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Results for “Synthetic Lethal Mutations”

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PubMed2001

Mutations in the TATA-binding protein, affecting transcriptional activation, show synthetic lethality with the TAF145 gene lacking the TAF N-terminal domain in Saccharomyces cerevisiae.

The general transcription factor TFIID, which is composed of the TATA box-binding protein (TBP) and a set of TBP-associated factors (TAFs), is crucial for both basal and regulated transcription by RNA polymerase II. The N-terminal small segment of yeast TAF145 (yTAF145) binds to TBP and thereby inhibits TBP function. To understand the physiological role of this inhibitory domain, which is designated as TAND (TAF N-terminal domain), we screened mutations, synthetically lethal with the TAF145 gene lacking TAND (taf145 Delta TAND), in Saccharomyces cerevisiae by exploiting a red/white colony-sectoring assay. Our screen yielded several recessive nsl (Delta TAND synthetic lethal) mutations, two of which, nsl1-1 and nsl1-2, define the same complementation group. The NSL1 gene was found to be identical to the SPT15 gene encoding TBP. Interestingly, both temperature-sensitive nsl1/spt15 alleles, which harbor the single amino acid substitutions, S118L and P65S, respectively, were defective in transcriptional activation in vivo. Several other previously characterized activation-deficient spt15 alleles also displayed synthetic lethal interactions with taf145 Delta TAND, indicating that TAND and TBP carry an overlapping but as yet unidentified function that is specifically required for transcriptional regulation.

Amino Acid Substitution↗

PubMed1993

Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth.

Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the beta subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.

Actins↗

PubMed2025

Mutations in tumor signaling, metastases, and synthetic lethality establish distinct patterns.

Effective identification of oncogenic mutations is essential for diagnosis, forecasting resistance, and metastasis in remission. It is required for an optimal drug regimen. We develop a framework to discover mutations that co-exist in different oncoproteins, and those that are excluded, likely encoding oncogene-induced senescence. First, mapping the proteins onto pathways assists combinatorial drug selections and helps to detect metastases. Second, it provides the molecular basis for synthetic lethality, to date investigated at the genome level. Our pan-cancer profiles of ~60,000 tumor sequences, detect 3424 co-existing tumor-specific mutations. Mapping them onto pathways indicates that they preferentially promote specific primary tumors. We uncover metastatic mutations and provide metastatic breast-cancer markers. This work not only clarifies the mechanistic basis of intratumor mutational diversity but usefully reveals markers for metastasis in patients' genomes and introduces a novel computational framework for detecting metastasis based on tumor mutational profiles. Mapping the mutations onto pathways provides an invaluable metastasis-targeting resource, guiding drug combinations.

Humans↗

PubMed2004

Functional distinction between Cln1p and Cln2p cyclins in the control of the Saccharomyces cerevisiae mitotic cycle.

Cln1p and Cln2p are considered as equivalent cyclins on the basis of sequence homology, regulation, and functional studies. Here we describe a functional distinction between the Cln1p and Cln2p cyclins in the control of the G1/S transition. Inactivation of CLN2, but not of CLN1, leads to a larger-than-normal cell size, whereas overexpression of CLN2, but not of CLN1, results in smaller-than-normal cells. Furthermore, mild ectopic expression of CLN2, but not of CLN1, suppresses the lethality of swi4swi6 and cdc28 mutant strains. In the absence of Cln1p, the kinetics of budding, initiation of DNA replication, and activation of the Start-transcription program are not affected; by contrast, loss of Cln2p causes a delay in bud emergence. A primary role for Cln2p but not for Cln1p in budding is reinforced by the observation that only the cln2 mutation is synthetic lethal with a cdc42 mutation, and only the cln2 mutant strain is hypersensitive to latrunculin B. In addition, we found that Cln1p showed a more prominent nuclear staining than Cln2p. Finally, chimeric proteins composed of Cln1p and Cln2p revealed that Cln2p integrity is required for its functional specificity.

Blotting, Northern↗

PubMed2005

The concept of synthetic lethality in the context of anticancer therapy.

