ABSTRACT
Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.
Subject(s)
Adenosine Triphosphatases/genetics , DNA Helicases/genetics , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Fungal , Homologous Recombination , Rad51 Recombinase/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Adenosine Triphosphatases/metabolism , Binding Sites , DNA Helicases/metabolism , DNA Repair Enzymes/metabolism , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , Protein Binding , Rad51 Recombinase/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Single Molecule Imaging , Red Fluorescent ProteinABSTRACT
As in human cells, yeast telomeres can be maintained in cells lacking telomerase activity by recombination-based mechanisms known as ALT (Alternative Lengthening of Telomeres). A hallmark of ALT human cancer cells are extrachromosomal telomeric DNA elements called C-circles, whose origin and function have remained unclear. Here, we show that extrachromosomal telomeric C-circles in yeast can be detected shortly after senescence crisis and concomitantly with the production of survivors arising from "type II" recombination events. We uncover that C-circles bind to the nuclear pore complex (NPC) and to the SAGA-TREX2 complex, similar to other non-centromeric episomal DNA. Disrupting the integrity of the SAGA/TREX2 complex affects both C-circle binding to NPCs and type II telomere recombination, suggesting that NPC tethering of C-circles facilitates formation and/or propagation of the long telomere repeats characteristic of type II survivors. Furthermore, we find that disruption of the nuclear diffusion barrier impairs type II recombination. These results support a model in which concentration of C-circles at NPCs benefits type II telomere recombination, highlighting the importance of spatial coordination in ALT-type mechanisms of telomere maintenance.
Subject(s)
Nuclear Pore , Saccharomyces cerevisiae , Cytoplasm , Humans , Nuclear Pore/genetics , Saccharomyces cerevisiae/genetics , Telomere/geneticsABSTRACT
Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.
Subject(s)
Gene Editing , Saccharomyces cerevisiae , Betaxanthins , Gene Editing/methods , Reproducibility of Results , Saccharomyces cerevisiae/geneticsABSTRACT
Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair damaged DNA, HR uses an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulation of DNA sequence transfer is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH), and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR. Several existing phenotypes in rdh54Δ strains suggest that Rdh54 may regulate effective exchange of DNA during HR. In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information during DNA repair. Our data indicate that RDH54 regulates DNA strand exchange by stabilizing Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 acts in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, favors non-crossover outcomes. From these results, we propose a model for how Rdh54 may kinetically regulate strand exchange during homologous recombination.
Subject(s)
Saccharomyces cerevisiae Proteins , Chromosomes/metabolism , DNA/genetics , DNA Helicases/genetics , DNA Repair/genetics , DNA Topoisomerases/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
PGM1-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.
Subject(s)
Phosphoglucomutase , Saccharomyces cerevisiae , Humans , Phosphoglucomutase/metabolism , Phosphoglucomutase/genetics , Phosphoglucomutase/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Proteolysis , Mutation, Missense , Ubiquitination , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/metabolism , Protein Stability , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/geneticsABSTRACT
Meiotic crossovers play a vital role in proper chromosome segregation and evolution of most sexually reproducing organisms. Meiotic recombination can be visually observed in Saccharomyces cerevisiae tetrads using linked spore-autonomous fluorescent markers placed at defined intervals within the genome, which allows for analysis of meiotic segregation without the need for tetrad dissection. To automate the analysis, we developed a deep learning-based image recognition and classification pipeline for high-throughput tetrad detection and meiotic crossover classification. As a proof of concept, we analyzed a large image data set from wild-type and selected gene knock-out mutants to quantify crossover frequency, interference, chromosome missegregation, and gene conversion events. The deep learning-based method has the potential to accelerate the discovery of new genes involved in meiotic recombination in S. cerevisiae such as the underlying factors controlling crossover frequency and interference.
Subject(s)
Crossing Over, Genetic , Deep Learning , Meiosis , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/classification , Meiosis/genetics , Chromosome Segregation , High-Throughput Screening Assays/methods , Image Processing, Computer-Assisted/methodsABSTRACT
DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)-mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error-free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error-free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR-mediated ICL repair is defective, and breaks are instead re-ligated by polymerase θ-dependent microhomology-mediated end-joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error-free ICL repair.
Subject(s)
Fanconi Anemia , DNA , DNA Damage , DNA Repair , DNA Replication , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , HumansABSTRACT
Mph1 is a member of the conserved FANCM family of DNA motor proteins that play key roles in genome maintenance processes underlying Fanconi anemia, a cancer predisposition syndrome in humans. Here, we identify Mte1 as a novel interactor of the Mph1 helicase in Saccharomyces cerevisiae. In vitro, Mte1 (Mph1-associated telomere maintenance protein 1) binds directly to DNA with a preference for branched molecules such as D loops and fork structures. In addition, Mte1 stimulates the helicase and fork regression activities of Mph1 while inhibiting the ability of Mph1 to dissociate recombination intermediates. Deletion of MTE1 reduces crossover recombination and suppresses the sensitivity of mph1Δ mutant cells to replication stress. Mph1 and Mte1 interdependently colocalize at DNA damage-induced foci and dysfunctional telomeres, and MTE1 deletion results in elongated telomeres. Taken together, our data indicate that Mte1 plays a role in regulation of crossover recombination, response to replication stress, and telomere maintenance.
