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.
Cyclic nucleotide-gated (CNG) ion channels, despite a significant homology with the highly selective K + channels, do not discriminate among monovalent alkali cations and are permeable also to several organic cations. We combined electrophysiology, molecular dynamics (MD) simulations, and X-ray crystallography to demonstrate that the pore of CNG channels is highly flexible. When a CNG mimic is crystallized in the presence of a variety of monovalent cations, including Na + , Cs + , and dimethylammonium (DMA + ), the side chain of Glu66 in the selectivity filter shows multiple conformations and the diameter of the pore changes significantly. MD simulations indicate that Glu66 and the prolines in the outer vestibule undergo large fluctuations, which are modulated by the ionic species and the voltage. This flexibility underlies the coupling between gating and permeation and the poor ionic selectivity of CNG channels.CNG channels | pore flexibility | X-ray crystallography | MD simulations I n K + selective channels, the opening and closing of the ion channel pore (gating) and the translocation of ions through the pore (permeation) are considered independent processes with distinct structural basis (1). Gating is controlled by the bundle crossing at the intracellular side, whereas permeation reflects ion-ion and ion-pore interactions within the selectivity filter (1-5). Based on these experimental observations, the current paradigm assumes that the 3D structure of the selectivity filter is relatively rigid during ion translocation and the mechanisms of ionic permeation can be deduced in essence from its crystal structure. This paradigm has been successfully applied to several K + channels (4, 6, 7). Cyclic nucleotide-gated (CNG) channels underlie sensory transduction in the retina and olfactory epithelium and share a high degree of homology with K + channels (8-10). In contrast to K + channels, CNG channels' primary gate is located at the selectivity filter (11), suggesting that the same protein region controls ion permeation and gating. In CNG channels the ionic species present inside the pore influences channel gating; however, the nature of this coupling is not well understood (12-16). In the presence of large cations, such as Rb + and Cs + , channel conductance and gating are also controlled by membrane voltage, and current-voltage relationships activated by 1 mM cGMP depend on the radius of the permeating ion (17, 18) (Fig. S1).Structural information on CNG channels is limited to a lowresolution electron microscopy map (19), partial crystal structures of the intracellular cyclic nucleotide-binding domains (20)(21)(22), and the crystal structure of a chimeric channel in which the CNG selectivity-filter sequence is engineered into a bacterial NaK channel, creating a CNG mimic (NaK2CNG; Fig. 1 A and B) that shares several properties of CNG channels (23). This CNG mimic provides a suitable model for understanding the properties of the pore underlying the low ionic selectivity and the coupling between gating and perme...
Abstractp15PAF 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, p15PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF–PCNA–DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF 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 p15PAF 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 p15PAF in DNA replication and suppression of DNA lesion bypass.
Edited by F. Peter GuengerichRecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and 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 RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc 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. Inductively coupled plasmaatomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix -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.
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