Two genes are synthetic lethal if mutation of either alone is compatible with viability but mutation of both leads to death. So, targeting a gene that is synthetic lethal to a cancer-relevant mutation should kill only cancer cells and spare normal cells. Synthetic lethality therefore provides a conceptual framework for the development of cancer-specific cytotoxic agents. This paradigm has not been exploited in the past because there were no robust methods for systematically identifying synthetic lethal genes. This is changing as a result of the increased availability of chemical and genetic tools for perturbing gene function in somatic cells.

Animals↗

PubMed1999

Synthetic lethal phenotypes caused by mutations affecting chromosome partitioning in Bacillus subtilis.

We investigated the genetic interactions between mutations affecting chromosome structure and partitioning in Bacillus subtilis. Loss-of-function mutations in spoIIIE (encoding a putative DNA translocase) and smc (involved in chromosome structure and partitioning) caused a synthetic lethal phenotype. We constructed a conditional mutation in smc and found that many of the spoIIIE smc double-mutant cells had a chromosome bisected by a division septum. The growth defect of the double mutant was exacerbated by a null mutation in the chromosome partitioning gene spo0J. These results suggest that mutants defective in nucleoid structure are unable to move chromosomes out of the way of the invaginating septum and that SpoIIIE is involved in repositioning these bisected chromosomes during vegetative growth.

Bacillus subtilis↗

PubMed1998

Characterization of NOT5 that encodes a new component of the Not protein complex.

The yeast HIS3 gene has two core promoters: TC, a TATA-less element and TR, a canonical TATA element. Four genes encode global negative regulators of transcription that preferentially repress TC-dependent transcription: NOT1 (CDC39), NOT2 (CDC36), NOT3 and NOT4 (SIG1, MOT2). Genetic and biochemical experiments suggest that the products of these genes are associated in a complex and regulate TFIID function. In this paper, we describe a new gene, NOT5, that also represses transcription of the HIS3 TATA-less promoter preferentially and encodes a protein whose N-terminal region is 44% identical to that of Not3p. Our results indicate that NOT5 is involved in Not function and encodes a product that is physically associated with the other Not proteins. First, overexpression of NOT3 or NOT4 suppresses mutations in NOT5. Secondly, mutations in NOT4 are synthetically lethal with mutations in NOT5. Thirdly, NOT5 interacts with NOT1 and NOT3 in the two-hybrid assay. Finally, Not1p, Not3p and Not4p co-immunoprecipitate with Not5p.

Amino Acid Sequence↗

PubMed1999

High-copy suppressor analysis reveals a physical interaction between Sec34p and Sec35p, a protein implicated in vesicle docking.

A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport. A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Delta mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER. Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function. In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi. This screen revealed that SEC34 specifically suppresses sec35-1. SEC34 encodes a hydrophilic protein of approximately 100 kDa. Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble. Sec34p and Sec35p stably associate with each other to form a multiprotein complex of approximately 480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.

Amino Acid Sequence↗

PubMed1998

Fission yeast Ste9, a homolog of Hct1/Cdh1 and Fizzy-related, is a novel negative regulator of cell cycle progression during G1-phase.

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role of ste9(+) in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10(ts), cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase.

Amino Acid Sequence↗

PubMed1998

Pom1p, a fission yeast protein kinase that provides positional information for both polarized growth and cytokinesis.

Schizosaccharomyces pombe cells have a well-defined pattern of polarized growth at the cell ends during interphase and divide symmetrically into two equal-sized daughter cells. We identified a gene, pom1, that provides positional information for both growth and division in S. pombe. pom1 mutants form functioning growth zones and division septa but show several abnormalities: (1) After division, cells initiate growth with equal frequencies from either the old or the new end; (2) most cells never switch to bipolar growth but instead grow exclusively at the randomly chosen end; (3) some cells mislocalize their growth axis altogether, leading to the formation of angled and branched cells; and (4) many cells misplace and/or misorient their septa, leading to asymmetric cell division. pom1 encodes a putative protein kinase that is concentrated at the new cell end during interphase, at both cell ends during mitosis, and at the septation site after mitosis. Small amounts of Pom1p are also found at the old cell end during interphase and associated with the actin ring during mitosis. Pom1p localization to the cell ends is independent of actin but requires microtubules and Tea1p. pom1 mutations are synthetically lethal with several other mutations that affect cytokinesis and/or the actin or microtubule cytoskeleton. Thus, Pom1p may position the growth and cytokinesis machineries by interaction with both the actin and microtubule cytoskeletons.