Subject(s)
Crossing Over, Genetic/genetics , DEAD-box RNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Telomere Homeostasis/genetics , Telomere-Binding Proteins/metabolism , DEAD-box RNA Helicases/genetics , Gene Deletion , Protein Transport , Saccharomyces cerevisiae Proteins/genetics , Stress, Physiological/genetics , Telomere-Binding Proteins/geneticsABSTRACT
TopBP1/Rad4/Dpb11 is an essential eukaryotic protein with important roles in DNA replication, DNA repair, DNA damage checkpoint activation, and chromosome segregation. TopBP1 serves as a scaffold to assemble protein complexes in a phosphorylation-dependent manner via its multiple BRCT-repeats. Recently, it has become clear that TopBP1 is repurposed to scaffold different processes dependent on cell cycle regulated changes in phosphorylation of client proteins. Here we review the functions of human TopBP1 in maintaining genome integrity during mitosis.
Subject(s)
Carrier Proteins/genetics , DNA-Binding Proteins/genetics , Genomics/methods , Mitosis/genetics , Nuclear Proteins/genetics , HumansABSTRACT
Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dubé (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present an integrated computational and experimental study that reveals that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant (ΔE510) forms perinuclear protein aggregates. A yeast-based stability screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature led to a stabilization of two FLCN missense proteins, and for one (R362C), function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization and resulting restoration of function may hold therapeutic potential of certain disease-linked variants. Our computational saturation scan encompassing both missense variants and single site deletions in FLCN may allow classification of rare FLCN variants of uncertain clinical significance.
Subject(s)
Birt-Hogg-Dube Syndrome/pathology , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Birt-Hogg-Dube Syndrome/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Computational Biology , Germ-Line Mutation , Humans , Loss of Function Mutation , Mutation, Missense , Protein Aggregates , Protein Binding/genetics , Protein Folding , Protein Stability , Proteolysis , Proto-Oncogene Proteins/genetics , Saccharomyces cerevisiae , Tumor Suppressor Proteins/genetics , Ubiquitin-Specific Peptidase 7/metabolismABSTRACT
Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double-strand break (DSB) repair, and the meiosis-specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis-specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real-time single-molecule imaging to probe how the Hed1- and Rad54-mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Helicases/metabolism , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Meiosis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Cycle Proteins/genetics , DNA Breaks, Double-Stranded , DNA Helicases/genetics , DNA Repair , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , Homologous Recombination , Meiosis/genetics , Mitosis , Rad51 Recombinase/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Single Molecule ImagingABSTRACT
Dipterocarpaceae are typical tropical plants (dipterocarp forests) that are famous for their high economic value because of their production of fragrant oleoresins, top-quality timber and usage in traditional Chinese medicine. Currently, the lack of Dipterocarpaceae genomes has been a limiting factor to decipher the fragrant oleoresin biosynthesis and gain evolutionary insights into high-quality wood formation in Dipterocarpaceae. We generated chromosome-level genome assemblies for two representative Dipterocarpaceae species viz. Dipterocarpus turbinatus Gaertn. f. and Hopea hainanensis Merr. et Chun. Our whole-genome duplication (WGD) analysis revealed that Dipterocarpaceae underwent a shared WGD event, which showed significant impacts on increased copy numbers of genes related to the biosynthesis of terpene, BAHD acyltransferases, fatty acid and benzenoid/phenylpropanoid, which probably confer to the formation of their characteristic fragrant oleoresin. Additionally, compared with common soft wood plants, the expansion of gene families was also found to be associated with wood formation, such as in CESA (cellulose synthase), CSLE (cellulose synthase-like protein E), laccase and peroxidase in Dipterocarpaceae genomes, which might also contribute to the formation of harder, stronger and high-density timbers. Finally, an integrative analysis on a combination of genomic, transcriptomic and metabolic data from different tissues provided further insights into the molecular basis of fragrant oleoresins biosynthesis and high-quality wood formation of Dipterocarpaceae. Our study contributes the first two representative genomes for Dipterocarpaceae, which are valuable genetic resources for further researches on the fragrant oleoresins and superior-quality timber, genome-assisted breeding and improvement, and conservation biology of this family.
