Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3–H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3–H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt- 2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion1. However, D2-I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation2-4. At present, it is unclear how DNA crosslinks are recognized and how D2-I functions in replication fork protection. Here, using single-molecule imaging, we show that D2-I is a sliding clamp that binds to and diffuses on double-stranded DNA. Notably, sliding D2-I stalls on encountering single-stranded-double-stranded (ss-ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions5. Using cryogenic electron microscopy, we determined structures of D2-I on DNA that show that stalled D2-I makes specific interactions with the ss-dsDNA junction that are distinct from those made by sliding D2-I. Thus, D2-I surveys dsDNA and, when it reaches an ssDNA gap, it specifically clamps onto ss-dsDNA junctions. Because ss-dsDNA junctions are found at stalled replication forks, D2-I can identify sites of DNA damage. Therefore, our data provide a unified molecular mechanism that reconciles the roles of D2-I in the recognition and protection of stalled replication forks in several DNA repair pathways.

DNA crosslinks block DNA replication and are repaired by the Fanconi Anemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion. However, D2-I is also known to play a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation. It is currently unclear how DNA crosslinks are recognized and how D2-I functions in replication fork protection. Using single-molecule microscopy, we show that D2-I is a sliding clamp that binds to and diffuses on double-stranded DNA.

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans orthologue of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt- 2 mutants have increased chromatin accessibility at these loci. We also show that human SPT2 controls the incorporation of new H3.3 into chromatin. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

Mutations in the adaptor protein PSTPIP1 cause a spectrum of autoinflammatory diseases, including PAPA and PAMI; however, the mechanism underlying these diseases remains unknown. Most of these mutations lie in PSTPIP1 F-BAR domain, which binds to LYP, a protein tyrosine phosphatase associated with arthritis and lupus. To shed light on the mechanism by which these mutations generate autoinflammatory disorders, we solved the structure of the F-BAR domain of PSTPIP1 alone and bound to the C-terminal homology segment of LYP, revealing a novel mechanism of recognition of Pro-rich motifs by proteins in which a single LYP molecule binds to the PSTPIP1 F-BAR dimer. The residues R228, D246, E250, and E257 of PSTPIP1 that are mutated in immunological diseases directly interact with LYP. These findings link the disruption of the PSTPIP1/LYP interaction to these diseases, and support a critical role for LYP phosphatase in their pathogenesis.

DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2–FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2–FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2–FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2–FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2–FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2–FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair. Sijacki et al. show how phosphorylation of FANCI by the ATR DNA damage kinase primes the FANCD2–FANCI clamp for ubiquitination. Phosphorylation promotes closure of the clamp, exposing a lysine for ubiquitination to initiate DNA cross-link repair.

Diffraction images of a crystal of the F-BAR domain of human PSTPIP1 (residues 1-289, Uniprot reference O43586-1) in complex with the CTH of LYP (residues 787-807, Uniprot Q9Y2R2-1). Data were collected on a single crystal at the beamline i03 of the Diamond Light Source synchrotron (Didcot, UK) using radiation of 0.99987 Å wavelength and a PILATUS3 6M detector. The dataset consists of 3 groups, each containing of 1800 images (0.1 degree oscillation per image), collected at three different positions of the same crystal. Crystal belongs to the space group P2(1)2(1)2(1) with unit cell dimensions a=48.0 Å, b=72.0 Å, c=205.0 Å. The asymmetric unit contains an homodimer of the F-BAR domain bound to a LYP-CTH (~53% solvent content), which is the biological complex. Diffraction data was notably anisotropic. The lowest resolution limit was 4.05 Å in the direction b* and the highest limits were 2.11 Å and 2.10 in the directions a* and c*, respectively. The structure derived form these data is published in: Manso, J.A., Marcos, T., Ruiz-Martín, V. Casas J, Alcón P, Sánchez Crespo M, Bayón Y, de Pereda JM, Alonso A PSTPIP1-LYP phosphatase interaction: structural basis and implications for autoinflammatory disorders. Cell. Mol. Life Sci. 79, 131 (2022). https://doi.org/10.1007/s00018-022-04173-w The structure is available at the PDB under the code 7AAM: https://www.ebi.ac.uk/pdbe/entry/pdb/7aam

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2–FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2–FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2–FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors. Structural analysis of the DNA crosslink repair factors FANCD2 and FANCI demonstrates that a complex of both factors adopts a closed conformation upon ubiquitination of FANCD2, trapping the complex on DNA.

Cas12a, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. In our recent publication in Cell, we combined cryoEM structures and single molecule FRET to provide a complete mechanistic understanding of endonuclease structural dynamics role in function and resetting(1). Cryo-EM readout provided the structures of intermediates of the cleavage reaction, and identified protein regions that sense the crRNA-DNA hybrid assembly triggering catalytic activation. Combined with single molecule readout of my group(2-4) and specifically FRET allowed us to directly observe the protein conformational dynamics along the entire reaction pathway. Parallel single molecule imaging provided the directionality of conformational transitions as well as the complete thermodynamic and kinetic characterisation of the conformational activation leading to function. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity degrading ssDNA molecules after activation and how other crRNAs displace the R-loop inside the protein after target DNA cleavage terminating indiscriminate ssDNA degradation. We proposed a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will recycle Cas12a specifically targeting new DNAs.

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2–FANCI4,5 by the FA core complex—a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair. The structure of the multiprotein Fanconi anaemia core complex, determined using cryo-electron microscopy and mass spectrometry, shows that the complex adopts an extended asymmetric structure and highlights the structural and functional asymmetry of the RING finger domains.

Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.

The purification, reconstitution and crystallization of a complex of F. novicida Cpf1 with CRISPR RNA and target DNA is described.

Bacteria and archaea use the CRISPR–Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR–Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5′-TTN-3′ protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro . Finally, the ternary FnCpf1–crRNA–DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C 222 1 , with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments.

Nature 546, 559–563 (2017); doi:10.1038/nature22398 In Fig. 1b of this Letter, owing to an error during the production process, the 5′ and 3′ labels at the bottom of the structure diagram were inadvertently reversed. This has been corrected in the online versions of the Letter.

CRISPR-Cas is a bacterial defense system against phage infection and nucleic acid invasion. Class 2 type II CRISPR-Cas9 has also been widely used for genome engineering. Here, we review novel insights into the CRISPR class 2 type V enzymes, specifically Cpf1 and C2c1, which display different DNA-recognition and cleavage characteristics than those of Cas9, the best-characterized member of class 2. Recent structures of these ribonucleoprotein complexes that capture several stages of the endonuclease reaction have provided molecular details of recognition, unzipping and cleavage of the target DNA, allowing their comparison with Cas9. A detailed understanding of these mechanisms is crucial for improving these genome engineering tools and expanding the genomic space that can be targeted.