Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination.

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.

A SAM-key domain required for enzymatic activity of the Fun30 nucleosome remodeler.

Fun30 is the prototype of the Fun30-SMARCAD1-ETL subfamily of nucleosome remodelers involved in DNA repair and gene silencing. These proteins appear to act as single-subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly understood. Using multiple sequence alignment and structure prediction, we identify an evolutionarily conserved domain that is modeled to contain a SAM-like fold with one long, protruding helix, which we term SAM-key. Deletion of the SAM-key within budding yeast Fun30 leads to a defect in DNA repair and gene silencing similar to that of the fun30Δ mutant. In vitro, Fun30 protein lacking the SAM-key is able to bind nucleosomes but is deficient in DNA-stimulated ATPase activity and nucleosome sliding and eviction. A structural model based on AlphaFold2 prediction and verified by crosslinking-MS indicates an interaction of the long SAM-key helix with protrusion I, a subdomain located between the two ATPase lobes that is critical for control of enzymatic activity. Mutation of the interaction interface phenocopies the domain deletion with a lack of DNA-stimulated ATPase activation and a nucleosome-remodeling defect, thereby confirming a role of the SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of allosteric activation for this class of enzymes.

Establishment and function of chromatin organization at replication origins.

The origin recognition complex (ORC) is essential for initiation of eukaryotic chromosome replication as it loads the replicative helicase-the minichromosome maintenance (MCM) complex-at replication origins1. Replication origins display a stereotypic nucleosome organization with nucleosome depletion at ORC-binding sites and flanking arrays of regularly spaced nucleosomes2-4. However, how this nucleosome organization is established and whether this organization is required for replication remain unknown. Here, using genome-scale biochemical reconstitution with approximately 300 replication origins, we screened 17 purified chromatin factors from budding yeast and found that the ORC established nucleosome depletion over replication origins and flanking nucleosome arrays by orchestrating the chromatin remodellers INO80, ISW1a, ISW2 and Chd1. The functional importance of the nucleosome-organizing activity of the ORC was demonstrated by orc1 mutations that maintained classical MCM-loader activity but abrogated the array-generation activity of ORC. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, in addition to its canonical role as the MCM loader, has a second crucial function as a master regulator of nucleosome organization at the replication origin, a crucial prerequisite for efficient chromosome replication.

Structural mechanism of extranucleosomal DNA readout by the INO80 complex.

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo-electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes.

Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions.

DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1-like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in over-replication and leads to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation.

A CDK-regulated chromatin segregase promoting chromosome replication.

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme-Yta7-entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase.

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.

Strand-specific ChIP-seq at DNA breaks distinguishes ssDNA versus dsDNA binding and refutes single-stranded nucleosomes.

In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-domain on damaged chromatin. The molecular organization of this ssDNA and the adjacent dsDNA domain is crucial during DSB signaling and repair. However, data regarding the presence of nucleosomes, the most basic chromatin components, in the ssDNA domain have been contradictory. Here, we use site-specific induction of DSBs and chromatin immunoprecipitation followed by strand-specific sequencing to analyze in vivo binding of key DSB repair and signaling proteins to either the ssDNA or dsDNA domain. In the case of nucleosomes, we show that recently proposed ssDNA nucleosomes are not a major, persistent species, but that nucleosome eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome and ssDNA-RPA domains during DSB repair.

MTBP phosphorylation controls DNA replication origin firing.

Faithful genome duplication requires regulation of origin firing to determine loci, timing and efficiency of replisome generation. Established kinase targets for eukaryotic origin firing regulation are the Mcm2-7 helicase, Sld3/Treslin/TICRR and Sld2/RecQL4. We report that metazoan Sld7, MTBP (Mdm2 binding protein), is targeted by at least three kinase pathways. MTBP was phosphorylated at CDK consensus sites by cell cycle cyclin-dependent kinases (CDK) and Cdk8/19-cyclin C. Phospho-mimetic MTBP CDK site mutants, but not non-phosphorylatable mutants, promoted origin firing in human cells. MTBP was also phosphorylated at DNA damage checkpoint kinase consensus sites. Phospho-mimetic mutations at these sites inhibited MTBP's origin firing capability. Whilst expressing a non-phospho MTBP mutant was insufficient to relieve the suppression of origin firing upon DNA damage, the mutant induced a genome-wide increase of origin firing in unperturbed cells. Our work establishes MTBP as a regulation platform of metazoan origin firing.

