Multipotent Genetic Suppression of Retrotransposon-Induced Mutations by Nxf1 through Fine-Tuning of Alternative Splicing

Cellular gene expression machinery has coevolved with molecular parasites, such as viruses and transposons, which rely on host cells for their expression and reproduction. We previously reported that a wild-derived allele of mouse Nxf1 (Tap), a key component of the host mRNA nuclear export machinery, suppresses two endogenous retrovirus-induced mutations and shows suggestive evidence of positive selection. Here we show that Nxf1^CAST suppresses a specific and frequent class of intracisternal A particle (IAP)-induced mutations, including Ap3d1^mh2J, a model for Hermansky-Pudlak syndrome, and Atcay^hes, an orthologous gene model for Cayman ataxia, among others. The molecular phenotype of suppression includes ∼two-fold increase in the level of correctly-spliced mRNA and a decrease in mutant-specific, alternatively-processed RNA accumulating from the inserted allele. Insertional mutations involving ETn and LINE elements are not suppressed, demonstrating a high degree of specificity to this suppression mechanism. These results implicate Nxf1 in some instances of pre-mRNA processing, demonstrate the useful range of Nxf1^CAST alleles for manipulating existing mouse models of disease, and specifically imply a low functional threshold for therapeutic benefit in Cayman ataxia.     

Inhibitor of Apoptosis Proteins (IAPs) and Their Antagonists Regulate Spontaneous and Tumor Necrosis Factor (TNF)-induced Proinflammatory Cytokine and Chemokine Production

Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings.     

Genetic interactions of modifier genes and modifiable alleles in Drosophila melanogaster

We have examined the effects of mutations in the six allele-specific modifier genes su(Hw), e(we), su(f), su(s), su(wa), and su(pr) on the expression of 18 modifiable alleles, situated at 11 loci. Ten of the modifiable alleles are associated with insertions of the gypsy retrotransposon and the others include alleles associated with insertions of copia and 412. We tested or retested 90 of the 108 possible combinations and examined the expression of modifiable alleles in flies mutant for pairs of modifier genes in various heterozygous and homozygous configurations. Our principal findings are: (1) a screen of 40,000 mutagenized X chromosomes yielded three new mutations in known modifier genes, but revealed no new modifier genes; (2) the modification effects of different mutations in a given modifier gene were qualitatively similar; (3) each of the six modifiers suppressed some modifiable alleles, enhanced others, and had no noticeable effect on still others; (4) the modifier genes could be placed in four classes, according to their effects on the gypsy-insertion alleles; and (5) the effects of mutations in different modifier genes combined additively. Implications of these results for models of modifier gene action are discussed.     

Inactivation of Nxf2 causes defects in male meiosis and age-dependent depletion of spermatogonia

In eukaryotes, mRNA is actively transported from nucleus to cytoplasm by a family of nuclear RNA export factors (NXF). While yeast harbors only one such factor (Mex67p), higher eukaryotes encode multiple NXFs. In mouse, four Nxf genes have been identified: Nxf1, Nxf2, Nxf3, and Nxf7. To date, the function of mouse Nxf genes has not been studied by targeted gene deletion in vivo. Here we report the generation of Nxf2 null mutant mice by homologous recombination in embryonic stem cells. Nxf2-deficient male mice exhibit fertility defects that differ between mouse strains. One third of Nxf2-deficient males on a mixed (C57BL/6 × 129) genetic background exhibit meiotic arrest and thus are sterile, whereas the remaining males are fertile. Disruption of Nxf2 in inbred (C57BL/6J) males impairs spermatogenesis, resulting in male subfertility, but causes no meiotic arrest. Testis weight and sperm output in C57BL/6J Nxf2^−/Y mice are sharply reduced. Mutant epididymal sperm exhibit diminished motility. Importantly, proliferation of spermatogonia in Nxf2^−/Y mice is significantly decreased. As a result, inactivation of Nxf2 causes depletion of germ cells in a substantial fraction of seminiferous tubules in aged mice. These studies demonstrate that Nxf2 plays a dual function in spermatogenesis: regulation of meiosis and maintenance of spermatogonial stem cells.     

Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function

Induction of apoptosis in Drosophila requires the activity of three closely linked genes, reaper, hid and grim. Here we show that the proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (diap1) protein. In a genetic modifier screen, both loss-of-function and gain-of-function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain-of-function mutations in diap1 strongly suppressed reaper-, hid- and grim-induced apoptosis. Sequence analysis of these alleles revealed that they were caused by single amino acid changes in the baculovirus IAP repeat domains of diap1, a domain implicated in binding REAPER, HID and GRIM. Significantly, the corresponding mutant DIAP1 proteins displayed greatly reduced binding of REAPER, HID and GRIM, indicating that REAPER, HID and GRIM kill by forming a complex with DIAP1. These data provide strong in vivo evidence for a previously published model of cell death regulation in Drosophila.     

Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.     

Unraveling Genetic Modifiers in the Gria4 Mouse Model of Absence Epilepsy

Absence epilepsy (AE) is a common type of genetic generalized epilepsy (GGE), particularly in children. AE and GGE are complex genetic diseases with few causal variants identified to date. Gria4 deficient mice provide a model of AE, one for which the common laboratory inbred strain C3H/HeJ (HeJ) harbors a natural IAP retrotransposon insertion in Gria4 that reduces its expression 8-fold. Between C3H and non-seizing strains such as C57BL/6, genetic modifiers alter disease severity. Even C3H substrains have surprising variation in the duration and incidence of spike-wave discharges (SWD), the characteristic electroencephalographic feature of absence seizures. Here we discovered extensive IAP retrotransposition in the C3H substrain, and identified a HeJ-private IAP in the Pcnxl2 gene, which encodes a putative multi-transmembrane protein of unknown function, resulting in decreased expression. By creating new Pcnxl2 frameshift alleles using TALEN mutagenesis, we show that Pcnxl2 deficiency is responsible for mitigating the seizure phenotype – making Pcnxl2 the first known modifier gene for absence seizures in any species. This finding gave us a handle on genetic complexity between strains, directing us to use another C3H substrain to map additional modifiers including validation of a Chr 15 locus that profoundly affects the severity of SWD episodes. Together these new findings expand our knowledge of how natural variation modulates seizures, and highlights the feasibility of characterizing and validating modifiers in mouse strains and substrains in the post-genome sequence era.     

Novel genes influencing the expression of the yellow locus and mdg4 (gypsy) in Drosophila melanogaster

Summary We used a system with a mobilized Stalker transposable element, sometimes in combination with P-M hybrid dysgenesis, in the search for new mutations interfering with the y ² mutation induced by mdg4 (gypsy) insertion into the yellow locus. A novel gene, modifier of mdg4, was detected in chromosome 3. The mutation mod(mdg4) either enhanced or suppressed phenotypic changes in different mutations induced by mdg4 insertions. Thus, mod(mdg4) seems to be involved in the control of mdg4 expression. Six other loci designated as enhancers of yellow were also detected. The e(y) ⁿ (with n from 1–6) mutations enhanced the expression of several y mutations induced by different insertions into the yellow locus. The major change is a damage of bristle and hair pigmentation which is not suppressed by su(Hw) mutations. On the other hand, e(y) ⁿ alleles do not interact with mdg4 induced mutations in other loci. All e(y) ⁿ genes are located in different regions of the X chromosome. One may speculate that e(y) ⁿ genes are involved in trans-regulation of the yellow locus and possibly of some other loci.     

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, and octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D₂O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. Our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.     

Molecular Identification of Collagen 17a1 as a Major Genetic Modifier of Laminin Gamma 2 Mutation-Induced Junctional Epidermolysis Bullosa in Mice

Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic jeb allele of laminin gamma 2 (Lamc2). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the Lamc2^jeb mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in Lamc2^jeb mice. This modifier is defined by variations in 1–3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the Lamc2^jeb mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder.     

