Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Critical importance of DNA binding for CSL protein functions in fission yeast.

CSL proteins [named after the homologs CBF1 (RBP-Jκ in mice), Suppressor of Hairless and LAG-1] are conserved transcription factors found in animals and fungi. In the fission yeast Schizosaccharomyces pombe, they regulate various cellular processes, including cell cycle progression, lipid metabolism and cell adhesion. CSL proteins bind to DNA through their N-terminal Rel-like domain and central ß-trefoil domain. Here, we investigated the importance of DNA binding for CSL protein functions in fission yeast. We created CSL protein mutants with disrupted DNA binding and found that the vast majority of CSL protein functions depend on intact DNA binding. Specifically, DNA binding is crucial for the regulation of cell adhesion, lipid metabolism, cell cycle progression, long non-coding RNA expression and genome integrity maintenance. Interestingly, perturbed lipid metabolism leads to chromatin structure changes, potentially linking lipid metabolism to the diverse phenotypes associated with CSL protein functions. Our study highlights the critical role of DNA binding for CSL protein functions in fission yeast.

Coordination of cohesin and DNA replication observed with purified proteins.

Two newly duplicated copies of genomic DNA are held together by the ring-shaped cohesin complex to ensure faithful inheritance of the genome during cell division1-3. Cohesin mediates sister chromatid cohesion by topologically entrapping two sister DNAs during DNA replication4,5, but how cohesion is established at the replication fork is poorly understood. Here, we studied the interplay between cohesin and replication by reconstituting a functional replisome using purified proteins. Once DNA is encircled before replication, the cohesin ring accommodates replication in its entirety, from initiation to termination, leading to topological capture of newly synthesized DNA. This suggests that topological cohesin loading is a critical molecular prerequisite to cope with replication. Paradoxically, topological loading per se is highly rate limiting and hardly occurs under the replication-competent physiological salt concentration. This inconsistency is resolved by the replisome-associated cohesion establishment factors Chl1 helicase and Ctf4 (refs. 6,7), which promote cohesin loading specifically during continuing replication. Accordingly, we found that bubble DNA, which mimics the state of DNA unwinding, induces topological cohesin loading and this is further promoted by Chl1. Thus, we propose that cohesin converts the initial electrostatic DNA-binding mode to a topological embrace when it encounters unwound DNA structures driven by enzymatic activities including replication. Together, our results show how cohesin initially responds to replication, and provide a molecular model for the establishment of sister chromatid cohesion.

Diverse modes of H3K36me3-guided nucleosomal deacetylation by Rpd3S.

Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation1-4. The Rpd3 small (Rpd3S) complex recognizes histone H3 trimethylation on lysine 36 (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions5-7. Here we solved the cryo-electron microscopy structures of Saccharomyces cerevisiae Rpd3S in its free and H3K36me3 nucleosome-bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3-Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex. Multivalent recognition of two H3K36me3 marks, nucleosomal DNA and linker DNAs by Eaf3, Sin3 and Rco1 positions the catalytic centre of Rpd3 next to the histone H4 N-terminal tail for deacetylation. In an alternative catalytic mode, combinatorial readout of unmethylated histone H3 lysine 4 and H3K36me3 by Rco1 and Eaf3 directs histone H3-specific deacetylation except for the registered histone H3 acetylated lysine 9. Collectively, our work illustrates dynamic and diverse modes of multivalent nucleosomal engagement and methylation-guided deacetylation by Rpd3S, highlighting the exquisite complexity of epigenetic regulation with delicately designed multi-subunit enzymatic machineries in transcription and beyond.

Nuclear pore complexes mediate subtelomeric gene silencing by regulating PCNA levels on chromatin.

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interactions, genetic interactions, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170Δ. The NPC, therefore, mediates subtelomeric gene silencing by regulating PCNA levels on DNA.

The Smc5/6 complex is a DNA loop-extruding motor.

