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 TopoIIα in XEE. (a) XEE were immunodepleted using antibodies against RanBP2, PIASy, or RanBP2/PIASy. The depleted extracts were incubated with 10,000 sperm nuclei/µl for 1 h at 25°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 TopoIIα, whereas PIASy ID eliminated TopoIIα SUMOylation. The arrow and bracket indicate unmodified and SUMO2/3-modified TopoIIα, 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: TopoIIα is shown in red, PIASy is shown in green, and SUMO2/3 is shown in blue in merged panel. TopoIIα colocalized with PIASy and SUMO2/3 at the centromeres. The addition of dnUbc9 eliminated SUMO2/3 modification but did not alter the localization of TopoIIα at the centromeres of mitotic chromosomes. Bars, 10 µm.
	Figure 2. PIASy is required for the efficient SUMOylation of TopoIIα and for the selection of SUMO paralogues. (a) Ubc9 dosage-dependent SUMOylation. T7 tagged-TopoIIα was incubated in a reaction containing various concentrations of Ubc9 (0–300 nM) in the presence of SUMO2. The amount of SUMO2-conjugated TopoIIα 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-TopoIIα was incubated as in panel a, except with various concentrations of PIASy (0–100 nM) and with the physiological concentration of Ubc9 (30 nM). PIASy efficiently facilitated SUMOylation of TopoIIα 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-TopoIIα 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 TopoIIα. (a) T7-TopoIIα 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 TopoIIα (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 Student’s t test. SUMO2 modification of TopoIIα decreased its decatenation activity.
	Figure 4. TopoIIα K660R, a candidate SUMOylation mutant, shows incomplete SUMOylation in XEE. (a) Schematic diagram of S. cerevisiae TopoIIα 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 TopoIIα 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 TopoIIα. The sequences near TopoIIα 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-TopoIIα antibody (−Topo). Efficiency of TopoIIα depletion was confirmed by comparison of mock-depleted (Cont.) to TopoIIα-depleted (−Topo) CSF extracts (left two lanes, labeled CSF extracts). WT nontagged TopoIIα (WT) or mutant TopoIIα, with substitution of arginine for Lys660 (K660R), was added to the TopoIIα-depleted extracts (−Topo). After 1 h incubation at 25°C, mitotic chromosomes were isolated and analyzed by anti-TopoIIα Western blotting. Analyzed chromosome samples were from mock-depleted (Cont.), TopoII-depleted (−Topo), and recombinant TopoIIα added-back extracts (−Topo+WT or –Topo+K660R). (c) Same examination as in b except that the recombinant TopoIIα 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 TopoIIα SUMOylation at Lys660 blocks SUMOylation-dependent inhibition of TopoIIα activity. (a) Unmodified TopoIIα WT and K660R proteins were incubated with kDNA to determine relative activity. K660R had ∼20 times less activity than WT. (b) Electrophoretic mobility shift assay. Unmodified TopoIIα 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 TopoIIα 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 TopoIIα samples were assayed for decatenation activity. (d and f) Representative results of decatenation activity assays with TopoIIα WT (d) and K660R (f) are shown. The mean decatenation activity from five independent experiments with TopoIIα WT (g) and four independent experiments with TopoIIα K660R (h) are displayed as the percentage of catenated kDNA remaining, with standard error (error bars). The strong inhibition of TopoIIα decatenation activity by SUMOylation was abolished in reactions using TopoIIα K660R. Positions of molecular mass standards (kD) are indicated on the sides of the gel blots.
	Figure 6. DNA binding of TopoIIα increases susceptibility of SUMOylation at Lys660. (a) TopoIIα 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 TopoIIα WT SUMOylation. (b) TopoIIα WT and TopoIIα K660R were subjected to in vitro reactions under the same condition as in a except for using 5 ng/µl of DNA. PARP1, a mitotic chromosomal SUMO2/3 substrate, was used as a control. A deficiency of TopoIIα K660R SUMOylation was observed in the presence of DNA compared with TopoIIα 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 TopoIIα resolves catenated DNA at the centromere and SUMOylation reduces the activity of TopoIIα that has completed the decatenation of DNA. Without SUMOylation, overly active TopoIIα could recatenate DNA at the centromere. (b) Regulation of the timing of decatenation. TopoIIα SUMOylation keeps centromeric TopoIIα temporally inert until anaphase, when decatenation of centromeric DNA must take place. Without proper deSUMOylation of TopoIIα, 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