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Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund-Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation-of-function alleles of SGS1 that suppress the slow growth of top3Delta and rmi1Delta cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild-type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1-D664Delta, unlike sgs1Delta, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication-associated X-shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1-D664Delta allele exhibits increased spontaneous RPA foci, suggesting that the persistent X-structures may contain single-stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.
Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress. The Mec1/Rad53/Dun1 DNA damage response kinase cascade exhibits multifaceted controls over RNR activity including the regulation of the RNR inhibitor, Sml1. After DNA damage, Sml1 is degraded leading to the up-regulation of dNTP pools by RNR. Here, we probe the requirements for Sml1 degradation and identify several sites required for in vivo phosphorylation and degradation of Sml1 in response to DNA damage. Further, in a strain containing a mutation in Rnr1, rnr1-W688G, mutation of these sites in Sml1 causes lethality. Degradation of Sml1 is dependent on the 26S proteasome. We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4.
Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.
The Shu complex, consisting of Rad51 paralogues, is an important regulator of homologous recombination, an error-free DNA repair pathway. Consequently, when members of this complex are disrupted, cells exhibit a mutator phenotype, sensitivity to DNA damage reagents and increased gross chromosomal rearrangements. Previously, we found that the Shu complex plays an important role in ribosomal DNA (rDNA) recombination when the Upstream Activating Factor (UAF) protein Uaf30 is disrupted. UAF30 encodes a protein needed for rDNA transcription and when deleted, rDNA recombination increases and the rDNA expands in a Shu1-dependent manner. Here we find using the uaf30-sensitized background that the central DNA repair protein Rad52, which is normally excluded from the nucleolus, frequently overlaps with the rDNA. This close association of Rad52 with the rDNA is dependent upon Shu1 in a uaf30 mutant. Previously, it was shown that in the absence of Rad52 sumoylation, Rad52 foci mislocalize to the nucleolus. Interestingly, here we find that using the uaf30 sensitized background the ability to regulate Rad52 sumoylation is important for Shu1 dependent rDNA recombination as well as Rad52 close association with rDNA. Our results suggest that in the absence of UAF30, the Shu complex plays a central role in Rad52 rDNA localization as long as Rad52 can be sumoylated. This discrimination is important for rDNA copy number homeostasis.
The yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling, and catalytic functions in the response to DNA damage. Xrs2, the eukaryotic-specific component of the complex, is required for nuclear import of Mre11 and Rad50 and to recruit the Tel1 kinase to damage sites. We show that nuclear-localized MR complex (Mre11-NLS) catalyzes homology-dependent repair without Xrs2, but MR cannot activate Tel1, and it fails to tether DSBs, resulting in sensitivity to genotoxins, replisome instability, and increased gross chromosome rearrangements (GCRs). Fusing the Tel1 interaction domain from Xrs2 to Mre11-NLS is sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells. Furthermore, Tel1 stabilizes Mre11-DNA association, and this stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Enforcing Tel1 recruitment to the nuclear MR complex fully rescues end tethering and stalled replication fork stability, and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.
The postreplication repair gene, HLTF, is often amplified and overexpressed in cancer. Here we model HLTF dysregulation through the functionally conserved Saccharomyces cerevisiae ortholog, RAD5. Genetic interaction profiling and landscape enrichment analysis of RAD5 overexpression (RAD5OE) reveals requirements for genes involved in recombination, crossover resolution, and DNA replication. While RAD5OE and rad5Δ both cause cisplatin sensitivity and share many genetic interactions, RAD5OE specifically requires crossover resolving genes and drives recombination in a region of repetitive DNA. Remarkably, RAD5OE induced recombination does not require other post-replication repair pathway members, or the PCNA modification sites involved in regulation of this pathway. Instead, the RAD5OE phenotype depends on a conserved domain necessary for binding 3' DNA ends. Analysis of DNA replication intermediates supports a model in which dysregulated Rad5 causes aberrant template switching at replication forks. The direct effect of Rad5 on replication forks in vivo, increased recombination, and cisplatin sensitivity predicts similar consequences for dysregulated HLTF in cancer.
