The TORC1 complex is a key regulator of cell growth and metabolism in The vacuole-associated EGO complex couples activation of TORC1 to the availability of amino acids, specifically glutamine and leucine. The EGO complex is also essential for reactivation of TORC1 following rapamycin-induced growth arrest and for its distribution on the vacuolar membrane. Pib2, a FYVE-containing phosphatidylinositol 3-phosphate (PI3P)-binding protein, is a newly discovered and poorly characterized activator of TORC1. Here, we show that Pib2 is required for reactivation of TORC1 following rapamycin-induced growth arrest. Pib2 is required for EGO complex-mediated activation of TORC1 by glutamine and leucine as well as for redistribution of Tor1 on the vacuolar membrane. Therefore, Pib2 and the EGO complex cooperate to activate TORC1 and connect phosphoinositide 3-kinase (PI3K) signaling and TORC1 activity.

Here, we investigate the role of the budding yeast Shu complex in promoting homologous recombination (HR) upon replication fork damage. We recently found that the Shu complex stimulates Rad51 filament formation during HR through its physical interactions with Rad55-Rad57. Unlike other HR factors, Shu complex mutants are primarily sensitive to replicative stress caused by MMS and not to more direct DNA breaks. Here, we uncover a novel role for the Shu complex in the repair of specific MMS-induced DNA lesions and elucidate the interplay between HR and translesion DNA synthesis. We find that the Shu complex promotes high-fidelity bypass of MMS-induced alkylation damage, such as N3-methyladenine, as well as bypassing the abasic sites generated after Mag1 removes N3-methyladenine lesions. Furthermore, we find that the Shu complex responds to ssDNA breaks generated in cells lacking the abasic site endonucleases. At each lesion, the Shu complex promotes Rad51-dependent HR as the primary repair/tolerance mechanism over error-prone translesion DNA polymerases. Together, our work demonstrates that the Shu complex's promotion of Rad51 pre-synaptic filaments is critical for high-fidelity bypass of multiple replication-blocking lesion.

The Saccharomyces cerevisiae Shu2 protein is an important regulator of Rad51, which promotes homologous recombination (HR). Shu2 functions in the Shu complex with Shu1 and the Rad51 paralogs Csm2 and Psy3. Shu2 belongs to the SWS1 protein family, which is characterized by its SWIM domain (CXC...X n ...CXH), a zinc-binding motif. In humans, SWS1 interacts with the Rad51 paralog SWSAP1. Using genetic and evolutionary analyses, we examined the role of the Shu complex in mitotic and meiotic processes across eukaryotic lineages. We provide evidence that the SWS1 protein family contains orthologous genes in early-branching eukaryote lineages (e.g., Giardia lamblia), as well as in multicellular eukaryotes including Caenorhabditis elegans and Drosophila melanogaster. Using sequence analysis, we expanded the SWIM domain to include an invariant alanine three residues after the terminal CXH motif (CXC. . .X n . . .CXHXXA). We found that the SWIM domain is conserved in all eukaryotic orthologs, and accordingly, in vivo disruption of the invariant residues within the canonical SWIM domain inhibits DNA damage tolerance in yeast and protein-protein interactions in yeast and humans. Furthermore, using evolutionary analyses, we found that yeast and Drosophila Shu2 exhibit strong coevolutionary signatures with meiotic proteins, and in yeast, its disruption leads to decreased meiotic progeny. Together our data indicate that the SWS1 family is an ancient and highly conserved eukaryotic regulator of meiotic and mitotic HR.KEYWORDS DNA repair; Shu complex; budding yeast; evolutionary rate covariation; homologous recombination H OMOLOGOUS recombination (HR) is an error-free DNA repair pathway that is essential both in mitotic cells to ensure DNA stability and in meiotic cells for faithful chromosome segregation. HR begins with double-strand break (DSB) formation and DNA end processing that give rise to 39 singlestranded DNA (ssDNA) overhangs (Heyer et al. 2010). A key step in HR is the formation of RecA-like filaments on these ssDNA ends. During mitosis in the budding yeast Saccharomyces cerevisiae, the RecA-like protein Rad51 coats the ssDNA, whereas during meiosis, both Rad51 and another RecA-like protein, Dmc1, form on ssDNA ends (Lin et al. 2006;Heyer et al. 2010). Formation of RecA-like filaments on the DNA is essential for the homology search and strand-invasion steps that define HR. Therefore, regulation of RecA filament formation is critical for accurate repair of DNA damage and chromosome segregation. Given their importance, both Rad51 and Dmc1 are extremely well-conserved descendants of the archaeal protein RADA (Diruggiero et al. 1999;Komori et al. 2000;Lin et al. 2006;Chintapalli et al. 2013).There are many proteins that both promote Rad51 filament formation and disassemble inappropriate filaments. Interestingly, in many organisms, the proteins that stabilize Rad51 filaments themselves share structural homology with Rad51 and evolved from the archaeal RADB homolog (Lin et al. 2006). In humans, these Rad51 paralogs...

