Chihiro Horigome

Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery

Chromatin mobility is thought to facilitate homology search during homologous recombination and to shift damage either towards or away from specialized repair compartments. However, unconstrained mobility of double-strand breaks could also promote deleterious chromosomal translocations. Here we use live time-lapse fluorescence microscopy to track the mobility of damaged DNA in budding yeast. We found that a Rad52–YFP focus formed at an irreparable double-strand break moves in a larger subnuclear volume than the undamaged locus. In contrast, Rad52–YFP bound at damage arising from a protein–DNA adduct shows no increase in movement. Mutant analysis shows that enhanced double-strand-break mobility requires Rad51, the ATPase activity of Rad54, the ATR homologue Mec1 and the DNA-damage-response mediator Rad9. Consistent with a role for movement in the homology-search step of homologous recombination, we show that recombination intermediates take longer to form in cells lacking Rad9.

SWR1 and INO80 Chromatin Remodelers Contribute to DNA Double-Strand Break Perinuclear Anchorage Site Choice

Persistent DNA double-strand breaks (DSBs) are recruited to the nuclear periphery in budding yeast. Both the Nup84 pore subcomplex and Mps3, an inner nuclear membrane (INM) SUN domain protein, have been implicated in DSB binding. It was unclear what, if anything, distinguishes the two potential sites of repair. Here, we characterize and distinguish the two binding sites. First, DSB-pore interaction occurs independently of cell-cycle phase and requires neither the chromatin remodeler INO80 nor recombinase Rad51 activity. In contrast, Mps3 binding is S and G2 phase specific and requires both factors. SWR1-dependent incorporation of Htz1 (H2A.Z) is necessary for break relocation to either site in both G1- and S-phase cells. Importantly, functional assays indicate that mutations in the two sites have additive repair defects, arguing that the two perinuclear anchorage sites define distinct survival pathways.

PolySUMOylation by Siz2 and Mms21 triggers relocation of DNA breaks to nuclear pores through the Slx5/Slx8 STUbL.

High-resolution imaging shows that persistent DNA damage in budding yeast localizes in distinct perinuclear foci for repair. The signals that trigger DNA double-strand break (DSB) relocation or determine their destination are unknown. We show here that DSB relocation to the nuclear envelope depends on SUMOylation mediated by the E3 ligases Siz2 and Mms21. In G1, a polySUMOylation signal deposited coordinately by Mms21 and Siz2 recruits the SUMO targeted ubiquitin ligase Slx5/Slx8 to persistent breaks. Both Slx5 and Slx8 are necessary for damage relocation to nuclear pores. When targeted to an undamaged locus, however, Slx5 alone can mediate relocation in G1-phase cells, bypassing the requirement for polySUMOylation. In contrast, in S-phase cells, monoSUMOylation mediated by the Rtt107-stabilized SMC5/6-Mms21 E3 complex drives DSBs to the SUN domain protein Mps3 in a manner independent of Slx5. Slx5/Slx8 and binding to pores favor repair by ectopic break-induced replication and imprecise end-joining.

Ribosome biogenesis factors bind a nuclear envelope SUN domain protein to cluster yeast telomeres

Two interacting ribosome biogenesis factors, Ebp2 and Rrs1, associate with Mps3, an essential inner nuclear membrane protein. Both are found in foci along the nuclear periphery, like Mps3, as well as in the nucleolus. Temperature‐sensitive ebp2 and rrs1 mutations that compromise ribosome biogenesis displace the mutant proteins from the nuclear rim and lead to a distorted nuclear shape. Mps3 is known to contribute to the S‐phase anchoring of telomeres through its interaction with the silent information regulator Sir4 and yKu. Intriguingly, we find that both Ebp2 and Rrs1 interact with the C‐terminal domain of Sir4, and that conditional inactivation of either ebp2 or rrs1 interferes with both the clustering and silencing of yeast telomeres, while telomere tethering to the nuclear periphery remains intact. Importantly, expression of an Ebp2–Mps3 fusion protein in the ebp2 mutant suppresses the defect in telomere clustering, but not its defects in growth or ribosome biogenesis. Our results suggest that the ribosome biogenesis factors Ebp2 and Rrs1 cooperate with Mps3 to mediate telomere clustering, but not telomere tethering, by binding Sir4.

