Topoisomerase I-dependent viability loss in saccharomyces cerevisiae mutants defective in both SUMO conjugation and DNA repair.

Siz1 and Siz2/Nfi1 are the two Siz/PIAS SUMO E3 ligases in Saccharomyces cerevisiae. Here we show that siz1Delta siz2Delta mutants fail to grow in the absence of the homologous recombination pathway or the Fen1 ortholog RAD27. Remarkably, the growth defects of mutants such as siz1Delta siz2Delta rad52Delta are suppressed by mutations in TOP1, suggesting that these growth defects are caused by topoisomerase I activity. Other mutants that affect SUMO conjugation, including a ulp1 mutant and the nuclear pore mutants nup60Delta and nup133Delta, show similar top1-suppressible synthetic defects with DNA repair mutants, suggesting that these phenotypes also result from reduced SUMO conjugation. siz1Delta siz2Delta mutants also display TOP1-independent genome instability phenotypes, including increased mitotic recombination and elongated telomeres. We also show that SUMO conjugation, TOP1, and RAD27 have overlapping roles in telomere maintenance. Top1 is sumoylated, but Top1 does not appear to be the SUMO substrate involved in the synthetic growth defects. However, sumoylation of certain substrates, including Top1 itself and Tri1 (YMR233W), is enhanced in the absence of Top1 activity. Sumoylation is also required for growth of top1Delta cells. These results suggest that the SUMO pathway has a complex effect on genome stability that involves several mechanistically distinct processes.

Multiple domains in Siz SUMO ligases contribute to substrate selectivity.

Saccharomyces cerevisiae contains two Siz/PIAS SUMO E3 ligases, Siz1 and Siz2/Nfi1, and one other known ligase, Mms21. Although ubiquitin ligases are highly substrate-specific, the degree to which SUMO ligases target distinct sets of substrates is unknown. Here we show that although Siz1 and Siz2 each have unique substrates in vivo, sumoylation of many substrates can be stimulated by either protein. Furthermore, in the absence of both Siz proteins, many of the same substrates are still sumoylated at low levels. Some of this residual sumoylation depends on MMS21. Siz1 targets its unique substrates through at least two distinct domains. Sumoylation of PCNA (proliferating cell nuclear antigen) and the splicing factor Prp45 requires part of the N-terminal region of Siz1, the ;PINIT' domain, whereas sumoylation of the bud neck-associated septin proteins Cdc3, Cdc11 and Shs1/Sep7 requires the C-terminal domain of Siz1, which is also sufficient for cell cycle-dependent localization of Siz1 to the bud neck. Remarkably, the non-sumoylated septins Cdc10 and Cdc12 also undergo Siz1-dependent sumoylation if they are fused to the short PsiKXE SUMO attachment-site sequence. Collectively, these results suggest that local concentration of the E3, rather than a single direct interaction with the substrate polypeptide, is the major factor in substrate selectivity by Siz proteins.

The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast.

The ubiquitin-related protein SUMO functions by becoming covalently attached to lysine residues in other proteins. Unlike ubiquitin, which is often linked to its substrates as a polyubiquitin chain, only one SUMO moiety is attached per modified site in most substrates. However, SUMO has recently been shown to form chains in vitro and in mammalian cells, with a lysine in the non-ubiquitin-like N-terminal extension serving as the major SUMO-SUMO branch site. To investigate the physiological function of SUMO chains, we generated Saccharomyces cerevisiae strains that expressed mutant SUMOs lacking various lysine residues. Otherwise wild-type strains lacking any of the nine lysines in SUMO were viable, had no obvious growth defects or stress sensitivities, and had SUMO conjugate patterns that did not differ dramatically from wild type. However, mutants lacking the SUMO-specific isopeptidase Ulp2 accumulated high molecular weight SUMO-containing species, which formed only when the N-terminal lysines of SUMO were present, suggesting that they contained SUMO chains. Furthermore SUMO branch-site mutants suppressed several of the phenotypes of ulp2delta, consistent with the possibility that some ulp2delta phenotypes are caused by accumulation of SUMO chains. We also found that a mutant SUMO whose non-ubiquitin-like N-terminal domain had been entirely deleted still carried out all the essential functions of SUMO. Thus, the ubiquitin-like domain of SUMO is sufficient for conjugation and all downstream functions required for yeast viability. Our data suggest that SUMO can form chains in vivo in yeast but demonstrate conclusively that chain formation is not required for the essential functions of SUMO in S. cerevisiae.

The role of apoptosis in response to photodynamic therapy: what, where, why, and how.

Photodynamic therapy (PDT), a treatment for cancer and for certain benign conditions, utilizes a photosensitizer and light to produce reactive oxygen in cells. PDT is primarily employed to kill tumor and other abnormal cells, so it is important to ask how this occurs. Many of the photosensitizers currently in clinical or pre-clinical studies of PDT localize in or have a major influence on mitochondria, and PDT is a strong inducer of apoptosis in many situations. The purpose of this review is to critically evaluate all of the recently published research on PDT-induced apoptosis, with a focus on studies providing mechanistic insights. Components of the mechanism whereby PDT causes cells to undergo apoptosis are becoming understood, as are the influences of several signal transduction pathways on the response. Future research should be directed to elucidating the role(s) of the multiple steps in apoptosis in directing damaged cells to an apoptotic vs. necrotic pathway and for producing tumor ablation in conjunction with tissue-level mechanisms operating in vivo.