Subject(s)
Dipterocarpaceae , Chromosomes , Dipterocarpaceae/genetics , Plant Breeding , Plant ExtractsABSTRACT
Clarifying the evolutionary processes underlying species diversification and adaptation is a key focus of evolutionary biology. Begonia (Begoniaceae) is one of the most species-rich angiosperm genera with c. 2000 species, most of which are shade-adapted. Here, we present chromosome-scale genome assemblies for four species of Begonia (B. loranthoides, B. masoniana, B. darthvaderiana and B. peltatifolia), and whole genome shotgun data for an additional 74 Begonia representatives to investigate lineage evolution and shade adaptation of the genus. The four genome assemblies range in size from 331.75 Mb (B. peltatifolia) to 799.83 Mb (B. masoniana), and harbor 22 059-23 444 protein-coding genes. Synteny analysis revealed a lineage-specific whole-genome duplication (WGD) that occurred just before the diversification of Begonia. Functional enrichment of gene families retained after WGD highlights the significance of modified carbohydrate metabolism and photosynthesis possibly linked to shade adaptation in the genus, which is further supported by expansions of gene families involved in light perception and harvesting. Phylogenomic reconstructions and genomics studies indicate that genomic introgression has also played a role in the evolution of Begonia. Overall, this study provides valuable genomic resources for Begonia and suggests potential drivers underlying the diversity and adaptive evolution of this mega-diverse clade.
Subject(s)
Begoniaceae , Begoniaceae/genetics , Evolution, Molecular , Genome , Phylogeny , Synteny/geneticsABSTRACT
Transcription-replication (T-R) conflicts are profound threats to genome integrity. However, whilst much is known about the existence of T-R conflicts, our understanding of the genetic and temporal nature of how cells respond to them is poorly established. Here, we address this by characterizing the early cellular response to transient T-R conflicts (TRe). This response specifically requires the DNA recombination repair proteins BLM and BRCA2 as well as a non-canonical monoubiquitylation-independent function of FANCD2. A hallmark of the TRe response is the rapid co-localization of these three DNA repair factors at sites of T-R collisions. We find that the TRe response relies on basal activity of the ATR kinase, yet it does not lead to hyperactivation of this key checkpoint protein. Furthermore, specific abrogation of the TRe response leads to DNA damage in mitosis, and promotes chromosome instability and cell death. Collectively our findings identify a new role for these well-established tumor suppressor proteins at an early stage of the cellular response to conflicts between DNA transcription and replication.
Subject(s)
DNA Replication , Recombinational DNA Repair , Transcription, Genetic , Ataxia Telangiectasia Mutated Proteins/metabolism , BRCA2 Protein/physiology , Cell Line , Cell Survival , Cyclin-Dependent Kinase 9/metabolism , DNA/metabolism , DNA Damage , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group D2 Protein/physiology , Humans , Mitosis/genetics , Promoter Regions, Genetic , RNA/metabolism , RNA Polymerase II/metabolism , RNA Splicing , RecQ Helicases/physiology , UbiquitinationABSTRACT
Clausena lansium (Lour.) Skeels (Rutaceae), recognized as wampee, is a widely distributed fruit tree which is utilized as a folk-medicine for treatment of several common diseases. However, the genomic information about this medicinally important species is still lacking. Therefore, we assembled the first genome of Clausena genus with a total length of 310.51 Mb and scaffold N50 of 2.24 Mb by using 10× Genomics technology. Further annotation revealed a total of 34,419 protein-coding genes, while repetitive elements covered 39.08% (121.36 Mb) of the genome. The Clausena and Citrus genus were found to diverge around 22 MYA, and also shared an ancient whole-genome triplication event with Vitis. Furthermore, multi-tissue transcriptomic analysis enabled the identification of genes involved in the synthesis of carbazole alkaloids. Altogether, these findings provided new insights into the genome evolution of Wampee species and highlighted the possible role of key genes involved in the carbazole alkaloids biosynthetic pathway.