Synergistic defect in 60S ribosomal subunit assembly caused by a mutation of Rrs1p, a ribosomal protein L11-binding protein, and 3′-extension of 5S rRNA in Saccharomyces cerevisiae

Rrs1p, a ribosomal protein L11-binding protein, has an essential role in biogenesis of 60S ribosomal subunits. We obtained conditionally synthetic lethal allele with the rrs1-5 mutation and determined that the mutation is in REX1, which encodes an exonuclease. The highly conserved leucine at 305 was substituted with tryptophan in rex1-1. The rex1-1 allele resulted in 3′-extended 5S rRNA. Polysome analysis revealed that rex1-1 and rrs1-5 caused a synergistic defect in the assembly of 60S ribosomal subunits. In vivo and in vitro binding assays indicate that Rrs1p interacts with the ribosomal protein L5–5S rRNA complex. The rrs1-5 mutation weakens the interaction between Rrs1p with both L5 and L11. These data suggest that the assembly of L5–5S rRNA on 60S ribosomal subunits coordinates with assembly of L11 via Rrs1p.

Rrs1p, a ribosomal protein L11-binding protein, is required for nuclear export of the 60S pre-ribosomal subunit in Saccharomyces cerevisiae.

Rrs1p is a ribosomal protein L11‐binding protein in Saccharomyces cerevisiae. We have obtained temperature‐sensitive rrs1 mutants by random PCR mutagenesis. [3H]Methionine pulse‐chase analysis reveals that the rrs1 mutations cause a defect in maturation of 25S rRNA. Ribosomal protein L25‐enhanced green fluorescent protein, a reporter of the 60S ribosomal subunit, concentrates in the nucleus with enrichment in the nucleolus when the rrs1 mutants are shifted to the restrictive temperature. These results suggest that Rrs1p stays on the pre‐60S particle from the early stage to very late stage of the large‐subunit maturation and is required for export of 60S subunits from the nucleolus to the cytoplasm.

A Ribosome Assembly Factor Ebp2p, the Yeast Homolog of EBNA1-Binding Protein 2, Is Involved in the Secretory Response

We have found that Ebp2p is essential for maturation of 25S rRNA and assembly of 60S pre-ribosomal subunits in Saccharomyces cerevisiae. We obtained three temperature-sensitive ebp2 mutants by PCR. Polysome analysis revealed that the synthesis of 60S ribosomal subunits was compromised in each of the ebp2 mutants at the restrictive temperature. The ebp2 alleles affected the transcriptional repression of both rRNA and ribosomal protein genes due to a secretion block. Fluorescence microscopy showed that a secretion block led to condensation of nucleolar Ebp2p, whereas that was not the case with the ebp2 mutant. These results suggest that Ebp2p is implicated in the secretory response, including changes in nucleolar architecture.

Genetic Interaction between Ribosome Biogenesis and Inositol Polyphosphate Metabolism in Saccharomyces cerevisiae

Rrs1 has an essential role in 60S ribosomal subunit assembly in Saccharomyces cerevisiae. We isolated a temperature-sensitive kcs1 mutant that suppresses the cold sensitivity of rrs1-1. The kcs1 allele, resulting in truncation of inositol 6 phosphate kinase domain, and kcs1 disruption suppress a defect of rrs1-1 in 60S ribosomal subunit assembly. These results suggest that inositol polyphosphate metabolism affects ribosome biogenesis in yeast.

Ribosome biogenesis factors working with a nuclear envelope SUN domain protein: New players in the solar system

The nucleolus, the most prominent structure observed in the nucleus, is often called a “ribosome factory.” Cells spend an enormous fraction of their resources to achieve the mass-production of ribosomes required by rapid growth. On the other hand, ribosome biogenesis is also tightly controlled, and must be coordinated with other cellular processes. Ribosomal proteins and ribosome biogenesis factors are attractive candidates for this link. Recent results suggest that some of them have functions beyond ribosome biogenesis. Here we review recent progress on ribosome biogenesis factors, Ebp2 and Rrs1, in yeast Saccharomyces cerevisiae. In this organism, Ebp2 and Rrs1 are found in the nucleolus and at the nuclear periphery. At the nuclear envelope, these proteins interact with a membrane-spanning SUN domain protein, Mps3, and play roles in telomere clustering and silencing along with the silent information regulator Sir4. We propose that a protein complex consisting Ebp2, Rrs1 and Mps3 is involved in a wide range of activities at the nuclear envelope.