Recent studies have demonstrated that the MYB oncogene is frequently duplicated in human T cell acute lymphoblastic leukemia (T-ALL). We find that the human MYB locus is flanked by 257-bp Alu repeats and that the duplication is mediated somatically by homologous recombination between the flanking Alu elements on sister chromatids. Nested long-range PCR analysis indicated a low frequency of homologous recombination leading to MYB tandem duplication in the peripheral blood mononuclear cells of approximately 50% of healthy individuals, none of whom had a MYB duplication in the germline. We conclude that Alu-mediated MYB tandem duplication occurs at low frequency during normal thymocyte development and is clonally selected during the molecular pathogenesis of human T-ALL.
During S phase in Saccharomyces cerevisiae, chromosomal loci become mobile in response to DNA double-strand breaks both at the break site (local mobility) and throughout the nucleus (global mobility). Increased nuclear exploration is regulated by the recombination machinery and the DNA damage checkpoint and is likely an important aspect of homology search. While mobility in response to DNA damage has been studied extensively in S phase, the response in interphase has not, and the question of whether homologous recombination proceeds to completion in G1 phase remains controversial. Here, we find that global mobility is triggered in G1 phase. As in S phase, global mobility in G1 phase is controlled by the DNA damage checkpoint and the Rad51 recombinase. Interestingly, despite the restriction of Rad52 mediator foci to S phase, Rad51 foci form at high levels in G1 phase. Together, these observations indicate that the recombination and checkpoint machineries promote global mobility in G1 phase, supporting the notion that recombination can occur in interphase diploids.
The sequence of the Saccharomyces cerevisiae RAD52 gene contains five potential translation start sites and protein-blot analysis typically detects multiple Rad52 species with different electrophoretic mobilities. Here we define the gene products encoded by RAD52. We show that the multiple Rad52 protein species are due to promiscuous choice of start codons as well as post-translational modification. Specifically, Rad52 is phosphorylated both in a cell cycle-independent and in a cell cycle-dependent manner. Furthermore, phosphorylation is dependent on the presence of the Rad52 C terminus, but not dependent on its interaction with Rad51. We also show that the Rad52 protein can be translated from the last three start sites and expression from any one of them is sufficient for spontaneous recombination and the repair of gamma-ray-induced double-strand breaks.
Spontaneous mitotic recombination is a potential source of genetic changes such as loss of heterozygosity and chromosome translocations, which may lead to genetic disease. In this study we have used a rad52 hyper-recombination mutant, rad52-Y66A, to investigate the process of spontaneous heteroallelic recombination in the yeast Saccharomyces cerevisiae. We find that spontaneous recombination has different genetic requirements, depending on whether the recombination event occurs between chromosomes or between chromosome and plasmid sequences. The hyper-recombination phenotype of the rad52-Y66A mutation is epistatic with deletion of MRE11, which is required for establishment of DNA damage-induced cohesion. Moreover, single-cell analysis of strains expressing YFP-tagged Rad52-Y66A reveals a close to wild-type frequency of focus formation, but with foci lasting 6 times longer. This result suggests that spontaneous DNA lesions that require recombinational repair occur at the same frequency in wild-type and rad52-Y66A cells, but that the recombination process is slow in rad52-Y66A cells. Taken together, we propose that the slow recombinational DNA repair in the rad52-Y66A mutant leads to a by-pass of the window-of-opportunity for sister chromatid recombination normally promoted by MRE11-dependent damage-induced cohesion thereby causing a shift towards interchromosomal recombination.
Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.