Subject(s)
Alkaloids , Clausena , Carbazoles , Clausena/genetics , Fruit , Molecular StructureABSTRACT
A key function of the cellular DNA damage response is to facilitate the bypass of replication fork-stalling DNA lesions. Template switch reactions allow such a bypass and involve the formation of DNA joint molecules (JMs) between sister chromatids. These JMs need to be resolved before cell division; however, the regulation of this process is only poorly understood. Here, we identify a regulatory mechanism in yeast that critically controls JM resolution by the Mus81-Mms4 endonuclease. Central to this regulation is a conserved complex comprising the scaffold proteins Dpb11 and Slx4 that is under stringent control. Cell cycle-dependent phosphorylation of Slx4 by Cdk1 promotes the Dpb11-Slx4 interaction, while in mitosis, phosphorylation of Mms4 by Polo-like kinase Cdc5 promotes the additional association of Mus81-Mms4 with the complex, thereby promoting JM resolution. Finally, the DNA damage checkpoint counteracts Mus81-Mms4 binding to the Dpb11-Slx4 complex. Thus, Dpb11-Slx4 integrates several cellular inputs and participates in the temporal program for activation of the JM-resolving nuclease Mus81.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Repair/physiology , DNA Replication , Endodeoxyribonucleases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Cycle , Endodeoxyribonucleases/genetics , Enzyme Activation/physiology , Mutation/genetics , Phosphorylation , Protein Binding , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Replication protein A (RPA) is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. RPA directly participates in DNA double-strand break repair by stimulating 5'-3' end resection by the Sgs1/BLM helicase and Dna2 endonuclease in vitro. Here we investigated the role of RPA in end resection in vivo, using a heat-inducible degron system that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. We found that RPA depletion eliminated both the Sgs1-Dna2- and Exo1-dependent extensive resection pathways and synergized with mre11Δ to prevent end resection. The short single-stranded DNA tails formed in the absence of RPA were unstable due to 3' strand loss and the formation of fold-back hairpin structures that required resection initiation and Pol32-dependent DNA synthesis. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements.
Subject(s)
DNA Helicases/metabolism , DNA, Fungal/metabolism , Exodeoxyribonucleases/metabolism , RecQ Helicases/metabolism , Replication Protein A/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Blotting, Western , DNA Breaks, Double-Stranded , DNA Helicases/genetics , DNA Repair , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Exodeoxyribonucleases/genetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Models, Genetic , Mutation , Nucleic Acid Conformation , Protein Binding , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , RecQ Helicases/genetics , Replication Protein A/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Telomeric regions of the genome are inherently difficult-to-replicate due to their propensity to generate DNA secondary structures and form nucleoprotein complexes that can impede DNA replication fork progression. Precisely how cells respond to DNA replication stalling within a telomere remains poorly characterized, largely due to the methodological difficulties in analysing defined stalling events in molecular detail. Here, we utilized a site-specific DNA replication barrier mediated by the 'Tus/Ter' system to define the consequences of DNA replication perturbation within a single telomeric locus. Through molecular genetic analysis of this defined fork-stalling event, coupled with the use of a genome-wide genetic screen, we identified an important role for the SUMO-like domain protein, Esc2, in limiting genome rearrangements at a telomere. Moreover, we showed that these rearrangements are driven by the combined action of the Mph1 helicase and the homologous recombination machinery. Our findings demonstrate that chromosomal context influences cellular responses to a stalled replication fork and reveal protective factors that are required at telomeric loci to limit DNA replication stress-induced chromosomal instability.
Subject(s)
DEAD-box RNA Helicases/genetics , DNA Replication/genetics , Nuclear Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Telomere/genetics , Cell Cycle Proteins , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Homologous Recombination/genetics , Nucleic Acid Conformation , Saccharomyces cerevisiae/geneticsABSTRACT
Common Chromosomal Fragile Sites (CFSs) are specific genomic regions prone to form breaks on metaphase chromosomes in response to replication stress. Moreover, CFSs are mutational hotspots in cancer genomes, showing that the mutational mechanisms that operate at CFSs are highly active in cancer cells. Orthologs of human CFSs are found in a number of other mammals, but the extent of CFS conservation beyond the mammalian lineage is unclear. Characterization of CFSs from distantly related organisms can provide new insight into the biology underlying CFSs. Here, we have mapped CFSs in an avian cell line. We find that, overall the most significant CFSs coincide with extremely large conserved genes, from which very long transcripts are produced. However, no significant correlation between any sequence characteristics and CFSs is found. Moreover, we identified putative early replicating fragile sites (ERFSs), which is a distinct class of fragile sites and we developed a fluctuation analysis revealing high mutation rates at the CFS gene PARK2, with deletions as the most prevalent mutation. Finally, we show that avian homologs of the human CFS genes despite their fragility have resisted the general intron size reduction observed in birds suggesting that CFSs have a conserved biological function.
Subject(s)
Avian Proteins/genetics , B-Lymphocytes/metabolism , Chromosome Fragile Sites , Fanconi Anemia Complementation Group D2 Protein/genetics , Transcription, Genetic , Ubiquitin-Protein Ligases/genetics , Animals , Avian Proteins/metabolism , B-Lymphocytes/pathology , Binding Sites , Cell Line, Transformed , Chickens , Chromosome Mapping , Conserved Sequence , DNA Replication , Fanconi Anemia Complementation Group D2 Protein/metabolism , Gene Expression Profiling , Gene Expression Regulation , Gene Ontology , Metaphase , Molecular Sequence Annotation , Mutation , Protein Binding , Ubiquitin-Protein Ligases/metabolismABSTRACT
RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1's helicase activity plays a crucial role in responding to persistent RNA-DNA hybrids.