DNA Double Strand Break Repair and Its Control by Nucleosome Remodeling.

DNA double strand breaks (DSBs) are repaired in eukaryotes by one of several cellular mechanisms. The decision-making process controlling DSB repair takes place at the step of DNA end resection, the nucleolytic processing of DNA ends, which generates single-stranded DNA overhangs. Dependent on the length of the overhang, a corresponding DSB repair mechanism is engaged. Interestingly, nucleosomes-the fundamental unit of chromatin-influence the activity of resection nucleases and nucleosome remodelers have emerged as key regulators of DSB repair. Nucleosome remodelers share a common enzymatic mechanism, but for global genome organization specific remodelers have been shown to exert distinct activities. Specifically, different remodelers have been found to slide and evict, position or edit nucleosomes. It is an open question whether the same remodelers exert the same function also in the context of DSBs. Here, we will review recent advances in our understanding of nucleosome remodelers at DSBs: to what extent nucleosome sliding, eviction, positioning and editing can be observed at DSBs and how these activities affect the DSB repair decision.

ESCRT recruitment by the S. cerevisiae inner nuclear membrane protein Heh1 is regulated by Hub1-mediated alternative splicing.

Misassembled nuclear pore complexes (NPCs) are removed by sealing off the surrounding nuclear envelope (NE), which is conducted by the endosomal sorting complexes required for transport (ESCRT) machinery. Recruitment of ESCRT proteins to the NE is mediated by the interaction between the ESCRT member Chm7 and the inner nuclear membrane protein Heh1, which belongs to the conserved LEM family. Increased ESCRT recruitment results in excessive membrane scission at damage sites but its regulation remains poorly understood. Here, we show that Hub1-mediated alternative splicing of HEH1 pre-mRNA, resulting in production of its shorter form Heh1-S, is critical for the integrity of the NE in Saccharomyces cerevisiae ESCRT-III mutants lacking Hub1 or Heh1-S display severe growth defects and accumulate improperly assembled NPCs. This depends on the interaction of Chm7 with the conserved MSC domain, which is only present in the longer variant Heh1-L. Heh1 variants assemble into heterodimers, and we demonstrate that a unique splice segment in Heh1-S suppresses growth defects associated with the uncontrolled interaction between Heh1-L and Chm7. Together, our findings reveal that Hub1-mediated splicing generates Heh1-S to regulate ESCRT recruitment to the NE.This article has an associated First Person interview with the first author of the paper.

A Selective Autophagy Pathway for Phase-Separated Endocytic Protein Deposits.

Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.

In-cell architecture of the nuclear pore and snapshots of its turnover.

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.

An advanced cell cycle tag toolbox reveals principles underlying temporal control of structure-selective nucleases.

Cell cycle tags allow to restrict target protein expression to specific cell cycle phases. Here, we present an advanced toolbox of cell cycle tag constructs in budding yeast with defined and compatible peak expression that allow comparison of protein functionality at different cell cycle phases. We apply this technology to the question of how and when Mus81-Mms4 and Yen1 nucleases act on DNA replication or recombination structures. Restriction of Mus81-Mms4 to M phase but not S phase allows a wildtype response to various forms of replication perturbation and DNA damage in S phase, suggesting it acts as a post-replicative resolvase. Moreover, we use cell cycle tags to reinstall cell cycle control to a deregulated version of Yen1, showing that its premature activation interferes with the response to perturbed replication. Curbing resolvase activity and establishing a hierarchy of resolution mechanisms are therefore the principal reasons underlying resolvase cell cycle regulation.

Selective autophagy degrades nuclear pore complexes.

Nuclear pore complexes (NPCs) are very large proteinaceous assemblies that consist of more than 500 individual proteins1,2. NPCs are essential for nucleocytoplasmic transport of different cellular components, and disruption of the integrity of NPCs has been linked to aging, cancer and neurodegenerative diseases3-7. However, the mechanism by which membrane-embedded NPCs are turned over is currently unknown. Here we show that, after nitrogen starvation or genetic interference with the architecture of NPCs, nucleoporins are rapidly degraded in the budding yeast Saccharomyces cerevisiae. We demonstrate that NPC turnover involves vacuolar proteases and the core autophagy machinery. Autophagic degradation is mediated by the cytoplasmically exposed Nup159, which serves as intrinsic cargo receptor and directly binds to the autophagy marker protein Atg8. Autophagic degradation of NPCs is therefore inducible, enabling the removal of individual NPCs from the nuclear envelope.