Host Insect Inhibitor-of-Apoptosis SfIAP Functionally Replaces Baculovirus IAP but Is Differentially Regulated by Its N-Terminal Leader

The inhibitor-of-apoptosis (IAP) proteins encoded by baculoviruses bear a striking resemblance to the cellular IAP homologs of their invertebrate hosts. By virtue of the acquired selective advantage of blocking virus-induced apoptosis, baculoviruses may have captured cellular IAP genes that subsequently evolved for virus-specific objectives. To compare viral and host IAPs, we defined antiapoptotic properties of SfIAP, the principal cellular IAP of the lepidopteran host Spodoptera frugiperda. We report here that SfIAP prevented virus-induced apoptosis as well as viral Op-IAP3 (which is encoded by the Orgyia pseudotsugata nucleopolyhedrovirus) when overexpressed from the baculovirus genome. Like Op-IAP3, SfIAP blocked apoptosis at a step prior to caspase activation. Both of the baculovirus IAP repeats (BIRs) were required for SfIAP function. Moreover, deletion of the C-terminal RING motif generated a loss-of-function SfIAP that interacted and dominantly interfered with wild-type SfIAP. Like Op-IAP3, wild-type SfIAP formed intracellular homodimers, suggesting that oligomerization is a functional requirement for both cellular and viral IAPs. SfIAP possesses a ∼100-residue N-terminal leader domain, which is absent among all viral IAPs. Remarkably, deletion of the leader yielded a fully functional SfIAP with dramatically increased protein stability. Thus, the SfIAP leader contains an instability motif that may confer regulatory options for cellular IAPs that baculovirus IAPs have evolved to bypass for maximal stability and antiapoptotic potency. Our findings that SfIAP and viral IAPs have common motifs, share multiple biochemical properties including oligomerization, and act at the same step to block apoptosis support the hypothesis that baculoviral IAPs were derived by acquisition of host insect IAPs.Apoptosis is a prevalent host cell response to virus infection. Representing an important antivirus defense, apoptotic cell death can limit multiplication and virus dissemination in the host. Thus, the mechanisms by which a host organism detects a viral intruder and initiates the apoptotic response are critical to the outcome of the infection for both the host and virus. The cellular inhibitor-of-apoptosis (IAP) proteins are important candidates for sensing virus infection and determining cell fate by virtue of their central position in the apoptosis pathway (reviewed in references 35, 36, and 44). Affirming their importance in regulation of apoptosis, IAPs are encoded by multiple DNA viruses, including baculoviruses, entomopoxviruses, iridoviruses, and African swine fever virus (reviewed in 3). Nonetheless, the molecular mechanisms by which viral IAPs regulate virus-induced apoptosis and how they biochemically differ from cellular IAPs are poorly understood.The IAPs were first discovered in baculoviruses because of their capacity to prevent virus-induced apoptosis and thereby facilitate virus multiplication (4, 8). The baculovirus IAPs bear a striking resemblance to the cellular IAPs carried by the host insects that they infect. Cellular IAPs are a highly conserved family of survival factors that regulate developmental and stress-induced apoptosis, as well as inflammation, the cell cycle, and other signaling processes (35, 38, 44). Importantly, misregulation or overexpression of IAPs is associated with neoplasia and tumor chemoresistance (24, 49). The IAPs are defined by the presence of one or more ∼80-residue baculovirus IAP repeat (BIR) domains. The BIRs consist of a conserved Zn²⁺-coordinating arrangement of Cys and His residues (CCHC) that interact with diverse proteins, including the cysteinyl aspartate-specific proteases called caspases that execute apoptosis (reviewed in 16 and 37). The antiapoptotic activity of some, but not all, IAPs is derived from their ability to bind and neutralize caspases (reviewed in 35 and 44). The BIRs also interact with proapoptotic factors that contain IAP binding motifs (IBMs). IBM-containing factors have the capacity to bind and dissociate the IAP-caspase complex, thereby liberating active caspases to execute apoptosis (16, 35, 36, 48). Many IAPs, including viral IAPs, also possess a C-terminal RING domain, which is a Zn²⁺-coordinating motif with E3-ubiquitin ligase activity, which can contribute to antiapoptotic activity (48).The best-studied baculovirus IAP is Op-IAP3, which is encoded by Orgyia pseudotsugata nucleopolyhedrovirus. This small IAP (268 residues) contains two BIRs and a C-terminal RING (Fig. ​(Fig.1A).1A). Both BIRs are required for Op-IAP3 antiapoptotic activity (19, 50, 53). Truncation of the Op-IAP3 RING creates a loss-of-function dominant inhibitor (19). Op-IAP3''s capacity to form a complex with this RING-lacking (RINGless) dominant inhibitor and with itself suggests that oligomerization is necessary for IAP function. Upon overexpression, Op-IAP3 blocks apoptosis triggered by diverse signals in cells from certain insects and mammals, suggesting that it acts through a conserved mechanism (7, 11, 15, 33, 51, 54, 56). In the baculovirus host moth Spodoptera frugiperda (Lepidoptera: Noctuidae), Op-IAP3 prevents apoptosis by blocking the activation of effector caspases (25, 32, 40). However, in contrast to host insect IAPs, Op-IAP3 fails to inhibit active caspases (45, 51, 54). Thus, the host cell target(s) and the mechanism by which they are neutralized by this viral IAP remain unclear.Open in a separate windowFIG. 1.SfIAP structure and mutagenesis. (A) Viral and cellular IAPs. Viral Op-IAP3 (268 residues) and SfIAP (377 residues) each contain two BIR motifs (black boxes) and an E3 ligase RING domain (cross-hatched box). Each representing a potential start site, four methionines (M1 to M4) exist in the N-terminal leader of SfIAP. (B) SfIAP^M4 mutations. SfIAP^M4 (281 residues) begins with the M4 methionine. SfIAP^M4ΔR (227 residues) lacks the C-terminal RING. Amino acid substitutions of Zn-coordinating residues are indicated. An epitope tag (HA) was inserted at the N terminus. (C) Marker rescue assay. The antiapoptotic activity of wild-type or mutated forms of SfIAP^M4 was assayed by virus marker rescue in which replication of p35-deficient vΔp35/lacZ was restored in proportion to the antiapoptotic activity of the mutated Sfiap gene acquired by integration of the SfIAP-encoding plasmid (2). Virus yields were determined by plaque assay using apoptosis-sensitive SF21 cells. Antiapoptotic activity is reported as the ratio of nonapoptotic, lacZ-expressing plaques produced by transfection of the indicated Sfiap to those produced by wild-type Sfiap. Values shown are the averages ± standard deviations obtained from triplicate transfections.Among the cellular IAPs, SfIAP from Spodoptera frugiperda is most closely related to viral Op-IAP3. SfIAP (Fig. ​(Fig.1A)1A) is 42% identical to Op-IAP3, with a higher degree of amino acid identity localized to its two BIRs and C-terminal RING (20). As the principal IAP in Spodoptera, SfIAP suppresses a constitutive push toward apoptosis (34); ablation of SfIAP leads to immediate apoptosis of cultured Spodoptera cells. Upon overexpression, SfIAP also rescues the multiplication of apoptosis-inducing baculoviruses and can prevent apoptosis in certain mammalian cell lines (20, 26). In contrast to viral Op-IAP3, SfIAP can bind and inhibit caspases, including Spodoptera frugiperda caspase-1 (Sf-caspase-1) and human caspase-9 (20, 45). Thus, despite their structural similarities, there exist fundamental differences in the biochemical activities of these two IAPs. Importantly, SfIAP fails to prevent baculovirus-induced apoptosis when produced at endogenous levels in permissive Spodoptera cells. Thus, it is expected that SfIAP also possesses regulatory motifs that respond to cellular signals triggered upon virus infection.SfIAP provides an unprecedented opportunity to investigate the functional and evolutionary relationships between host and viral IAPs and to test the intriguing hypothesis that viral IAPs were acquired by host gene capture (21). We have investigated the biochemical properties of SfIAP as a means to define its molecular mechanisms and to test its relatedness to viral IAPs. We report here that SfIAP shares many biochemical and functional features with viral IAPs. Like Op-IAP3, overexpressed SfIAP prevented virus-induced apoptosis at a step upstream of caspase activation by a mechanism that required BIR1, BIR2, and the RING. SfIAP formed a complex with itself and with a RINGless dominant inhibitor, suggesting that oligomerization is also required for function of cellular IAPs. Unlike viral IAPs, SfIAP possesses an N-terminal leader, which modulates intracellular SfIAP levels and may respond to apoptotic signals to regulate cell survival. Our data are consistent with a model in which baculoviruses acquired a host cell IAP and modified it for virus-specific needs, thereby increasing virus fitness by preventing virus-induced apoptosis.     