Structural maintenance of chromosomes (SMC) protein complexes are essential for the spatial organization of chromosomes1. Whereas cohesin and condensin organize chromosomes by extrusion of DNA loops, the molecular functions of the third eukaryotic SMC complex, Smc5/6, remain largely unknown2. Using single-molecule imaging, we show that Smc5/6 forms DNA loops by extrusion. Upon ATP hydrolysis, Smc5/6 reels DNA symmetrically into loops at a force-dependent rate of one kilobase pair per second. Smc5/6 extrudes loops in the form of dimers, whereas monomeric Smc5/6 unidirectionally translocates along DNA. We also find that the subunits Nse5 and Nse6 (Nse5/6) act as negative regulators of loop extrusion. Nse5/6 inhibits loop-extrusion initiation by hindering Smc5/6 dimerization but has no influence on ongoing loop extrusion. Our findings reveal functions of Smc5/6 at the molecular level and establish DNA loop extrusion as a conserved mechanism among eukaryotic SMC complexes.

Fission yeast Swi2 designates cell-type specific donor and stimulates Rad51-driven strand exchange for mating-type switching.

A haploid of the fission yeast Schizosaccharomyces pombe expresses either the P or M mating-type, determined by the active, euchromatic, mat1 cassette. Mating-type is switched by Rad51-driven gene conversion of mat1 using a heterochromatic donor cassette, mat2-P or mat3-M. The Swi2-Swi5 complex, a mating-type switching factor, is central to this process by designating a preferred donor in a cell-type-specific manner. Swi2-Swi5 selectively enables one of two cis-acting recombination enhancers, SRE2 adjacent to mat2-P or SRE3 adjacent to mat3-M. Here, we identified two functionally important motifs in Swi2, a Swi6 (HP1 homolog)-binding site and two DNA-binding AT-hooks. Genetic analysis demonstrated that the AT-hooks were required for Swi2 localization at SRE3 to select the mat3-M donor in P cells, while the Swi6-binding site was required for Swi2 localization at SRE2 to select mat2-P in M cells. In addition, the Swi2-Swi5 complex promoted Rad51-driven strand exchange in vitro. Taken together, our results show how the Swi2-Swi5 complex would localize to recombination enhancers through a cell-type specific binding mechanism and stimulate Rad51-driven gene conversion at the localization site.

Tissue Cultivation, Preparation, and Extraction of High Molecular Weight DNA for Single-Molecule Genome Sequencing of Plant-Associated Fungi.

Extraction of high-quality, high molecular weight DNA is a critical step for sequencing an organism's genome. For fungi, DNA extraction is often complicated by co-precipitation of secondary metabolites, the most destructive being polysaccharides, polyphenols, and melanin. Different DNA extraction protocols and clean-up methods have been developed to address challenging materials and contaminants; however, the method of fungal cultivation and tissue preparation also plays a critical role to limit the production of inhibitory compounds prior to extraction. Here, we provide protocols and guidelines for (i) fungal tissue cultivation and processing with solid media containing a cellophane overlay or in liquid media, (ii) DNA extraction with customized recommendations for taxonomically and ecologically diverse plant-associated fungi, and (iii) assessing DNA quantity and quality for downstream genome sequencing with single-molecule technology such as PacBio.

Transcriptome analysis reveals gene expression changes of the basidiomycetous yeast Apiotrichum mycotoxinivorans in response to ochratoxin A exposure.

Ochratoxin A (OTA) is one of the most common and deleterious mycotoxins found in food and feedstuffs worldwide; however, Apiotrichum mycotoxinivorans can detoxify OTA. Our results show that A. mycotoxinivorans GUM1709 efficiently degraded OTA, but it caused the accumulation of intracellular reactive oxygen species. The main aim of this study was to identify potential OTA-detoxifying enzymes and to explore the effects of OTA on A. mycotoxinivorans GMU1709. RNA-seq data revealed that 1643 and 1980 genes were significantly upregulated and downregulated, respectively, after OTA exposure. Functional enrichment analyses indicated that OTA exposure enhanced defense capability, protein transport, endocytosis, and energy metabolism; caused ribosomal stress; suppressed DNA replication and transcription; inhibited cell growth and division; and promoted cell death. The integration of secretome, gene expression, and molecular docking analyses revealed that two carboxypeptidase homologues (members of the metallocarboxypeptidase family) were most likely responsible for the detoxification of both extracellular and intracellular OTA. Superoxide dismutase and catalase were the main genes activated in response to oxidative stress. In addition, analysis of key genes associated with cell division and apoptosis showed that OTA exposure inhibited mitosis and promoted cell death. This study revealed the possible OTA response and detoxification mechanisms in A. mycotoxinivorans.