The CKS1B gene located on chromosome 1q21 is frequently amplified in breast, lung, and liver cancers. CKS1B codes for a conserved regulatory subunit of cyclin-CDK complexes that function at multiple stages of cell cycle progression. We used a high throughput screening protocol to mimic cancer-related overexpression in a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential only when CKS1 is overexpressed, a synthetic dosage lethal (SDL) interaction. Mutations in multiple genes affecting mitotic entry and mitotic exit are highly enriched in the set of SDL interactions. The interactions between Cks1 and the mitotic entry checkpoint genes require the inhibitory activity of Swe1 on the yeast cyclin-dependent kinase (CDK), Cdc28. In addition, the SDL interactions of overexpressed CKS1 with mutations in the mitotic exit network are suppressed by modulating expression of the CDK inhibitor Sic1. Mutation of the polo-like kinase Cdc5, which functions in both the mitotic entry and mitotic exit pathways, is lethal in combination with overexpressed CKS1 Therefore we investigated the effect of targeting the human Cdc5 ortholog, PLK1, in breast cancers with various expression levels of human CKS1B Growth inhibition by PLK1 knockdown correlates with increased CKS1B expression in published tumor cell data sets, and this correlation was confirmed using shRNAs against PLK1 in tumor cell lines. In addition, we overexpressed CKS1B in multiple cell lines and found increased sensitivity to PLK1 knockdown and PLK1 drug inhibition. Finally, combined inhibition of WEE1 and PLK1 results in less apoptosis than predicted based on an additive model of the individual inhibitors, showing an epistatic interaction and confirming a prediction of the yeast data. Thus, identification of a yeast SDL interaction uncovers conserved genetic interactions that can affect human cancer cell viability.
Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell.
Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.
The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. We discovered a link to ribosomal DNA (rDNA) recombination when we found an interaction between one member of the Shu complex, SHU1, and UAF30, a component of the upstream activating factor complex (UAF), which regulates rDNA transcription. In the absence of Uaf30, rDNA copy number increases, and this increase depends on several functional subunits of the Shu complex. Furthermore, in the absence of Uaf30, we find that Shu1 and Srs2, an anti-recombinase DNA helicase with which the Shu complex physically interacts, act in the same pathway regulating rDNA recombination. In addition, Shu1 modulates Srs2 recruitment to both induced and spontaneous foci correlating with a decrease in Rad51 foci, demonstrating that the Shu complex is an important regulator of Srs2 activity. Last, we show that Shu1 regulation of Srs2 to double-strand breaks is not restricted to the rDNA, indicating a more general function for the Shu complex in the regulation of Srs2. We propose that the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2.
During homologous recombination, cells must coordinate repair, DNA damage checkpoint signaling, and movement of chromosomal loci to facilitate homology search. In Saccharomyces cerevisiae, increased movement of damaged loci (local mobility) and undamaged loci (global mobility) precedes homolog pairing in mitotic cells. How cells modulate chromosome mobility in response to DNA damage remains unclear. Here, we demonstrate that global chromosome mobility is regulated by the Rad51 recombinase and its mediator, Rad52. Surprisingly, rad51Δ rad52Δ cells display checkpoint-dependent constitutively increased mobility, indicating that a regulatory circuit exists between recombination and checkpoint machineries to govern chromosomal mobility. We found that the requirement for Rad51 in this circuit is distinct from its role in recombination and that interaction with Rad52 is necessary to alleviate inhibition imposed by mediator recruitment to ssDNA. Thus, interplay between recombination factors and the checkpoint restricts increased mobility until recombination proteins are assembled at damaged sites.
Rad52 is essential for all homologous recombination and DNA double strand break repair events in Saccharomyces cerevisiae. This protein is multifunctional and contains several domains that allow it to interact with DNA as well as with different repair proteins. However, it has been unclear how Rad52 enters the nucleus. In the present study, we have used a combination of mutagenesis and sequence analysis to show that Rad52 from S. cerevisiae contains a single functional pat7 type NLS essential for its nuclear localization. The region containing the NLS seems only to be involved in nuclear transport as it plays no role in repair of MMS-induced DNA damage. The NLS in Rad52 is weak, as monomeric protein species that harbor this NLS are mainly located in the cytosol. In contrast, multimeric protein complexes wherein each subunit contains a single NLS(Rad52) sort efficiently to the nucleus. Based on the results we propose a model where the additive effect of multiple NLS(Rad52) sequences in a Rad52 ring-structure ensures efficient nuclear localization of Rad52.
Recruitment of the homologous recombination machinery to sites of double-strand breaks is a cell cycle-regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B-type cyclin/CDK1 activity. Induction of the intra-S-phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU-treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra-S-phase checkpoint. We propose that B-type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.
Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.
Mutations in KRAS are frequent in human cancer, yet effective targeted therapeutics for these cancers are still lacking. Attempts to drug the MEK kinases downstream of KRAS have had limited success in clinical trials. Understanding the specific genomic vulnerabilities of KRAS-driven cancers may uncover novel patient-tailored treatment options.
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