Cleavage of the Drosophila screw prodomain is critical for a dynamic BMP morphogen gradient in embryogenesis

Dorsoventral patterning of the Drosophila embryo is regulated by graded distribution of bone morphogenetic proteins (BMPs) composed of two ligands, decapentaplegic (Dpp) a BMP2/4 ortholog and screw (Scw) a BMP5/6/7/8 family member. scw^E1 encodes an unusual allele that was isolated as a dominant enhancer of partial loss-of-function mutations in dpp. However, the molecular mechanisms that underlie this genetic interaction remain to be addressed. Here we show that scw^E1 contains a mutation at the furin cleavage site within the prodomain that is crucial for ligand production. Furthermore, our data show that Scw^E1 preferentially forms heterodimers with Dpp rather than homotypic dimers, providing a possible explanation for the dominant negative phenotype of scw^E1 alleles. The unprocessed prodomain of Scw^E1 remains in a complex with the Dpp:Scw heterodimer, and thus could interfere with interaction of the ligand with the extracellular matrix, or the kinetics of processing/secretion of the ligand in vivo. These data reveal novel mechanisms by which post-translational regulation of Scw can modulate Dpp signaling activity.     

Selection of dinB Alleles Suppressing Survival Loss upon dinB Overexpression in Escherichia coli

Escherichia coli strains overproducing DinB undergo survival loss; however, the mechanisms regulating this phenotype are poorly understood. Here we report a genetic selection revealing DinB residues essential to effect this loss-of-survival phenotype. The selection uses strains carrying both an antimutator allele of DNA polymerase III (Pol III) α-subunit (dnaE915) and either chromosomal or plasmid-borne dinB alleles. We hypothesized that dnaE915 cells would respond to DinB overproduction differently from dnaE⁺ cells because the dnaE915 allele is known to have an altered genetic interaction with dinB⁺ compared to its interaction with dnaE⁺. Notably, we observe a loss-of-survival phenotype in dnaE915 strains with either a chromosomal catalytically inactive dinB(D103N) allele or a low-copy-number plasmid-borne dinB⁺ upon DNA damage treatment. Furthermore, we find that the loss-of-survival phenotype occurs independently of DNA damage treatment in a dnaE915 strain expressing the catalytically inactive dinB(D103N) allele from a low-copy-number plasmid. The selective pressure imposed resulted in suppressor mutations that eliminated growth defects. The dinB intragenic mutations examined were either base pair substitutions or those that we inferred to be loss of function (i.e., deletions and insertions). Further analyses of selected novel dinB alleles, generated by single-base-pair substitutions in the dnaE915 strain, indicated that these no longer effect loss of survival upon overproduction in dnaE⁺ strains. These mutations are mapped to specific areas of DinB; this permits us to gain insights into the mechanisms underlying the DinB-mediated overproduction loss-of-survival phenotype.     

Molecular cloning of the SUF2 frameshift suppressor gene from Saccharomyces cerevisiae

A genetic approach to the molecular cloning of frameshift suppressor genes from yeast is described. These suppressors act by suppressing +1 G:C base-pair insertion mutations in glycine or proline codons. The cloning regimen involves an indirect screen for yeast transformants which harbor a functional suppressor gene inserted into the autonomously replicating “shuttle” vector YEp13, followed by transfer of the hybrid plasmid from yeast into Escherichia coli. Using this procedure a 10.7-kb DNA fragment carrying the SUF2 frameshift suppressor gene has been isolated. This suppressor acts specifically on +1 G:C insertions in proline codons. When inserted into an integrative vehicle and reintroduced into yeast by transformation, this fragment integrates by homologous recombination in the region of the SUF2 locus on chromosome III. A large proportion of the fragment overlaps with another cloned DNA segment which carries the closely linked CDC10 gene. The SUF2 fragment carries at least two tRNA genes. The SUF2 gene and one of the tRNA genes are located on a 0.85-kb restriction fragment within the 10.7-kb segment. A method is also described for the isolation of DNA fragments carrying alternative alleles of the SUF2 locus. Using this procedure, the wild-type suf2⁺ allele has been cloned.     

Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

The Schizosaccharomyces pombe pfh1⁺ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1⁺ gene suppress a temperature-sensitive allele of cdc24⁺, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1⁺ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1⁺ and dna2⁺ (or cdc24⁺) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol δ-mediated lagging strand displacement DNA synthesis.