Nucleosome Positioning on Large Tandem DNA Repeats of the '601' Sequence Engineered in Saccharomyces cerevisiae.

The artificial 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, application of this property in vivo has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro, analysis of nucleosome occupancy in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

Nucleosome recognition and DNA distortion by the Chd1 remodeler in a nucleotide-free state.

Chromatin remodelers are ATP-dependent enzymes that reorganize nucleosomes within all eukaryotic genomes. Here we report a complex of the Chd1 remodeler bound to a nucleosome in a nucleotide-free state, determined by cryo-EM to 2.3 Å resolution. The remodeler stimulates the nucleosome to absorb an additional nucleotide on each strand at two different locations: on the tracking strand within the ATPase binding site and on the guide strand one helical turn from the ATPase motor. Remarkably, the additional nucleotide on the tracking strand is associated with a local transformation toward an A-form geometry, explaining how sequential ratcheting of each DNA strand occurs. The structure also reveals a histone-binding motif, ChEx, which can block opposing remodelers on the nucleosome and may allow Chd1 to participate in histone reorganization during transcription.

Deciphering the mechanism of processive ssDNA digestion by the Dna2-RPA ensemble.

Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors outcompete RPA to access ssDNA. Using the budding yeast Saccharomyces cerevisiae as a model system, we find that Dna2 - a key nuclease in DNA replication and repair - employs a bimodal interface to act with RPA both in cis and in trans. The cis-activity makes RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA delivers its bound ssDNA to Dna2. On the other hand, activity in trans is mediated by an acidic patch on Dna2, which enables it to function with a sub-optimal amount of RPA, or to overcome DNA secondary structures. The trans-activity mode is not required for cell viability, but is necessary for effective double strand break (DSB) repair.

Relationship between delayed luminescence emission and mitochondrial status in Saccharomyces cerevisiae.

Delayed luminescence (DL) is gradually used in various detection of biological systems as a rapid detection technique, however, its biological mechanism was still not clear. In this study, a new model of DL detection system for liquid biological samples is established to investigate the DL emission of Saccharomyces cerevisiae cells cultured in different glucose concentrations. We analyzed the relationship between the DL emission and cell growth, cell vitality, mitochondrial morphology, mitochondrial DNA (mtDNA) copy number, adenosine triphosphate (ATP), oxygen consumption rate (OCR), as well as mitochondria membrane potential (MMP) in S. cerevisiae cells cultured with 0.01, 0.05, 0.15, 3, 10 and 20 g/L glucose respectively. It was found that the DL emission had strong correlation with mitochondrial morphology, OCR, and MMP. The results suggested that DL is an indicator of mitochondria status under different glucose supply conditions, and may be an effective method to detect mitochondrial metabolism related disorders.

Stimulation of adaptive gene amplification by origin firing under replication fork constraint.

Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 can cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32 and Pif1. CUP1 amplification is also critically dependent on late-firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after activation, leaving a tract of H3K56-acetylated chromatin that promotes secondary recombination events during elongation after replication fork re-start events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ.

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.

AoATG5 plays pleiotropic roles in vegetative growth, cell nucleus development, conidiation, and virulence in the nematode-trapping fungus Arthrobotrys oligospora.