Quantitative mechanisms of DNA damage sensing and signaling.

DNA damage occurs abundantly during normal cellular proliferation. This necessitates that cellular DNA damage response and checkpoint pathways monitor the cellular DNA damage load and that DNA damage signaling is quantitative. Yet, how DNA lesions are counted and converted into a quantitative response remains poorly understood. We have recently obtained insights into this question investigating DNA damage signaling elicited by single-stranded DNA (ssDNA). Intriguingly, our findings suggest that local and global DNA damage signaling react differentially to increasing amounts of DNA damage. In this mini-review, we will discuss these findings and put them into perspective of current knowledge on the DNA damage response.

A SUMO-dependent pathway controls elongating RNA Polymerase II upon UV-induced damage.

RNA polymerase II (RNAPII) is the workhorse of eukaryotic transcription and produces messenger RNAs and small nuclear RNAs. Stalling of RNAPII caused by transcription obstacles such as DNA damage threatens functional gene expression and is linked to transcription-coupled DNA repair. To restore transcription, persistently stalled RNAPII can be disassembled and removed from chromatin. This process involves several ubiquitin ligases that have been implicated in RNAPII ubiquitylation and proteasomal degradation. Transcription by RNAPII is heavily controlled by phosphorylation of the C-terminal domain of its largest subunit Rpb1. Here, we show that the elongating form of Rpb1, marked by S2 phosphorylation, is specifically controlled upon UV-induced DNA damage. Regulation of S2-phosphorylated Rpb1 is mediated by SUMOylation, the SUMO-targeted ubiquitin ligase Slx5-Slx8, the Cdc48 segregase as well as the proteasome. Our data suggest an RNAPII control pathway with striking parallels to known disassembly mechanisms acting on defective RNA polymerase III.

Nucleosome Remodeling by Fun30SMARCAD1 in the DNA Damage Response.

Many cellular pathways are dedicated to maintain the integrity of the genome. In eukaryotes, the underlying DNA transactions occur in the context of chromatin. Cells utilize chromatin and its dynamic nature to regulate those genome integrity pathways. Accordingly, chromatin becomes restructured and modified around DNA damage sites. Here, we review the current knowledge of a chromatin remodeler Fun30SMARCAD1, which plays a key role in genome maintenance. Fun30SMARCAD1 promotes DNA end resection and the repair of DNA double-stranded breaks (DSBs). Notably, however, Fun30SMARCAD1 plays additional roles in maintaining heterochromatin and promoting transcription. Overall, Fun30SMARCAD1 is involved in distinct processes and the specific roles of Fun30SMARCAD1 at DSBs, replication forks and sites of transcription appear discordant at first view. Nonetheless, a picture emerges in which commonalities within these context-dependent roles of Fun30SMARCAD1 exist, which may help to gain a more global understanding of chromatin alterations induced by Fun30SMARCAD1.

Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance.

Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance, and transcription. However, how STUbLs select specific substrates among myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term "ubiquitin hotspots". Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and to be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation.

Quantitative sensing and signalling of single-stranded DNA during the DNA damage response.

The DNA damage checkpoint senses the presence of DNA lesions and controls the cellular response thereto. A crucial DNA damage signal is single-stranded DNA (ssDNA), which is frequently found at sites of DNA damage and recruits the sensor checkpoint kinase Mec1-Ddc2. However, how this signal - and therefore the cell's DNA damage load - is quantified, is poorly understood. Here, we use genetic manipulation of DNA end resection to induce quantitatively different ssDNA signals at a site-specific double strand break in budding yeast and identify two distinct signalling circuits within the checkpoint. The local checkpoint signalling circuit leading to γH2A phosphorylation is unresponsive to increased amounts of ssDNA, while the global checkpoint signalling circuit, which triggers Rad53 activation, integrates the ssDNA signal quantitatively. The global checkpoint signal critically depends on the 9-1-1 and its downstream acting signalling axis, suggesting that ssDNA quantification depends on at least two sensor complexes.