Mechanism of homology search expansion during recombinational DNA break repair in Saccharomyces cerevisiae.

Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination (HR). How it operates in cells remains elusive. We developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search that is orchestrated by chromatin organizers, long-range resection, end-tethering, and specialized genetic elements and that exploits the stiff NPF structure conferred by Rad51 oligomerization.

Mechanisms controlling replication fork stalling and collapse at topoisomerase 1 cleavage complexes.

Topoisomerase 1 cleavage complexes (Top1-ccs) comprise a DNA-protein crosslink and a single-stranded DNA break that can significantly impact the DNA replication machinery (replisome). Consequently, inhibitors that trap Top1-ccs are used extensively in research and clinical settings to generate DNA replication stress, yet how the replisome responds upon collision with a Top1-cc remains obscure. By reconstituting collisions between budding yeast replisomes, assembled from purified proteins, and site-specific Top1-ccs, we have uncovered mechanisms underlying replication fork stalling and collapse. We find that stalled replication forks are surprisingly stable and that their stability is influenced by the template strand that Top1 is crosslinked to, the fork protection complex proteins Tof1-Csm3 (human TIMELESS-TIPIN), and the convergence of replication forks. Moreover, nascent-strand mapping and cryoelectron microscopy (cryo-EM) of stalled forks establishes replisome remodeling as a key factor in the initial response to Top1-ccs. These findings have important implications for the use of Top1 inhibitors in research and in the clinic.

Airborne DNA reveals predictable spatial and seasonal dynamics of fungi.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions1,2. To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores3. The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms4,5.

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.

Methylation of histone H3 at lysine 37 by Set1 and Set2 prevents spurious DNA replication.

DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination.

Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by different mechanisms: noncrossovers by synthesis-dependent strand annealing and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution parental strand contributions to recombinants at a model locus. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes involving branch migration as an integral feature, one that can be separated from repair of the break itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.

Damage-induced chromatome dynamics link Ubiquitin ligase and proteasome recruitment to histone loss and efficient DNA repair.

Eukaryotic cells package their genomes around histone octamers. In response to DNA damage, checkpoint activation in yeast induces core histone degradation resulting in 20%-40% reduction in nucleosome occupancy. To gain insight into this process, we developed a new approach to analyze the chromatin-associated proteome comprehensively before and after damage. This revealed extensive changes in protein composition after Zeocin-induced damage. First, core histones and the H1 homolog Hho1 were partially lost from chromatin along with replication, transcription, and chromatin remodeling machineries, while ubiquitin ligases and the proteasome were recruited. We found that the checkpoint- and INO80C-dependent recruitment of five ubiquitin-conjugating factors (Rad6, Bre1, Pep5, Ufd4, and Rsp5) contributes to core and linker histone depletion, reducing chromatin compaction and enhancing DNA locus mobility. Importantly, loss of Rad6/Bre1, Ufd4/TRIP12, and Pep5/VPS11 compromise DNA strand invasion kinetics during homology-driven repair. Thus we provide a comprehensive overview of a functionally relevant genome-wide chromatin response to DNA damage.

A composite DNA element that functions as a maintainer required for epigenetic inheritance of heterochromatin.

Epigenetic inheritance of heterochromatin requires DNA-sequence-independent propagation mechanisms, coupling to RNAi, or input from DNA sequence, but how DNA contributes to inheritance is not understood. Here, we identify a DNA element (termed "maintainer") that is sufficient for epigenetic inheritance of pre-existing histone H3 lysine 9 methylation (H3K9me) and heterochromatin in Schizosaccharomyces pombe but cannot establish de novo gene silencing in wild-type cells. This maintainer is a composite DNA element with binding sites for the Atf1/Pcr1 and Deb1 transcription factors and the origin recognition complex (ORC), located within a 130-bp region, and can be converted to a silencer in cells with lower rates of H3K9me turnover, suggesting that it participates in recruiting the H3K9 methyltransferase Clr4/Suv39h. These results suggest that, in the absence of RNAi, histone H3K9me is only heritable when it can collaborate with maintainer-associated DNA-binding proteins that help recruit the enzyme responsible for its epigenetic deposition.

