SUMO-targeted ubiquitin ligases in genome stability

We identify the SUMO-Targeted Ubiquitin Ligase (STUbL) family of proteins and propose that STUbLs selectively ubiquitinate sumoylated proteins and proteins that contain SUMO-like domains (SLDs). STUbL recruitment to sumoylated/SLD proteins is mediated by tandem SUMO interaction motifs (SIMs) within the STUbLs N-terminus. STUbL-mediated ubiquitination maintains sumoylation pathway homeostasis by promoting target protein desumoylation and/or degradation. Thus, STUbLs establish a novel mode of communication between the sumoylation and ubiquitination pathways. STUbLs are evolutionarily conserved and include: Schizosaccharomyces pombe Slx8-Rfp (founding member), Homo sapiens RNF4, Dictyostelium discoideum MIP1 and Saccharomyces cerevisiae Slx5-Slx8. Cells lacking Slx8-Rfp accumulate sumoylated proteins, display genomic instability, and are hypersensitive to genotoxic stress. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1, demonstrating the specificity of STUbLs as regulators of sumoylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8-Rfp, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 and its human homolog NIP45, which contain SLDs, are candidate STUbL targets. Consistently, Rad60 and Slx8-Rfp mutants have similar DNA repair defects.     

A SUMO-targeted ubiquitin ligase is involved in the degradation of the nuclear pool of the SUMO E3 ligase Siz1

The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5∆ cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase.     

Disruption of SUMO-targeted ubiquitin ligases Slx5–Slx8/RNF4 alters RecQ-like helicase Sgs1/BLM localization in yeast and human cells

RecQ-like helicases are a highly conserved protein family that functions during DNA repair and, when mutated in humans, is associated with cancer and/or premature aging syndromes. The budding yeast RecQ-like helicase Sgs1 has important functions in double-strand break (DSB) repair of exogenously induced breaks, as well as those that arise endogenously, for example during DNA replication. To further investigate Sgs1's regulation, we analyzed the subcellular localization of a fluorescent fusion of Sgs1 upon DNA damage. Consistent with a role in DSB repair, Sgs1 recruitment into nuclear foci in asynchronous cultures increases after ionizing radiation (IR) and after exposure to the alkylating agent methyl methanesulfonate (MMS). Yet, despite the importance of Sgs1 in replicative damage repair and in contrast to its elevated protein levels during S-phase, we find that the number of Sgs1 foci decreases upon nucleotide pool depletion by hydroxyurea (HU) treatment and that this negative regulation depends on the intra S-phase checkpoint kinase Mec1. Importantly, we identify the SUMO-targeted ubiquitin ligase (STUbL) complex Slx5–Slx8 as a negative regulator of Sgs1 foci, both spontaneously and upon replicative damage. Slx5–Slx8 regulation of Sgs1 foci is likely conserved in eukaryotes, since expression of the mammalian Slx5–Slx8 functional homologue, RNF4, restores Sgs1 focus number in slx8 cells and furthermore, knockdown of RNF4 leads to more BLM foci in U-2 OS cells. Our results point to a model where RecQ-like helicase subcellular localization is regulated by STUbLs in response to DNA damage, presumably to prevent illegitimate recombination events.     

NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres

Nijmegen breakage syndrome, a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. The NBS1 gene product, p95 (NBS1 or nibrin) forms a complex with Rad50 and Mre11. Cells deficient in the formation of this complex are defective in DNA double-strand break repair, cell cycle checkpoint control, and telomere length maintenance. How the NBS1 complex is involved in telomere length maintenance remains unclear. Here we show that the C-terminal region of NBS1 interacts directly with a telomere repeat binding factor, TRF1, by both yeast two-hybrid and in vivo DNA-coimmunoprecipitation assays. NBS1 and Mre11 colocalize with TRF1 at promyelocytic leukemia (PML) nuclear bodies in immortalized telomerase-negative cell lines, but rarely in telomerase-positive cell lines. The translocation of NBS1 to PML bodies occurs specifically during late S to G(2) phases of the cell cycle and coincides with active DNA synthesis in these NBS1-containing PML bodies. These results suggest that NBS1 may be involved in alternative lengthening of telomeres in telomerase-negative immortalized cells.     

Purification of the yeast Slx5-Slx8 protein complex and characterization of its DNA-binding activity

SLX5 and SLX8 encode RING-finger proteins that were previously identified based on their requirement for viability in yeast cells lacking Sgs1 DNA helicase. Slx5 and Slx8 proteins are known to be required for genome stability and to physically interact in yeast extracts; however, their biochemical functions are unknown. To address this question we purified and characterized recombinant Slx5 and Slx8 proteins. Here we show that Slx5 and Slx8 form a heterodimeric complex with double-stranded DNA (dsDNA)-binding activity. Individually, only the Slx8 subunit displays this activity. Structure–function studies indicate that the DNA-binding activity requires only the N-terminal 160 amino acids of Slx8, but not its C-terminal RING-finger domain. Alleles of SLX8 that express the RING-finger domain alone show almost complete complementation in yeast indicating that this DNA-binding domain is not essential for this in vivo function. Consistent with these findings we show that Slx5 immunolocalizes to the nucleus and that a portion of the Slx8 protein co-fractionates with chromatin. These results suggest that Slx5–Slx8 may act directly on DNA to promote genome stability.     

Nuclear bodies: multifunctional companions of the genome

It has become increasingly apparent that gene expression is regulated by the functional interplay between spatial genome organization and nuclear architecture. Within the nuclear environment a variety of distinct nuclear bodies exist. They are dynamic, self-organizing structures that do not assemble as pre-formed entities but rather emerge as a direct reflection of specific activities associated with gene expression and genome maintenance. Here I summarize recent findings on functions of some of the most prominent nuclear bodies, including the nucleolus, Cajal body, PML nuclear body, Polycomb group body and the 53BP1 nuclear body. The emerging view is that their organization is orchestrated by similar principles, and they function in fundamental cellular processes involved in homeostasis, differentiation, development and disease.     

Genome stability is guarded by yeast Rtt105 through multiple mechanisms

Ty1 mobile DNA element is the most abundant and mutagenic retrotransposon present in the genome of the budding yeast Saccharomyces cerevisiae. Protein regulator of Ty1 transposition 105 (Rtt105) associates with large subunit of RPA and facilitates its loading onto a single-stranded DNA at replication forks. Here, we dissect the role of RTT105 in the maintenance of genome stability under normal conditions and upon various replication stresses through multiple genetic analyses. RTT105 is essential for viability in cells experiencing replication problems and in cells lacking functional S-phase checkpoints and DNA repair pathways involving homologous recombination. Our genetic analyses also indicate that RTT105 is crucial when cohesion is affected and is required for the establishment of normal heterochromatic structures. Moreover, RTT105 plays a role in telomere maintenance as its function is important for the telomere elongation phenotype resulting from the Est1 tethering to telomeres. Genetic analyses indicate that rtt105Δ affects the growth of several rfa1 mutants but does not aggravate their telomere length defects. Analysis of the phenotypes of rtt105Δ cells expressing NLS-Rfa1 fusion protein reveals that RTT105 safeguards genome stability through its role in RPA nuclear import but also by directly affecting RPA function in genome stability maintenance during replication.     

p53 and p21(Waf1) Are Recruited to Distinct PML‐Containing Nuclear Foci in Irradiated and Nutlin‐3a‐Treated U2OS Cells

Promyelocytic leukemia nuclear bodies (PML‐NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML‐NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53‐dependent growth inhibition. ALT‐associated PML bodies (APBs) are specialized PML‐NBs that include telomere‐repeat binding‐factor TRF1 and are exclusively in telomerase‐negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell‐cycle and p53 responses in ALT‐positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin‐3a. Both IR and Nutlin‐3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin‐3a. p53 was recruited to PML‐NBs 3–4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML‐NBs marked sites of apparently unrepaired DNA double‐strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin‐3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin‐3a‐treated cells. These data indicate (1) p53 is recruited to PML‐NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53–p21 pathway activation increases the percentage of APB‐positive cells, (3) p21 and p53 are recruited to ALT‐associated PML‐NBs after Nutlin‐3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance. J. Cell. Biochem. 111: 1280–1290, 2010. © 2010 Wiley‐Liss, Inc.     

