanes 2 and 4) at both permissive (25°C) (lanes 1 and 2) and nonpermissive (36°C) (lanes 3 and 4) temperatures. Cells were lysed and analyzed on SDS-PAGE and immunoblotted with anti-hTOP1 antibodies as described in Materials and Methods. (B) The formation of hTOP1-Smt3p conjugates was examined in the UBC9 wild-type strain Y0007 as described in A.
Figure 4
Figure 4
CPT induces SUMO-1 conjugation to hTOP1 in HeLa cells. HeLa cells were treated with 10 μM CPT (+) or DMSO (−) for 15 min. Cells were lysed and analyzed as described in Materials and Methods. (A) Samples were analyzed on 5% SDS-PAGE and immunoblotted with anti-hTOP1 antibodies. Duplicate samples were loaded. Lanes 1 and 2 were samples from cells treated with CPT. Lanes 3 and 4 were samples from cells without CPT treatment. (B) The same membrane filter was stripped and reblotted with anti-SUMO-1 antibodies.
Figure 5
Figure 5
Overexpression of hUBC9 enhances SUMO-1 conjugation to hTOP1 in HeLa cells treated with CPT. (A) Tet-Off HeLa cells were transfected with pTRE-hUBC9 and the stable transfectant (clone 2) was selected by Northern blot screening. Clone 2 was cultured in the presence (+) or absence (−) of tetracycline for 48 h before CPT treatment (10 μM for 10 min). The same amount of samples (based on protein concentrations) were analyzed on 5% SDS-PAGE and immunoblotted with anti-hTOP1 antibodies. (B) The same membrane filter from (A) was stripped and reblotted with anti-SUMO-1 antibodies. (C) The same membrane filter from (B) was stripped and reblotted with anti-hTOP2α antibodies.
Figure 6
Figure 6
Binding of hTOP1 to GST-hUBC9. (A) hTOP1 binds to GST-hUBC9 in a GST pull down assay. The GST pull down assay was performed as described in Materials and Methods. Lanes 1 and 2 were lysates from GST bead fractions. Lanes 3 and 4 were lysates from GST-hUBC9 bead fractions. NE indicates that nuclear extracts were used in the pull down assay (lanes 1 and 3). “TOP1” indicates that purified recombinant hTOP1 was used in the pull down assay (lanes 2 and 4). (B) hTOP1 binds to GST-hUBC9 in a coimmunoprecipitation assay. The coimmunoprecipitation assay was performed as descried in Materials and Methods. Purified GST-hUBC9 is shown in lane 1. Lanes 2 (bead) and 4 (supernatant): anti-HA antibody (control antibody) was used in coimmunoprecipitation. Lanes 3 (bead) and 5 (supernatant): anti-hTOP1 antibodies were used in coimmunoprecipitation.
Figure 7
Figure 7
UBC9 and Smt3p affect CPT sensitivity in yeast expressing hTOP1. (A) An ubc9 ts mutant is hypersensitive to CPT treatment. Yeast strains Y0007 (wild-type) and Y0174 (ubc9ts) were transformed with YCpGAL1-hTOP1. The CPT sensitivity were determined at 30°C as described in Materials and Methods. (B) The effect of Smt3p overexpression on CPT sensitivity. hTOP1 and Smt3p (wild-type or G97 mutant) were coinduced in JN362a cells by 2% galactose. CPT sensitivity was determined at 30°C as described in Materials and Methods.
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