Actins↗

PubMed2006

A novel dnaC mutation that suppresses priB rep mutant phenotypes in Escherichia coli K-12.

The loading of a replisome in prokaryotic and eukaryotic cells at an origin of DNA replication and during replication restart is a highly ordered and regulated process. During replication restart in Escherichia coli, the PriA, PriB, PriC, DnaT and Rep proteins form multiple pathways that bind to repaired replication forks. These complexes are then recognized by DnaC as sites to load DnaB, the replicative helicase. Several dnaC mutations have been isolated that suppress phenotypes of some replication restart mutants. A new dnaC mutation (dnaC824) is reported here that efficiently suppresses priB rep mutant phenotypes. Furthermore, it is shown that dnaC824 will suppress phenotypes of priB priA300, rep priA300 and priB priC strains. Unlike other dnaC suppressors, it can only weakly suppress the absence of priA. Others have reported a different type of dnaC mutation, dnaC1331, is able to mimic priB mutant phenotypes. This is supported herein by showing that like dnaC1331, a priB mutation is synthetically lethal with a dam mutation and this can be rescued by a mutH mutation. Furthermore, priB dam lethality can also be suppressed by dnaC824. Like a priB mutation, a dnaC1331 mutation causes a priA2::kan-like phenotype when combined with priA300. Lastly, we show that dnaC824 is dominant to wild type and that dnaC1331 is recessive to wild type. Several models are discussed for the action of these mutant dnaC proteins in replication restart.

DNA Replication↗

PubMed1999

A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.

To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

DNA Repair↗

PubMed1998

Interaction of Bnr1p with a novel Src homology 3 domain-containing Hof1p. Implication in cytokinesis in Saccharomyces cerevisiae.

Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in cytokinesis or establishment of cell polarity in a variety of organisms. We have shown that the FH proteins Bni1p and Bnr1p are potential targets of the Rho family small GTP-binding proteins and bind to an actin-binding protein, profilin, at their proline-rich FH1 domains to regulate reorganization of the actin cytoskeleton in the yeast Saccharomyces cerevisiae. We found here that a novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w, interacted with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of Bnr1p and the SH3 domain of Ymr032wp. Ymr032wp weakly bound to Bni1p. Ymr032wp was homologous to cdc15p, which is involved in cytokinesis in Schizosaccharomyces pombe, and we named this gene HOF1 (homolog of cdc 15). Both Bnr1p and Hof1p were localized at the bud neck, and both the bnr1 and hof1 mutations showed synthetic lethal interactions with the bni1 mutation. The hof1 mutant cells showed phenotypes similar to those of the septin mutants, indicating that HOF1 is involved in cytokinesis. These results indicate that Bnr1p directly interacts with Hof1p as well as with profilin to regulate cytoskeletal functions in S. cerevisiae.

Actins↗

PubMed1999

Sli15 associates with the ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae.

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. We show here that sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole-associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation.

Aurora Kinases↗

PubMed1995

Suppressors of a Saccharomyces cerevisiae pkc1 mutation identify alleles of the phosphatase gene PTC1 and of a novel gene encoding a putative basic leucine zipper protein.

The PKC1 gene product, protein kinase C, regulates a mitogen-activated protein kinase (MAPK) cascade, which is implicated in cell wall metabolism. Previously, we identified the pkc1-4 allele in a screen for mutants with increased rates of recombination, indicating that PKC1 may also regulate DNA metabolism. The pkc1-4 allele also conferred a temperature-sensitive (ts) growth defect. Extragenic suppressors were isolated that suppress both the ts and hyperrecombination phenotypes conferred by the pkc1-4 mutation. Eight of these suppressors for into two complementation groups, designated KCS1 and KCS2. KCS1 was cloned and found to encode a novel protein with homology to the basic leucine zipper family of transcription factors. KCS2 is allelic with PTC1, a previously identified type 2C serine/threonine protein phosphatase. Although mutation of either KCS1 or PTC1 causes little apparent phenotype, the kcs1 delta ptc1 delta double mutant fails to grow at 30 degrees. Furthermore, the ptc1 deletion mutation is synthetically lethal in combination with a mutation in MPK1, which encodes a MAPK homologue proposed to act in the PKC1 pathway. Because PTC1 was initially isolated as a component of the Hog1p MAPK pathway, it appears that these two MAPK cascades share a common regulatory feature.

Alleles↗

PubMed2000

EXO1 and MSH6 are high-copy suppressors of conditional mutations in the MSH2 mismatch repair gene of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Msh2p, a central component in mismatch repair, forms a heterodimer with Msh3p to repair small insertion/deletion mismatches and with Msh6p to repair base pair mismatches and single-nucleotide insertion/deletion mismatches. In haploids, a msh2Delta mutation is synthetically lethal with pol3-01, a mutation in the Poldelta proofreading exonuclease. Six conditional alleles of msh2 were identified as those that conferred viability in pol3-01 strains at 26 degrees but not at 35 degrees. DNA sequencing revealed that mutations in several of the msh2(ts) alleles are located in regions with previously unidentified functions. The conditional inviability of two mutants, msh2-L560S pol3-01 and msh2-L910P pol3-01, was suppressed by overexpression of EXO1 and MSH6, respectively. Partial suppression was also observed for the temperature-sensitive mutator phenotype exhibited by msh2-L560S and msh2-L910P strains in the lys2-Bgl reversion assay. High-copy plasmids bearing mutations in the conserved EXO1 nuclease domain were unable to suppress msh2-L560S pol3-01 conditional lethality. These results, in combination with a genetic analysis of msh6Delta pol3-01 and msh3Delta pol3-01 strains, suggest that the activity of the Msh2p-Msh6p heterodimer is important for viability in the presence of the pol3-01 mutation and that Exo1p plays a catalytic role in Msh2p-mediated mismatch repair.

14-3-3 Proteins↗

PubMed1998

Prohibitin family members interact genetically with mitochondrial inheritance components in Saccharomyces cerevisiae.

Phb2p, a homolog of the tumor suppressor protein prohibitin, was identified in a genetic screen for suppressors of the loss of Mdm12p, a mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance in Saccharomyces cerevisiae. Phb2p and its homolog, prohibitin (Phb1p), were localized to the mitochondrial inner membrane and characterized as integral membrane proteins which depend on each other for their stability. In otherwise wild-type genetic backgrounds, null mutations in PHB1 and PHB2 did not confer any obvious phenotypes. However, loss of function of either PHB1 or PHB2 in cells with mitochondrial DNA deleted led to altered mitochondrial morphology, and phb1 or phb2 mutations were synthetically lethal when combined with a mutation in any of three mitochondrial inheritance components of the mitochondrial outer membrane, Mdm12p, Mdm10p, and Mmm1p. These results provide the first evidence of a role for prohibitin in mitochondrial inheritance and in the regulation of mitochondrial morphology.

Fungal Proteins↗

PubMed2005

Requirements for replication restart proteins during constitutive stable DNA replication in Escherichia coli K-12.

Constitutive stable DNA replication (cSDR) is a mechanism for replisome loading in Escherichia coli K-12. This occurs in a dnaA-independent fashion in an rnhA mutant. cSDR is dependent on recA, priA, and transcription. In this report, it is shown that dnaA rnhA mutants using cSDR for initiation of their DNA replication additionally require priB, but not priC, for viability. Two subtle priA missense mutations either eliminated the ability to grow using cSDR (priA301 C479Y) or resulted in very small colonies (priA300 K230R). DnaC809, a priA suppressor, failed to allow priA or priB mutants to grow using cSDR to initiate DNA replication. Furthermore, unlike dnaC(+) strains, dnaC809 strains require priC for cSDR. DnaC809,820, a priC-independent suppressor of priA2::kan phenotypes, allowed priA and priC (but not priB) mutants to grow using cSDR to initiate DNA replication. It is also shown that rep and rnhA mutations are synthetically lethal. DnaC809 and dnaC809,820 mutations suppress this lethality. Rep is further shown to be required for cSDR in a dnaC809 strain. A model whereby these different sets of replication restart proteins interact preferentially with substrates associated with either RecA or SSB during replication restart and cSDR, respectively, is proposed.

Bacterial Proteins↗