SUMOrganization of the nucleus

In the eukaryotic nucleus, gene expression and maintenance of genome integrity are tightly controlled at multiple levels, from the molecular details to the higher-order structure of the genome. The nucleus contains spatially and functionally distinct compartments in which these fundamental processes are carried out. While the dynamics and functions of some nuclear subdomains, like the nucleolus, have been well studied, other domains, like the PML-nuclear bodies, remain enigmatic. Recent evidence has now implicated the SUMOylation pathway as an important player in subnuclear architecture, particularly in the assembly of PML-nuclear bodies. Related functions include the organization of chromatin loops and maintenance of rDNA repeat stability. Consequently, complete loss of SUMO modification profoundly affects nuclear organization and cell viability.     

End‐joining inhibition at telomeres requires the translocase and polySUMO‐dependent ubiquitin ligase Uls1

In eukaryotes, permanent inhibition of the non‐homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non‐essential Swi2/Snf2‐related translocase and a Small Ubiquitin‐related Modifier (SUMO)‐Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere–telomere fusions. Uls1 requirement is alleviated by the absence of poly‐SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly‐SUMO conjugates. We propose that one of Uls1 functions is to clear non‐functional poly‐SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly‐SUMOylated proteins on DNA in eukaryotes.     

The Nijmegen breakage syndrome gene and its role in genome stability

NBS1 is the key regulator of the RAD50/MRE11/NBS1 (R/M/N) protein complex, a sensor and mediator for cellular DNA damage response. NBS1 potentiates the enzymatic activity of MRE11 and directs the R/M/N complex to sites of DNA damage, where it forms nuclear foci by interacting with phosphorylated H2AX. The R/M/N complex also activates the ATM kinase, which is a major kinase involved in the activation of DNA damage signal pathways. The ATM requires the R/M/N complex for its own activation following DNA damage, and for conformational change to develop a high affinity for target proteins. In addition, association of NBS1 with PML, the promyelocytic leukemia protein, is required to form nuclear bodies, which have various functions depending on their location and composition. These nuclear bodies function not only in response to DNA damage, but are also involved in telomere maintenance when they are located on telomeres. In this review, we describe the role of NBS1 in the maintenance of genetic stability through the activation of cell-cycle checkpoints, DNA repair, and protein relocation.     

PML nuclear bodies and chromatin dynamics: catch me if you can!

Eukaryotic cells compartmentalize their internal milieu in order to achieve specific reactions in time and space. This organization in distinct compartments is essential to allow subcellular processing of regulatory signals and generate specific cellular responses. In the nucleus, genetic information is packaged in the form of chromatin, an organized and repeated nucleoprotein structure that is a source of epigenetic information. In addition, cells organize the distribution of macromolecules via various membrane-less nuclear organelles, which have gathered considerable attention in the last few years. The macromolecular multiprotein complexes known as Promyelocytic Leukemia Nuclear Bodies (PML NBs) are an archetype for nuclear membrane-less organelles. Chromatin interactions with nuclear bodies are important to regulate genome function. In this review, we will focus on the dynamic interplay between PML NBs and chromatin. We report how the structure and formation of PML NBs, which may involve phase separation mechanisms, might impact their functions in the regulation of chromatin dynamics. In particular, we will discuss how PML NBs participate in the chromatinization of viral genomes, as well as in the control of specific cellular chromatin assembly pathways which govern physiological mechanisms such as senescence or telomere maintenance.     

The G-Quadruplex Ligand Telomestatin Impairs Binding of Topoisomerase IIIα to G-Quadruplex-Forming Oligonucleotides and Uncaps Telomeres in ALT Cells

In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIα (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed.     