Autophagy is an evolutionarily conserved process in eukaryotes, which is regulated by autophagy-related genes (ATGs). Arthrobotrys oligospora is a representative species of nematode-trapping (NT) fungi that can produce special traps for nematode predation. To elucidate the biological roles of autophagy in NT fungi, we characterized an orthologous Atg protein, AoAtg5, in A. oligospora. We found that AoATG5 deletion causes a significant reduction in vegetative growth and conidiation, and that the transcript levels of several sporulation-related genes were significantly downregulated during sporulation stage. In addition, the cell nuclei were significantly reduced in the ΔAoATG5 mutant, and the transcripts of several genes involved in DNA biosynthesis, repair, and ligation were significantly upregulated. In ΔAoATG5 mutants, the autophagic process was significantly impaired, and trap formation and nematocidal activity were significantly decreased. Comparative transcriptome analysis results showed that AoAtg5 is involved in the regulation of multiple cellular processes, such as autophagy, nitrogen metabolism, DNA biosynthesis and repair, and vesicular transport. In summary, our results suggest that AoAtg5 is essential for autophagy and significantly contributes to vegetative growth, cell nucleus development, sporulation, trap formation, and pathogenicity in A. oligospora, thus providing a basis for future studies focusing on related mechanisms of autophagy in NT fungi.

Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events.

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ∼50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20-40 nm at forces of 1.0-0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ∼200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.

Pif1 Activity is Modulated by DNA Sequence and Structure.

The gene encoding the Pif1 helicase was first discovered in a Saccharomyces cerevisiae genetic screen as a mutant that reduces recombination between mitochondrial respiratory mutants and was subsequently rediscovered in a screen for genes affecting the telomere length in the nucleus. It is now known that Pif1 is involved in numerous aspects of DNA metabolism. All known functions of Pif1 rely on binding to DNA substrates followed by ATP hydrolysis, coupling the energy released to translocation along DNA to unwind duplex DNA or alternative DNA secondary structures. The interaction of Pif1 with higher-order DNA structures, like G-quadruplex DNA, as well as the length of single-stranded (ss)DNA necessary for Pif1 loading have been widely studied. Here, to test the effects of ssDNA length, sequence, and structure on Pif1's biochemical activities in vitro, we used a suite of oligonucleotide-based substrates to perform a basic characterization of Pif1 ssDNA binding, ATPase activity, and helicase activity. Using recombinant, untagged S. cerevisiae Pif1, we found that Pif1 preferentially binds to structured G-rich ssDNA, but the preferred binding substrates failed to maximally stimulate ATPase activity. In helicase assays, significant DNA unwinding activity was detected at Pif1 concentrations as low as 250 pM. Helicase assays also demonstrated that Pif1 most efficiently unwinds DNA fork substrates with unstructured ssDNA tails. As the chemical step size of Pif1 has been determined to be 1 ATP per translocation or unwinding event, this implies that the highly structured DNA inhibits conformational changes in Pif1 that couple ATP hydrolysis to DNA translocation and unwinding.

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

Redirecting meiotic DNA break hotspot determinant proteins alters localized spatial control of DNA break formation and repair.

During meiosis, DNA double-strand breaks (DSBs) are formed at high frequency at special chromosomal sites, called DSB hotspots, to generate crossovers that aid proper chromosome segregation. Multiple chromosomal features affect hotspot formation. In the fission yeast S. pombe the linear element proteins Rec25, Rec27 and Mug20 are hotspot determinants - they bind hotspots with high specificity and are necessary for nearly all DSBs at hotspots. To assess whether they are also sufficient for hotspot determination, we localized each linear element protein to a novel chromosomal site (ade6 with lacO substitutions) by fusion to the Escherichia coli LacI repressor. The Mug20-LacI plus lacO combination, but not the two separate lac elements, produced a strong ade6 DSB hotspot, comparable to strong endogenous DSB hotspots. This hotspot had unexpectedly low ade6 recombinant frequency and negligible DSB hotspot competition, although like endogenous hotspots it manifested DSB interference. We infer that linear element proteins must be properly placed by endogenous functions to impose hotspot competition and proper partner choice for DSB repair. Our results support and expand our previously proposed DSB hotspot-clustering model for local control of meiotic recombination.