SUMO wrestles breaks to the nuclear ring's edge

The accurate repair of DNA double-strand breaks (DSBs) is essential for cell survival and maintenance of genome integrity. In most cases, cells counteract DSBs by employing 2 highly conserved repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). When these pathways are impaired due to a lack of homologous donor sequence or conditions that block end-to-end ligation, alternative repair occurs, such as break-induced replication (BIR) or else imprecise or microhomology-mediated end-joining. Since these alternative pathways tend to be highly mutagenic, the choice of the repair pathway is controlled on at least 3 levels: by cell cycle stage, the chromatin context of the damage and the subnuclear position of the breaks. In budding yeast, persistent DSBs are recruited to the nuclear periphery and associate with nuclear pores through the Nup84 subcomplex or with an inner nuclear membrane SUN domain protein called Mps3. The cell cycle stage influences target site choice: pores are used in both G1 and S/G2-phases of cell cycle, while Mps3 binding only occurs in S/G2-phase cells. In S-phase cells, collapsed or stalled replication forks at extended triplet repeats, as well as eroded telomeres, were shown to shift to nuclear pores. Importantly, these 2 perinuclear binding sites differentially affected the repair outcome. Mps3 appears to sequester resected DSBs and thereby inhibits aberrant recombination events, whereas nuclear pores are implicated in the non-canonical repair pathways such as BIR and imprecise end-joining (see reviews and Horigome et al). There is, however, some cross-talk between the sites, as Mps3 may contribute to proper pore assembly, complicating the interpretation of repair data based on mps3 mutants. Several recent papers highlight the importance of SUMO (small ubiquitin-like modifier) as a driver for perinuclear anchoring of DSBs. The nuclear pore harbors the SUMO protease Ulp1 and Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL) and an earlier genetic study revealed that nuclear pores, Slx5/Slx8, and the proteasome act on the same pathway in DNA repair. Extensive SUMOylation events occur in response to DNA breaks in multiple species, such that factors involved in various pathways of repair become modified. Intriguingly, Horigome et al showed that the target of DSB relocation at the nuclear envelope depends on the nature of SUMOylation mediated by the E3 ligases Siz2 and Mms21. In G1and S-phase cells, a polySUMOylation chain deposited coordinately by Mms21 and Siz2 recruits the Slx5/Slx8 STUbL to persistent breaks. Then Slx5 mediates binding to the nuclear pore subcomplex, Nup84. Slx5 alone can shift DNA to pores when it is targeted to a tagged locus through a DNA binding domain, even in the absence of damage. This artificial targeting of Slx5 bypasses the need for polySUMOylation for relocation. Nonetheless, at endogenous breaks and shortened telomeres, both SUMOylation and Slx8 are needed to stabilize Slx5 binding and allow the damaged site to shift to the Nup84. In S-phase cells, monoSUMOylation mediated by the SMC5/6-Mms21 E3 complex correlated with the association of resected DSBs with the SUN domain protein, Mps3, and this can occur in the absence of Slx5. Moreover, the targeted binding of a polymer of SUMO residues (4 head-to-tail linked Smt3 residues) to an undamaged chromatin locus, allowed it to bind to pores, while the targeting of a single Smt3 residue (mono-SUMO), shifted the same locus to Mps3. Importantly, the polySUMO-dependent relocation to pores still required Slx5, arguing that this STUbL and its SUMO interacting motifs must recognize a polySUMO chain to mediate relocation (Fig. 1). The question arises as to whether one or multiple SUMOylation targets are crucial for the relocation. This may well depend on the type of damage. At eroded telomeres RPA was shown to be a SUMOylation target. Since it recruits Slx5/Slx8, it was proposed to be involved in targeting the telomere to nuclear pores for an alternative pathway of repair. In Drosophila, Ryu et al. showed that DSBs in heterochromatin shift away from the compacted chromatin domain and bind to either the nuclear pore (Nup107 or Nup160) and/or the SUN domain proteins (Koi or Spag4) in a SUMOylationand STUbL (Dgrn)-dependent manner. As in yeast, both nuclear pores and the SUN domain proteins work in concert with Smc5/6 and its targeted SUMO ligase Mms21 (Nse2), yet the 2 perinuclear binding sites act independently from each other. The recruitment of the fly Slx5/Slx8 homolog (Dgrn) requires SUMO ligases Nse2 and dPIAS, which modifies