Ryu H et al. (2010), PIASy-dependent SUMOylation regulates DNA topoi...

XB-ART-42585

J Cell Biol 2010 Nov 15;1914:783-94. doi: 10.1083/jcb.201004033.

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PIASy-dependent SUMOylation regulates DNA topoisomerase IIalpha activity.

Ryu H , Furuta M , Kirkpatrick D , Gygi SP , Azuma Y .

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DNA topoisomerase IIα (TopoIIα) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of TopoIIα activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of TopoIIα strongly inhibits TopoIIα decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that TopoIIα is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of TopoIIα, which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of TopoIIα activity. Together, our findings provide evidence for the regulation of TopoIIα activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of TopoIIα.

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Species referenced: Xenopus laevis
Genes referenced: parp1 pias4 ranbp2 sumo1 sumo2 ube2i

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	Figure 1. PIASy but not RanBP2 is required for SUMO2/3 conjugation of TopoIIÎ± in XEE. (a) XEE were immunodepleted using antibodies against RanBP2, PIASy, or RanBP2/PIASy. The depleted extracts were incubated with 10,000 sperm nuclei/Âµl for 1 h at 25Â°C. Non- or mock (IgG)-depleted extracts were also subjected to the same procedure. Isolated chromosomes from each reaction were analyzed by Western blotting (WB) for the indicated protein. Immunodepletion (ID) of RanBP2 had no effect on the SUMOylation of TopoIIÎ±, whereas PIASy ID eliminated TopoIIÎ± SUMOylation. The arrow and bracket indicate unmodified and SUMO2/3-modified TopoIIÎ±, respectively. Positions of molecular mass standards (kD) are indicated on the left. (b) The mitotic chromosomes were prepared as in Materials and methods and were analyzed by immunostaining with the indicated antibodies: TopoIIÎ± is shown in red, PIASy is shown in green, and SUMO2/3 is shown in blue in merged panel. TopoIIÎ± colocalized with PIASy and SUMO2/3 at the centromeres. The addition of dnUbc9 eliminated SUMO2/3 modification but did not alter the localization of TopoIIÎ± at the centromeres of mitotic chromosomes. Bars, 10 Âµm.
	Figure 2. PIASy is required for the efficient SUMOylation of TopoIIÎ± and for the selection of SUMO paralogues. (a) Ubc9 dosage-dependent SUMOylation. T7 tagged-TopoIIÎ± was incubated in a reaction containing various concentrations of Ubc9 (0â300 nM) in the presence of SUMO2. The amount of SUMO2-conjugated TopoIIÎ± was similar to that seen in XEE only when 300 nM Ubc9 was added. (b) Time course experiment with physiological (30 nM) and higher (300 nM) concentration of Ubc9. (c) PIASy dosage-dependent SUMOylation. T7-TopoIIÎ± was incubated as in panel a, except with various concentrations of PIASy (0â100 nM) and with the physiological concentration of Ubc9 (30 nM). PIASy efficiently facilitated SUMOylation of TopoIIÎ± under conditions using 30 nM Ubc9, where SUMOylation had barely appeared in the absence of PIASy. SUMOylation was saturated using >60 nM PIASy. (d) Time course experiment of PIASy-dependent SUMOylation. The reactions were performed with physiological (10 nM) or higher (100 nM) concentrations of PIASy in the presence of 30 nM Ubc9. (e) T7-TopoIIÎ± was incubated with either SUMO1 (s1) or SUMO2 (s2) in the presence of PIASy as indicated. PIASy showed a preference for SUMO2 over SUMO1. Positions of molecular mass standards (kD) are indicated on the sides of the gel blots.
	Figure 3. SUMO modification affects the decatenation activity of TopoIIÎ±. (a) T7-TopoIIÎ± was incubated with various combinations of Ubc9/PIASy as indicated to obtain a series of SUMOylation profiles. All control reactions (Cont.) were performed with 60 nM Ubc9/10 nM PIASy and SUMO2-G, which could not be conjugated. The samples were analyzed by Western blotting for the T7 tag. The arrow indicates maximal SUMO modification of TopoIIÎ± (seen in 30/30 and 60/10). Positions of molecular mass standards (kD) are indicated on the right. (b) Representative data of decatenation assay. Samples in a were further incubated with decatenation buffer that contained kDNA for 10 or 20 min, and the products were resolved in an agarose gel. Decatenated and linearized markers are designated. (c) Band intensity data from five independent experiments performed as in b are presented as the percentage of catenated kDNA remaining after a 20-min incubation, with standard error (error bars) and probability value from a Studentâs t test. SUMO2 modification of TopoIIÎ± decreased its decatenation activity.
