The Impact of Rare Human Variants on Barrier-To-Auto-Integration Factor 1 (Banf1) Structure and Function

Barrier-to-Autointegration Factor 1 (Banf1/BAF) is a critical component of the nuclear envelope and is involved in the maintenance of chromatin structure and genome stability. Banf1 is a small DNA binding protein that is conserved amongst multicellular eukaryotes. Banf1 functions as a dimer, and binds non-specifically to the phosphate backbone of DNA, compacting the DNA in a looping process. The loss of Banf1 results in loss of nuclear envelope integrity and aberrant chromatin organisation. Significantly, mutations in Banf1 are associated with the severe premature ageing syndrome, Néstor–Guillermo Progeria Syndrome. Previously, rare human variants of Banf1 have been identified, however the impact of these variants on Banf1 function has not been explored. Here, using in silico modelling, biophysical and cell-based approaches, we investigate the effect of rare human variants on Banf1 structure and function. We show that these variants do not significantly alter the secondary structure of Banf1, but several single amino acid variants in the N- and C-terminus of Banf1 impact upon the DNA binding ability of Banf1, without altering Banf1 localisation or nuclear integrity. The functional characterisation of these variants provides further insight into Banf1 structure and function and may aid future studies examining the potential impact of Banf1 function on nuclear structure and human health.

Convertible and Constrained Nucleotides: The 2’-Deoxyribose 5’-C-Functionalization Approach, a French Touch

Many strategies have been developed to modulate the biological or biotechnical properties of oligonucleotides by introducing new chemical functionalities or by enhancing their affinity and specificity while restricting their conformational space. Among them, we review our approach consisting of modifications of the 5’-C-position of the nucleoside sugar. This allows the introduction of an additional chemical handle at any position on the nucleotide chain without disturbing the Watson–Crick base-pairing. We show that 5’-C bromo or propargyl convertible nucleotides (CvN) are accessible in pure diastereoisomeric form, either for nucleophilic displacement or for CuAAC conjugation. Alternatively, the 5’-carbon can be connected in a stereo-controlled manner to the phosphate moiety of the nucleotide chain to generate conformationally constrained nucleotides (CNA). These allow the precise control of the sugar/phosphate backbone torsional angles. The consequent modulation of the nucleic acid shape induces outstanding stabilization properties of duplex or hairpin structures in accordance with the preorganization concept. Some biological applications of these distorted oligonucleotides are also described. Effectively, the convertible and the constrained approaches have been merged to create constrained and convertible nucleotides (C2NA) providing unique tools to functionalize and stabilize nucleic acids.

Crystal structure of the complex of DNA with the C-terminal domain of TYE7 from Saccharomyces cerevisiae

TYE7, a bHLH (basic helix–loop–helix) transcription factor from Saccharomyces cerevisiae, is involved in the regulation of many genes, including glycolytic genes. Meanwhile, accumulating evidence indicates that TYE7 also functions as a cyclin and is linked to sulfur metabolism. Here, the structure of TYE7 (residues 165–291) complexed with its specific DNA was determined by X-ray crystallography. Structural analysis and comparison revealed that His185 and Glu189 are conserved in base recognition. However, Arg193 is also involved in base recognition in the structures that were compared. In the structure in this study, Arg193 in chain A has two conformations and makes a salt bridge with the phosphate backbone structure. In addition, a series of corresponding electrophoretic mobility shift assays were performed to better understand the DNA-binding mechanism of the bHLH domain of TYE7.