The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination.

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.

An extrinsic motor directs chromatin loop formation by cohesin.

The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations-whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation.

MRX Increases Chromatin Accessibility at Stalled Replication Forks to Promote Nascent DNA Resection and Cohesin Loading.

The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.

Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork.

The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 "grip" duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.

ATP Hydrolysis by the SNF2 Domain of Dnmt5 Is Coupled to Both Specific Recognition and Modification of Hemimethylated DNA.

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast.

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not repaired in a timely manner. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including stabilized topoisomerase-1 cleavage complexes (Top1ccs). Here, we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1 wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S phase-dependent manner to assist in the eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitizes cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among the potential Ddi1 targets, we found the core component of Pol II and show that its genotoxin-induced degradation is impaired in ddi1. We propose that the Ddi1 protease contributes to DPC proteolysis.

Cohesin Causes Replicative DNA Damage by Trapping DNA Topological Stress.

DNA topological stress inhibits DNA replication fork (RF) progression and contributes to DNA replication stress. In Saccharomyces cerevisiae, we demonstrate that centromeric DNA and the rDNA array are especially vulnerable to DNA topological stress during replication. The activity of the SMC complexes cohesin and condensin are linked to both the generation and repair of DNA topological-stress-linked damage in these regions. At cohesin-enriched centromeres, cohesin activity causes the accumulation of DNA damage, RF rotation, and pre-catenation, confirming that cohesin-dependent DNA topological stress impacts on normal replication progression. In contrast, at the rDNA, cohesin and condensin activity inhibit the repair of damage caused by DNA topological stress. We propose that, as well as generally acting to ensure faithful genetic inheritance, SMCs can disrupt genome stability by trapping DNA topological stress.

Measuring DNA mechanics on the genome scale.

Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions1. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop 'loop-seq'-a high-throughput assay to measure the propensity for DNA looping-and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors2. Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a 'mechanical code' with broad functional implications.

Promoter Distortion and Opening in the RNA Polymerase II Cleft.

Transcription initiation requires opening of promoter DNA in the RNA polymerase II (Pol II) pre-initiation complex (PIC), but it remains unclear how this is achieved. Here we report the cryo-electron microscopic (cryo-EM) structure of a yeast PIC that contains underwound, distorted promoter DNA in the closed Pol II cleft. The DNA duplex axis is offset at the upstream edge of the initially melted DNA region (IMR) where DNA opening begins. Unstable IMRs are found in a subset of yeast promoters that we show can still initiate transcription after depletion of the transcription factor (TF) IIH (TFIIH) translocase Ssl2 (XPB in human) from the nucleus in vivo. PIC-induced DNA distortions may thus prime the IMR for melting and may explain how unstable IMRs that are predicted in promoters of Pol I and Pol III can open spontaneously. These results suggest that DNA distortion in the polymerase cleft is a general mechanism that contributes to promoter opening.

RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast.

The classic view of nucleosome organization at active promoters is that two well-positioned nucleosomes flank a nucleosome-depleted region (NDR). However, this view has been recently disputed by contradictory reports as to whether wider (≳150 bp) NDRs instead contain unstable, micrococcal nuclease-sensitive ("fragile") nucleosomal particles. To determine the composition of fragile particles, we introduce CUT&RUN.ChIP, in which targeted nuclease cleavage and release is followed by chromatin immunoprecipitation. We find that fragile particles represent the occupancy of the RSC (remodeling the structure of chromatin) nucleosome remodeling complex and RSC-bound, partially unwrapped nucleosomal intermediates. We also find that general regulatory factors (GRFs) bind to partially unwrapped nucleosomes at these promoters. We propose that RSC binding and its action cause nucleosomes to unravel, facilitate subsequent binding of GRFs, and constitute a dynamic cycle of nucleosome deposition and clearance at the subset of wide Pol II promoter NDRs.

DNA Polymerase Delta Synthesizes Both Strands during Break-Induced Replication.

Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect.