Gene duplication and deletion caused by over-replication at a fork barrier.

Replication fork stalling can provoke fork reversal to form a four-way DNA junction. This remodelling of the replication fork can facilitate repair, aid bypass of DNA lesions, and enable replication restart, but may also pose a risk of over-replication during fork convergence. We show that replication fork stalling at a site-specific barrier in fission yeast can induce gene duplication-deletion rearrangements that are independent of replication restart-associated template switching and Rad51-dependent multi-invasion. Instead, they resemble targeted gene replacements (TGRs), requiring the DNA annealing activity of Rad52, the 3'-flap nuclease Rad16-Swi10, and mismatch repair protein Msh2. We propose that excess DNA, generated during the merging of a canonical fork with a reversed fork, can be liberated by a nuclease and integrated at an ectopic site via a TGR-like mechanism. This highlights how over-replication at replication termination sites can threaten genome stability in eukaryotes.

Rad52's DNA annealing activity drives template switching associated with restarted DNA replication.

It is thought that many of the simple and complex genomic rearrangements associated with congenital diseases and cancers stem from mistakes made during the restart of collapsed replication forks by recombination enzymes. It is hypothesised that this recombination-mediated restart process transitions from a relatively accurate initiation phase to a less accurate elongation phase characterised by extensive template switching between homologous, homeologous and microhomologous DNA sequences. Using an experimental system in fission yeast, where fork collapse is triggered by a site-specific replication barrier, we show that ectopic recombination, associated with the initiation of recombination-dependent replication (RDR), is driven mainly by the Rad51 recombinase, whereas template switching, during the elongation phase of RDR, relies more on DNA annealing by Rad52. This finding provides both evidence and a mechanistic basis for the transition hypothesis.

The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.

The PCNA unloader Elg1 promotes recombination at collapsed replication forks in fission yeast.

Protein-DNA complexes can impede DNA replication and cause replication fork collapse. Whilst it is known that homologous recombination is deployed in such instances to restart replication, it is unclear how a stalled fork transitions into a collapsed fork at which recombination proteins can load. Previously we established assays in Schizosaccharomyces pombe for studying recombination induced by replication fork collapse at the site-specific protein-DNA barrier RTS1 (Nguyen et al., 2015). Here, we provide evidence that efficient recruitment/retention of two key recombination proteins (Rad51 and Rad52) to RTS1 depends on unloading of the polymerase sliding clamp PCNA from DNA by Elg1. We also show that, in the absence of Elg1, reduced recombination is partially suppressed by deleting fbh1 or, to a lesser extent, srs2, which encode known anti-recombinogenic DNA helicases. These findings suggest that PCNA unloading by Elg1 is necessary to limit Fbh1 and Srs2 activity, and thereby enable recombination to proceed.

Factors affecting template switch recombination associated with restarted DNA replication.

Homologous recombination helps ensure the timely completion of genome duplication by restarting collapsed replication forks. However, this beneficial function is not without risk as replication restarted by homologous recombination is prone to template switching (TS) that can generate deleterious genome rearrangements associated with diseases such as cancer. Previously we established an assay for studying TS in Schizosaccharomyces pombe (Nguyen et al., 2015). Here, we show that TS is detected up to 75 kb downstream of a collapsed replication fork and can be triggered by head-on collision between the restarted fork and RNA Polymerase III transcription. The Pif1 DNA helicase, Pfh1, promotes efficient restart and also suppresses TS. A further three conserved helicases (Fbh1, Rqh1 and Srs2) strongly suppress TS, but there is no change in TS frequency in cells lacking Fml1 or Mus81. We discuss how these factors likely influence TS.

Inter-Fork Strand Annealing causes genomic deletions during the termination of DNA replication.

Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.

Recombination occurs within minutes of replication blockage by RTS1 producing restarted forks that are prone to collapse.

The completion of genome duplication during the cell cycle is threatened by the presence of replication fork barriers (RFBs). Following collision with a RFB, replication proteins can dissociate from the stalled fork (fork collapse) rendering it incapable of further DNA synthesis unless recombination intervenes to restart replication. We use time-lapse microscopy and genetic assays to show that recombination is initiated within ∼ 10 min of replication fork blockage at a site-specific barrier in fission yeast, leading to a restarted fork within ∼ 60 min, which is only prevented/curtailed by the arrival of the opposing replication fork. The restarted fork is susceptible to further collapse causing hyper-recombination downstream of the barrier. Surprisingly, in our system fork restart is unnecessary for maintaining cell viability. Seemingly, the risk of failing to complete replication prior to mitosis is sufficient to warrant the induction of recombination even though it can cause deleterious genetic change.

Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen Cryptococcus neoformans.

With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.2 Šresolution.

Is the nickel-dependent urease complex of Cryptococcus the pathogen's Achilles' heel?

The nitrogen-scavenging enzyme urease has been coopted in a variety of pathogenic organisms as a virulence factor, most notoriously to neutralize stomach acid and establish infection by the gastric pathogen Helicobacter pylori. The opportunistic fungal pathogen Cryptococcus neoformans also utilizes urease as a virulence factor, only in this case to invade the central nervous system (CNS) via the blood-brain barrier and cause life-threatening meningoencephalitis. A recent study [A. Singh, R. Panting, A. Varma, T. Saijo, K. Waldron, A. Jong, P. Ngamskulrungroj, Y. Chan, J. Rutherford, K. Kwon-Chung, mBio 4(3):e00220-13] genetically and biochemically characterizes the accessory proteins required for successful activation of the urease protein complex, including the essential nickel cofactor. The accessory proteins Ure4, Ure6, and Ure7 are all essential for urease function. Ure7 appears to combine the roles of two bacterial accessory proteins: it incorporates both the GTPase activity and nickel chaperone properties of UreE, a bacterial protein whose homolog is missing in the fungi. An accompanying nickel transporter, Nic1, is responsible for most, but not all, nickel uptake into the fungal cell. Mutants of the core urease protein Ure1, accessory protein Ure7, and transporter Nic1 are all attenuated for invasion of the CNS of mice, and urease activity may directly affect integrity of the tight junction of the endothelial cells of the blood-brain barrier, the network of proteins that limits paracellular permeability. This work highlights the potential of urease, its accessory proteins, and nickel transport as potential chemotherapeutic targets.

Nitrogen regulation of virulence in clinically prevalent fungal pathogens.

The habitats of fungal pathogens range from environmental to commensal, and the nutrient content of these different niches varies considerably. Upon infection of humans, nutrient availability changes significantly depending on the site and pathophysiology of infection. Nonetheless, a common feature enabling successful establishment in these niches is the ability to metabolise available nutrients including sources of nitrogen, carbon and essential metals such as iron. In particular, nitrogen source utilisation influences specific morphological transitions, sexual and asexual sporulation and virulence factor production. All these physiological changes confer selective advantages to facilitate fungal survival, proliferation and colonisation. The three most well-studied components of the nitrogen regulatory circuit that commonly impact fungal pathogenesis are the ammonium permeases (the nitrogen availability sensor candidate), ureases (a nitrogen-scavenging enzyme) and GATA transcription factors (global regulators of nitrogen catabolism). In certain species, the ammonium permease induces a morphological switch from yeast to invasive filamentous growth forms or infectious spores, while in others, urease is a bona fide virulence factor. In all species studied thus far, transcription of the ammonium permease and urease-encoding genes is modulated by GATA factors. Fungal pathogens therefore integrate the expression of different virulence-associated phenotypes into the regulatory network controlling nitrogen catabolism.

Characterization of the complete uric acid degradation pathway in the fungal pathogen Cryptococcus neoformans.

Degradation of purines to uric acid is generally conserved among organisms, however, the end product of uric acid degradation varies from species to species depending on the presence of active catabolic enzymes. In humans, most higher primates and birds, the urate oxidase gene is non-functional and hence uric acid is not further broken down. Uric acid in human blood plasma serves as an antioxidant and an immune enhancer; conversely, excessive amounts cause the common affliction gout. In contrast, uric acid is completely degraded to ammonia in most fungi. Currently, relatively little is known about uric acid catabolism in the fungal pathogen Cryptococcus neoformans even though this yeast is commonly isolated from uric acid-rich pigeon guano. In addition, uric acid utilization enhances the production of the cryptococcal virulence factors capsule and urease, and may potentially modulate the host immune response during infection. Based on these important observations, we employed both Agrobacterium-mediated insertional mutagenesis and bioinformatics to predict all the uric acid catabolic enzyme-encoding genes in the H99 genome. The candidate C. neoformans uric acid catabolic genes identified were named: URO1 (urate oxidase), URO2 (HIU hydrolase), URO3 (OHCU decarboxylase), DAL1 (allantoinase), DAL2,3,3 (allantoicase-ureidoglycolate hydrolase fusion protein), and URE1 (urease). All six ORFs were then deleted via homologous recombination; assaying of the deletion mutants' ability to assimilate uric acid and its pathway intermediates as the sole nitrogen source validated their enzymatic functions. While Uro1, Uro2, Uro3, Dal1 and Dal2,3,3 were demonstrated to be dispensable for virulence, the significance of using a modified animal model system of cryptococcosis for improved mimicking of human pathogenicity is discussed.