The RecQ helicase family is critical during DNA damage repair, and mutations in these proteins are associated with Bloom, Werner, or Rothmund-Thompson syndromes in humans, leading to cancer predisposition and/or premature aging. In the budding yeast Saccharomyces cerevisiae, mutations in the RecQ homolog, SGS1, phenocopy many of the defects observed in the human syndromes. One challenge to studying RecQ helicases is that their disruption leads to a pleiotropic phenotype. Using yeast, we show that the separation-of-function allele of SGS1, sgs1-D664Δ, has impaired activity at DNA ends, resulting in a resection processivity defect. Compromising Sgs1 resection function in the absence of the Sae2 nuclease causes slow growth, which is alleviated by making the DNA ends accessible to Exo1 nuclease. Furthermore, fluorescent microscopy studies reveal that, when Sgs1 resection activity is compromised in sae2Δ cells, Mre11 repair foci persist. We suggest a model where the role of Sgs1 in end resection along with Sae2 is important for removing Mre11 from DNA ends during repair. REPAIR of DNA damage is essential for maintenance of genomic integrity. When DNA repair is compromised, mutations and genetic alterations that are the hallmarks of cancer cells can occur. One group of proteins important for repair of DNA damage is the RecQ family of DNA helicases. In humans, mutations in three of the five RecQ-like proteins (BLM, WRN, RTS) are associated with heritable cancer predisposition diseases and/or premature aging (Bloom, Werner, or Rothmund-Thomson syndromes, respectively). In the budding yeast Saccharomyces cerevisiae, the RecQ helicase, Sgs1, when mutated, exhibits many aspects of the phenotype associated with alterations in its human homologs such as genomic instability and premature aging (reviewed in Bernstein et al. 2010).Sgs1 and its homologs function in the homologous recombination (HR) DNA repair pathway. When a doublestrand break (DSB) occurs, the broken DNA ends are bound by the Mre11-Rad50-Xrs2 (MRX)/MRE11-RAD50-NBS1 (MRN) complex in yeast and humans, respectively (hereafter, the yeast nomenclature is written first and the human protein second). Subsequently, Sae2/CtIP, together with MRX/MRN, initiate resection of the DNA ends, thus preventing the binding of the Ku70-Ku80 heterodimer and inhibiting nonhomologous end joining (NHEJ) (Mimitou and Symington 2010;Foster et al. 2011;. Sae2 is essential to remove covalent adducts, such as Spo11, from DNA ends, but its function in resection initiation at endonuclease-induced DSBs can be bypassed by downstream nucleases. The absence of Mre11 leads to a greater delay in resection initiation at endonucleaseinduced DSBs than that observed for Sae2 due to the role of the MRX complex in recruiting Sgs1, Dna2, and Exo1 to DNA ends (Mimitou and Symington 2010;Shim et al. 2010). Subsequent long-range resection of the 39 overhangs can then occur by one of two pathways: the first utilizes the Sgs1/BLM helicase in conjunction with the endonuclease Dna2/DNA2 and the second u...

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.