	Figure 4. TopoIIÎ± K660R, a candidate SUMOylation mutant, shows incomplete SUMOylation in XEE. (a) Schematic diagram of S. cerevisiae TopoIIÎ± primary structure. Domains are denoted by color. This panel was modified from Schoeffler and Berger (2008), with permission from Cambridge University Press. TOPRIM, Topoisomerase-primase fold domain; WHD, Winged-helix domain; Tower, adjacent domain to WHD. The black bar indicates the catalytic tyrosine (Y782) for DNA cleavage in the WHD domain. Lys660 in X. laevis TopoIIÎ± was designated as a potential SUMOylation site by mass spectrometric analysis. The approximate position of the candidate lysine is shown by a green star in the DNA gate domain of TopoIIÎ±. The sequences near TopoIIÎ± Lys660 from X. laevis (xl), Homo sapiens (hs), and S. cerevisiae (sc) are conserved (indicated with bold and underlined text). (b) XEE were immunodepleted using nonspecific IgG (Cont.) or an anti-TopoIIÎ± antibody (âˆ’Topo). Efficiency of TopoIIÎ± depletion was confirmed by comparison of mock-depleted (Cont.) to TopoIIÎ±-depleted (âˆ’Topo) CSF extracts (left two lanes, labeled CSF extracts). WT nontagged TopoIIÎ± (WT) or mutant TopoIIÎ±, with substitution of arginine for Lys660 (K660R), was added to the TopoIIÎ±-depleted extracts (âˆ’Topo). After 1 h incubation at 25Â°C, mitotic chromosomes were isolated and analyzed by anti-TopoIIÎ± Western blotting. Analyzed chromosome samples were from mock-depleted (Cont.), TopoII-depleted (âˆ’Topo), and recombinant TopoIIÎ± added-back extracts (âˆ’Topo+WT or â€“Topo+K660R). (c) Same examination as in b except that the recombinant TopoIIÎ± proteins had a T7 tag at the N terminus. Both nontagged and T7-tagged K660R mutant showed subtle but reproducible reduction in higher shifted bands (indicated by brackets) of SUMOylation compared with WT. Positions of molecular mass standards (kD) are indicated on the sides of the gel blots.
	Figure 5. The elimination of TopoIIÎ± SUMOylation at Lys660 blocks SUMOylation-dependent inhibition of TopoIIÎ± activity. (a) Unmodified TopoIIÎ± WT and K660R proteins were incubated with kDNA to determine relative activity. K660R had â¼20 times less activity than WT. (b) Electrophoretic mobility shift assay. Unmodified TopoIIÎ± WT and K660R were incubated with plasmid DNA to determine relative DNA binding affinity. Both WT and K660R displayed similar binding affinity to DNA. oc and cc stand for open and closed circle, respectively. (c and e) The TopoIIÎ± WT and K660R were SUMO2-modified in vitro with 60 nM of Ubc9 and 30 nM of PIASy. Control reactions (Cont.) using the same condition except for SUMO2-G were also performed. Non-SUMOylated or SUMOylated TopoIIÎ± samples were assayed for decatenation activity. (d and f) Representative results of decatenation activity assays with TopoIIÎ± WT (d) and K660R (f) are shown. The mean decatenation activity from five independent experiments with TopoIIÎ± WT (g) and four independent experiments with TopoIIÎ± K660R (h) are displayed as the percentage of catenated kDNA remaining, with standard error (error bars). The strong inhibition of TopoIIÎ± decatenation activity by SUMOylation was abolished in reactions using TopoIIÎ± K660R. Positions of molecular mass standards (kD) are indicated on the sides of the gel blots.
	Figure 6. DNA binding of TopoIIÎ± increases susceptibility of SUMOylation at Lys660. (a) TopoIIÎ± WT in vitro SUMOylation reactions were performed with or without DNA. The samples were analyzed with anti-T7 tag antibody Western blots. The presence of DNA in the SUMOylation reactions significantly stimulates TopoIIÎ± WT SUMOylation. (b) TopoIIÎ± WT and TopoIIÎ± K660R were subjected to in vitro reactions under the same condition as in a except for using 5 ng/Âµl of DNA. PARP1, a mitotic chromosomal SUMO2/3 substrate, was used as a control. A deficiency of TopoIIÎ± K660R SUMOylation was observed in the presence of DNA compared with TopoIIÎ± WT. Positions of molecular mass standards (kD) are indicated on the left.
	Figure 7. Implications of SUMOylation in regulating the resolution of centromeric DNA. (a) Regulating the amount of catenated centromeric DNA. Active TopoIIÎ± resolves catenated DNA at the centromere and SUMOylation reduces the activity of TopoIIÎ± that has completed the decatenation of DNA. Without SUMOylation, overly active TopoIIÎ± could recatenate DNA at the centromere. (b) Regulation of the timing of decatenation. TopoIIÎ± SUMOylation keeps centromeric TopoIIÎ± temporally inert until anaphase, when decatenation of centromeric DNA must take place. Without proper deSUMOylation of TopoIIÎ±, decatenation of centromeric DNA will be compromised.

References [+] :

Agostinho, Conjugation of human topoisomerase 2 alpha with small ubiquitin-like modifiers 2/3 in response to topoisomerase inhibitors: cell cycle stage and chromosome domain specificity. 2008, Pubmed, Xenbase