Comparative Genomics of Serial Isolates of Cryptococcus neoformans Reveals Gene Associated With Carbon Utilization and Virulence.

The opportunistic fungal pathogen Cryptococcus neoformans is a leading cause of mortality among the human immunodeficiency virus/acquired immunodeficiency syndrome population and is known for frequently causing life-threatening relapses. To investigate the potential contribution of in-host microevolution to persistence and relapse, we have analyzed two serial isolates obtained from a patient with acquired immunodeficiency syndrome who suffered an initial and relapse episode of cryptococcal meningoencephalitis. Despite being identical by multilocus sequence typing, the isolates differ phenotypically, exhibiting changes in key virulence factors, nutrient acquisition, metabolic profiles, and the ability to disseminate in an animal model. Whole-genome sequencing uncovered a clonal relationship, with only a few unique differences. Of these, two key changes are expected to explain the phenotypic differences observed in the relapse isolate: loss of a predicted AT-rich interaction domain protein and changes in copy number of the left and right arms of chromosome 12. Gene deletion of the predicted transcriptional regulator produced changes in melanin, capsule, carbon source use, and dissemination in the host, consistent with the phenotype of the relapse isolate. In addition, the deletion mutant displayed altered virulence in the murine model. The observed differences suggest the relapse isolate evolved subsequent to penetration of the central nervous system and may have gained dominance following the administration of antifungal therapy. These data reveal the first molecular insights into how the Cryptococcus neoformans genome changes during infection of humans and the manner in which microevolution progresses in this deadly fungal pathogen.

Reactive oxygen species homeostasis and virulence of the fungal pathogen Cryptococcus neoformans requires an intact proline catabolism pathway.

Degradation of the multifunctional amino acid proline is associated with mitochondrial oxidative respiration. The two-step oxidation of proline is catalyzed by proline oxidase and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase, which produce P5C and glutamate, respectively. In animal and plant cells, impairment of P5C dehydrogenase activity results in P5C-proline cycling when exogenous proline is supplied via the actions of proline oxidase and P5C reductase (the enzyme that converts P5C to proline). This proline is oxidized by the proline oxidase-FAD complex that delivers electrons to the electron transport chain and to O2, leading to mitochondrial reactive oxygen species (ROS) overproduction. Coupled activity of proline oxidase and P5C dehydrogenase is therefore important for maintaining ROS homeostasis. In the genome of the fungal pathogen Cryptococcus neoformans, there are two paralogs (PUT1 and PUT5) that encode proline oxidases and a single ortholog (PUT2) that encodes P5C dehydrogenase. Transcription of all three catabolic genes is inducible by the presence of proline. However, through the creation of deletion mutants, only Put5 and Put2 were found to be required for proline utilization. The put2Δ mutant also generates excessive mitochondrial superoxide when exposed to proline. Intracellular accumulation of ROS is a critical feature of cell death; consistent with this fact, the put2Δ mutant exhibits a slight, general growth defect. Furthermore, Put2 is required for optimal production of the major cryptococcal virulence factors. During murine infection, the put2Δ mutant was discovered to be avirulent; this is the first report highlighting the importance of P5C dehydrogenase in enabling pathogenesis of a microorganism.

Ploidy variation as an adaptive mechanism in human pathogenic fungi.

Changes in ploidy have a profound and usually negative influence on cellular viability and proliferation, yet the vast majority of cancers and tumours exhibit an aneuploid karyotype. Whether this genomic plasticity is a cause or consequence of malignant transformation remains uncertain. Systemic fungal pathogens regularly develop aneuploidies in a similar manner during human infection, often far in excess of the natural rate of chromosome nondisjunction. As both processes fundamentally represent cells evolving under selective pressures, this suggests that changes in chromosome number may be a concerted mechanism to adapt to the hostile host environment. Here, we examine the mechanisms by which aneuploidy and polyploidy are generated in the fungal pathogens Candida albicans and Cryptococcus neoformans and investigate whether these represent an adaptive strategy under severe stress through the rapid generation of large-scale mutations. Insights into fungal ploidy changes, strategies for tolerating aneuploidies and proliferation during infection may yield novel targets for both antifungal and anticancer therapies.

Smg1 haploinsufficiency predisposes to tumor formation and inflammation.

SMG1 is a member of the phosphoinositide kinase-like kinase family of proteins that includes ATM, ATR, and DNA-PK, proteins with known roles in DNA damage and cellular stress responses. SMG1 has a well-characterized role in nonsense-mediated decay as well as suggested roles in the DNA damage response, resistance to oxidative stress, regulation of hypoxic responses, and apoptosis. To understand the roles of SMG1 further, we generated a Genetrap Smg1 mouse model. Smg1 homozygous KO mice were early embryonic lethal, but Smg1 heterozygous mice showed a predisposition to a range of cancers, particularly lung and hematopoietic malignancies, as well as development of chronic inflammation. These mice did not display deficiencies in known roles of SMG1, including nonsense-mediated decay. However, they showed elevated basal tissue and serum cytokine levels, indicating low-level inflammation before the development of tumors. Smg1 heterozygous mice also showed evidence of oxidative damage in tissues. These data suggest that the inflammation observed in Smg1 haploinsufficiency contributes to susceptibility to cancer and that Smg1-deficient animals represent a model of inflammation-enhanced cancer development.

Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology.

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.

De novo GTP biosynthesis is critical for virulence of the fungal pathogen Cryptococcus neoformans.

We have investigated the potential of the GTP synthesis pathways as chemotherapeutic targets in the human pathogen Cryptococcus neoformans, a common cause of fatal fungal meningoencephalitis. We find that de novo GTP biosynthesis, but not the alternate salvage pathway, is critical to cryptococcal dissemination and survival in vivo. Loss of inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway results in slow growth and virulence factor defects, while loss of the cognate phosphoribosyltransferase in the salvage pathway yielded no phenotypes. Further, the Cryptococcus species complex displays variable sensitivity to the IMPDH inhibitor mycophenolic acid, and we uncover a rare drug-resistant subtype of C. gattii that suggests an adaptive response to microbial IMPDH inhibitors in its environmental niche. We report the structural and functional characterization of IMPDH from Cryptococcus, revealing insights into the basis for drug resistance and suggesting strategies for the development of fungal-specific inhibitors. The crystal structure reveals the position of the IMPDH moveable flap and catalytic arginine in the open conformation for the first time, plus unique, exploitable differences in the highly conserved active site. Treatment with mycophenolic acid led to significantly increased survival times in a nematode model, validating de novo GTP biosynthesis as an antifungal target in Cryptococcus.

Characterization of an Nmr homolog that modulates GATA factor-mediated nitrogen metabolite repression in Cryptococcus neoformans.

Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis.

A unique chromosomal rearrangement in the Cryptococcus neoformans var. grubii type strain enhances key phenotypes associated with virulence.

UNLABELLED: The accumulation of genomic structural variation between closely related populations over time can lead to reproductive isolation and speciation. The fungal pathogen Cryptococcus is thought to have recently diversified, forming a species complex containing members with distinct morphologies, distributions, and pathologies of infection. We have investigated structural changes in genomic architecture such as inversions and translocations that distinguish the most pathogenic variety, Cryptococcus neoformans var. grubii, from the less clinically prevalent Cryptococcus neoformans var. neoformans and Cryptococcus gattii. Synteny analysis between the genomes of the three Cryptococcus species/varieties (strains H99, JEC21, and R265) reveals that C. neoformans var. grubii possesses surprisingly few unique genomic rearrangements. All but one are relatively small and are shared by all molecular subtypes of C. neoformans var. grubii. In contrast, the large translocation peculiar to the C. neoformans var. grubii type strain is found in all tested subcultures from multiple laboratories, suggesting that it has possessed this rearrangement since its isolation from a human clinical sample. Furthermore, we find that the translocation directly disrupts two genes. The first of these encodes a novel protein involved in metabolism of glucose at human body temperature and affects intracellular levels of trehalose. The second encodes a homeodomain-containing transcription factor that modulates melanin production. Both mutations would be predicted to increase pathogenicity; however, when recreated in an alternate genetic background, these mutations do not affect virulence in animal models. The type strain of C. neoformans var. grubii in which the majority of molecular studies have been performed is therefore atypical for carbon metabolism and key virulence attributes. IMPORTANCE: The fungal pathogen Cryptococcus is a major cause of mortality among the immunocompromised population, primarily in AIDS patients of sub-Saharan Africa. Most research into the particular variety of Cryptococcus responsible for the vast majority of infections, Cryptococcus neoformans var. grubii, is performed using the type strain isolated in 1978 from a Hodgkin's disease patient from North Carolina. We have determined that this particular isolate contains a chromosomal translocation that directly interrupts two genes, which all descendants of this strain from various research laboratories appear to possess. Disruption of these two genes affects multiple virulence factors of Cryptococcus, particularly the ability to grow at human body temperature, which could have wide-ranging implications for molecular genetic studies and virulence assays using this important strain.