Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a ‘cogwheel’ mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA–DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme.

Significance: Cyclic nucleotide-gated (CNG) channels underlie sensory transduction in photoreceptors and olfactory epithelium and share a high degree of homology with K ⁺ channels. However, these channels conduct Na ⁺ and K ⁺ differently: although K ⁺ channels discriminate with high accuracy Na ⁺ from K ⁺ , CNG channels do not discriminate among different cations. By combining electrophysiology, molecular dynamics simulations, and X-ray crystallography we found that the pore region exhibits a dynamic structure. We show that ( i ) the selectivity filter can adapt to large and small ions with a different geometry and ( ii ) the pore diameter critically depends on the ion within. We conclude that the pore of CNG channels is highly flexible and that this flexibility is at the basis of their poor ionic selectivity.

P15^PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15^PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15^PAF–PCNA–DNA ternary complexes. Crystallography and MD simulations show that, when p15^PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15^PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15^PAF from the ring wall. Thus, p15^PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15^PAF impairs primer synthesis by pol η–PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15^PAF in DNA replication and suppression of DNA lesion bypass.

Multiheme cytochromes c (cyts c) are c-type cyts characterized by non-standard structural and spectroscopic properties. The relative disposition of the heme cofactors in the core of these proteins is conserved and they can be classified from their geometry in two main groups. In one group the porphyrin planes are arranged in a perpendicular fashion, while in the other they are parallel. Orientation of the heme groups is a key factor that regulates the intramolecular electron transfer pathway. A 16.5 kDa diheme cyt c, isolated from the bacterium Shewanella baltica OS155 (Sb-DHC), was cloned and expressed in E. coli and its structure was investigated by X-ray crystallography. Using high-resolution data (1.14 Å) collected at ELETTRA (Trieste), the crystal structure, with an orthorhombic cell (a = 40.81, b = 42.97, c = 82.07 Å), was solved using the homologous diheme from Rhodobacter sphaeroides (Rs-DHC) as the initial model. The electron density map of the refined structure (R_fact of 13.8% and R_free of 15.4%) shows a two domain structure connected by a central unstructured region (N72-G87). The Sb-DHC, like its homologue (Rs-DHC), folds into a new cyt c class: the N-terminal globular domain, with its three α-helices, belongs to class I of c-type cyts, while the C-terminal domain includes a rare π-helix. The metal centre of the c-type heme groups is axially coordinated by two His residues and it is covalently bound to the protein through two Cys bonds.

Extracellular vesicles (EVs) have enormous potential for the implementation of liquid biopsy and as effective drug delivery means, but the fulfilment of these expectations requires overcoming at least two bottlenecks relative to their purification, namely the finalization of reliable and affordable protocols for: (i) EV sub-population selective isolation and (ii) the scalability of their production/isolation from complex biological fluids. In this work, we demonstrated that these objectives can be achieved by a conceptually new affinity chromatography platform composed of a macroporous epoxy monolith matrix functionalized with anti-CD63 nanobodies with afflux of samples and buffers regulated through a pump. Such a system successfully captured and released integral EVs from urine samples and showed negligible unspecific binding for circulating proteins. Additionally, size discrimination of eluted EVs was achieved by different elution approaches (competitive versus pH-dependent). The physical characteristics of monolith material and the inexpensive production of recombinant nanobodies make scaling-up the capture unit feasible and affordable. Additionally, the availability of nanobodies for further specific EV biomarkers will allow for the preparation of monolithic affinity filters selective for different EV subclasses.

RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a C-terminal region (RQC) found in the other RecQ paralogues; such region consists of a Zn domain and a winged-helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. ICP-AES detected the unusual presence of two zinc clusters within the Zn domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative Zn ligands and the aromatic residue predicted to be at the tip of the winged helix β hairpin), showed a decrease in DNA binding, unwinding and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.