Phosphorylation of Slx4 by Mec1 and Tel1 regulates the single-strand annealing mode of DNA repair in budding yeast

Budding yeast (Saccharomyces cerevisiae) Slx4 is essential for cell viability in the absence of the Sgs1 helicase and for recovery from DNA damage. Here we report that cells lacking Slx4 have difficulties in completing DNA synthesis during recovery from replisome stalling induced by the DNA alkylating agent methyl methanesulfonate (MMS). Although DNA synthesis restarts during recovery, cells are left with unreplicated gaps in the genome despite an increase in translesion synthesis. In this light, epistasis experiments show that SLX4 interacts with genes involved in error-free bypass of DNA lesions. Slx4 associates physically, in a mutually exclusive manner, with two structure-specific endonucleases, Rad1 and Slx1, but neither of these enzymes is required for Slx4 to promote resistance to MMS. However, Rad1-dependent DNA repair by single-strand annealing (SSA) requires Slx4. Strikingly, phosphorylation of Slx4 by the Mec1 and Tel1 kinases appears to be essential for SSA but not for cell viability in the absence of Sgs1 or for cellular resistance to MMS. These results indicate that Slx4 has multiple functions in responding to DNA damage and that a subset of these are regulated by Mec1/Tel1-dependent phosphorylation.     

A SUMO-interacting motif activates budding yeast ubiquitin ligase Rad18 towards SUMO-modified PCNA

SUMO-targeted ubiquitin ligases (STUbLs) recognize sumoylated proteins as substrates for ubiquitylation and have been implicated in several aspects of DNA repair and the damage response. However, few physiological STUbL substrates have been identified, and the relative importance of SUMO binding versus direct interactions with the substrate remains a matter of debate. We now present evidence that the ubiquitin ligase Rad18 from Saccharomyces cerevisiae, which monoubiquitylates the sliding clamp protein proliferating cell nuclear antigen (PCNA) in response to DNA damage, exhibits the hallmarks of a STUbL. Although not completely dependent on sumoylation, Rad18’s activity towards PCNA is strongly enhanced by the presence of SUMO on the clamp. The stimulation is brought about by a SUMO-interacting motif in Rad18, which also mediates sumoylation of Rad18 itself. Our results imply that sumoylated PCNA is the physiological ubiquitylation target of budding yeast Rad18 and suggest a new mechanism by which the transition from S phase-associated sumoylation to damage-induced ubiquitylation of PCNA is accomplished.     

The SUMO-targeted ubiquitin ligase subunit Slx5 resides in nuclear foci and at sites of DNA breaks

The Slx5/Slx8 protein complex, a heterodimeric SUMO-targeted ubiquitin ligase, plays an important role in genomic integrity. Slx5/Slx8 is believed to interact with sumoylated proteins that reside in the nuclei of budding yeast cells. In this complex, Slx5, owing to at least two SUMO interacting motifs (SIMs), has been proposed to be the targeting subunit of the Slx8 ubiquitin ligase. However, little is known about the exact subnuclear localization and targets of Slx5/Slx8. In this study we show that Slx5, but not Slx8, forms prominent nuclear foci. The formation of these foci depends on SUMO and a SIM in Slx5. Therefore, we investigated the subnuclear localization and potential chromatin association of Slx5. Using co-localization studies in live cells and fixed chromatin, we were able to localize Slx5 to DNA damage induced foci of Rad52 and Rad9, two proteins involved in the cellular response to DNA damage. Subsequent chromatin immunoprecipitation (ChIP) studies revealed that Slx5 is associated with HO endonuclease induced chromosome breaks. Surprisingly, real-time PCR analysis of Slx5 ChIPs revealed that the level of Slx5 at HO breaks in an slx8 deletion background is reduced about 4-fold. These results indicate that the DNA-damage targeting of Slx5/Slx8 depends on formation of the heterodimer and that this occurs at a subset of nuclear foci also containing DNA damage repair and checkpoint factors.     

A SIM-ultaneous role for SUMO and ubiquitin

Ubiquitin and ubiquitin-like proteins (Ubls) share a beta-GRASP fold and have key roles in cellular growth and suppression of genome instability. Despite their common fold, SUMO and ubiquitin are classically portrayed as distinct, and they can have antagonistic roles. Recently, a new family of proteins, the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligases (STUbLs), which directly connect sumoylation and ubiquitylation, has been discovered. Uniquely, STUbLs use SUMO-interaction motifs (SIMs) to recognize their sumoylated targets. STUbLs are global regulators of protein sumoylation levels, and cells lacking STUbLs display genomic instability and hypersensitivity to genotoxic stress. The human STUbL, RNF4, is implicated in several diseases including cancer, highlighting the importance of characterizing the cellular functions of STUbLs.