Understanding the Origin of Structural Diversity of DNA Double Helix

Deciphering the contribution of DNA subunits to the variability of its 3D structure represents an important step toward the elucidation of DNA functions at the atomic level. In the pursuit of that goal, our previous studies revealed that the essential conformational characteristics of the most populated “canonic” BI and AI conformational families of Watson–Crick duplexes, including the sequence dependence of their 3D structure, preexist in the local energy minima of the elemental single-chain fragments, deoxydinucleoside monophosphates (dDMPs). Those computations have uncovered important sequence-dependent regularity in the superposition of neighbor bases. The present work expands our studies to new minimal fragments of DNA with Watson–Crick nucleoside pairs that differ from canonic families in the torsion angles of the sugar-phosphate backbone (SPB). To address this objective, computations have been performed on dDMPs, cdDMPs (complementary dDMPs), and minimal fragments of SPBs of respective systems by using methods of molecular and quantum mechanics. These computations reveal that the conformations of dDMPs and cdDMPs having torsion angles of SPB corresponding to the local energy minima of separate minimal units of SPB exhibit sequence-dependent characteristics representative of canonic families. In contrast, conformations of dDMP and cdDMP with SPB torsions being far from the local minima of separate SPB units exhibit more complex sequence dependence.

Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding

G-quadruplexes (G4s) are unusual DNA structures and can stall DNA replication, causing genomic instability for the cell. Although the solved crystal structure of the DHX36 helicase demonstrated that G4 was specifically targeted by a DHX36-specific motif (DSM), lack of complete structural details for general G4-resolving helicases without specific target motifs remains a barrier to the complete understanding of the molecular basis underlying the recognition and unfolding of G4s. Herein, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif) complexed with a G4, thereby mimicking the physiological G4 formed during DNA replication. Strictly different from the previous determined G4-helicase structure of DHX36, our structure revealed that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B constituting a G4-Recognizing Surface (GRS). The overall topology of the G4 structure solved in this work maintains its three-layered propeller-type G4 topology, with no significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 were differentially recognized by GRS residues mainly through electrostatic, ionic interactions and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Our structure explains how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.

Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the øX174 Capsid

Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging is specific to single-stranded (ss) DNA viruses. ssDNA genome packaging combines elements found in both double-stranded (ds) DNA and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA’s negative phosphate backbone. øX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a øXG4J chimera, in which the øX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, øXα3J. Unlike øXG4J, øXα3J efficiently packaged DNA but produced non-infectious particles. These particles displayed a reduced ability to attach to host cells, suggesting internal DNA organization could distort the capsid’s outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and post-packaging phenomena. Importance ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit øX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid’s outer surface.

Structure of the mini-RNA-guided endonuclease CRISPR-CasΦ3

CasΦ is a novel family of miniaturized RNA-guided endonucleases from phages 1,2. These novel ribonucleoproteins (RNPs) provide a compact scaffold gathering all key activities of a genome editing tool2. Here, we provide the first structural insight into CasΦ singular DNA targeting and cleavage mechanism by determining the cryoEM structure of CasΦ3 with the triple strand R-loop generated after DNA cleavage. The structure reveals the unique machinery for target unwinding to form the crRNA-DNA hybrid and cleaving the target DNA. The protospacer adjacent motif (PAM) is recognised by the target strand (T-strand) and non-target strand (NT-strand) PAM interacting domains (TPID and NPID). Unwinding occurs after insertion of the conserved α1 helix disrupting the dsDNA, thus facilitating the crRNA-DNA hybrid formation. The NT-strand is funnelled towards the RuvC catalytic site, while a long helix of TPID separates the displaced NT-strand and the crRNA-DNA hybrid avoiding DNA re-annealing. The crRNA-DNA hybrid is directed to the stop (STP) domain that splits the hybrid guiding the T-strand towards the RuvC active site. The conserved RuvC insertion of the CasΦ family is extended along the hybrid, interacting with the phosphate backbone of the crRNA. A cluster of hydrophobic residues anchors the RuvC insertion in a cavity of the STP domain. The assembly of the hybrid promotes the shortening of the RuvC insertion, thus pulling the STP towards the RuvC active site to activate catalysis. These findings illustrate why CasΦ unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. Site-directed mutagenesis in key residues support CasΦ3 target DNA and non-specific ssDNA cutting mechanism. Our analysis provides new avenues to redesign the compact CRISPR-CasΦ nucleases for genome editing.