SIZ1/SIZ2 Control of Chromosome Transmission Fidelity Is Mediated by the Sumoylation of Topoisomerase II

Abstract

The Smt3 (SUMO) protein is conjugated to substrate proteins through a cascade of E1, E2, and E3 enzymes. In budding yeast, the E3 step in sumoylation is largely controlled by Siz1p and Siz2p. Analysis of Siz ÿ cells shows that SUMO E3 is required for minichromosome segregation and thus has a positive role in maintaining the fidelity of mitotic transmission of genetic information. Sumoylation of the carboxy-terminus of Top2p, a known SUMO target, is mediated by Siz1p and Siz2p both in vivo and in vitro. Sumoylation in vitro reveals that Top2p is an extremely potent substrate for Smt3p conjugation and that chromatin-bound Top2p can still be sumoylated, unlike many other SUMO substrates. By combining mutations in the TOP2 sumoylation sites and the SIZ1 and SIZ2 genes we demonstrate that the minichromosome segregation defect and dicentric minichromosome stabilization, both characteristic for Smt3p-E3-deficient cells, are mediated by the lack of Top2p sumoylation in these cells. A role for Smt3p-modification as a signal for Top2p targeting to pericentromeric regions was suggested by an analysis of Top2p-Smt3p fusion. We propose a model for the positive control of the centromeric pool of Top2p, required for high segregation fidelity, by Smt3p modification.

In vivo modeling of polysumoylation uncovers targeting of Topoisomerase II to the nucleolus via optimal level of SUMO modification

“…S. cerevisiae strains were derived from W303-1A or S288c, as indicated in Table 2. Manipulation of yeast cells, microscopy, and biochemical techniques were essentially as described in Takahashi et al (2006). For microscopic imaging, the diploid strains carrying specific fluorescent markers, i.e., heterozygous fusions of mRFP to NIC96, SIK1, and SPC42 genes, were used for the integration of TOP2:4SMT3:GFP and TOP2:5SMT3:GFP constructs (Tables 1 and 2).…”

“…To insert SMT3-coding sequences into TOP2, two primers (ttgcgcatgctagcTCATCACCATCATCATCACATGCATGACTACAAG and CTAATACGTAactagtCCAATCTGTTCTCTGTGAGCCTCAAT) were used to amplify the poly-histidine and FLAG-tagged SMT3 (HF:SMT3; Takahashi et al 2006), without the last glycine codon required for Smt3p processing in vivo. The amplified fragment was digested with NheI and SpeI and inserted into the SpeI site of the 5′-truncated TOP2 gene (cloned into pTS901IU or pTS901IL with 5HA or green fluorescent protein (GFP) tags, respectively; Sasaki et al 2000).…”

“…To replace the wild-type SMT3 gene with the GFP-tagged versions HFG-SMT3 (GFP tag) expressed from the native SMT3 promoter, the targeting constructs were generated on the basis of the HF-SMT3::LEU2 integrative plasmid pAS924 (Takahashi et al 2006). The polymerase chain reaction (PCR)-generated GFP fragment (SpeI-NheI) was inserted into the SpeI site between the SMT3 promoter and the SMT3 ORF in pAS924 (Takahashi et al 2006).…”

“…However, biological adequacy of such fusions remains questionable because of unnatural positioning of the fused SUMO and its presence in only a single copy. In our pervious work, we employed a more biologically relevant fusion design, by introducing a single SUMO inside the Top2p in the close vicinity of the natural target site (Takahashi et al 2006). Such a fusion targeted Top2p to yeast centromeres (Takahashi et al 2006).…”

“…Top2p has a number of rather distinct roles in the cell, most notably in deoxyribonucleic acid (DNA) replication, transcription, and chromosome segregation (Wang 2002), making it tempting to test whether polysumoylation is specifically related to one of these functions. In this report, we attempted to track the artificially poly-sumoylated (SUMO-chain-modified) pool of Topoisomerase II in vivo using the fusion principle introduced in Takahashi et al (2006). In vivo modeling of Top2p polysumoylation allowed us to approach several unresolved questions about Top2p sumoylation.…”