The purpose of the present study was to compare the effect of 3 types of titanium reinforced e-PTFE membranes (GTRM #1, #2, and #3) on bone regeneration and tissue integration. GTRM #1 was a standard titanium reinforced membrane, GTRM #2 was a prototype characterized by a more open total surface microstructure and GTRM #3 was a prototype characterized by two extremely porous external layers separated by a totally occlusive internal layer. The membranes were used to cover saddle type ridge defects surgically created in the mandible of 6 dogs. After a healing period of 6 months, all the membrane protected sites demonstrated almost complete osseous healing of the defects. GTRM #3 specimens showed the most favourable biologic response, in fact they were characterized by a larger surface of contact between the membrane and the regenerated bone, but from a clinical point of view GTRM #3 material was too difficult to remove. The results of this study suggest that an extremely open porous microstructure, in combination with a totally occlusive barrier, may provide significant improvements in regenerative outcomes. However, these design characteristics may be applied only to resorbable devices, which do not require removal.
Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.
During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.
During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Flap endonuclease 1 (FEN1), but the structural basis of this mechanism is unknown. We present cryo-EM structures of human Lig1 bound to nicked DNA and PCNA in absence and presence of FEN1. Lig1 and PCNA form a two-ring complex encircling the DNA substrate. The ligase DNA binding domain (DBD) dynamically tethers Lig1 to one PCNA protomer, leaving one protomer fully accessible for FEN1 engagement. Ligation assays argue that Lig1 binding to PCNA is critical for substrate handoff from FEN1 preassembled with nicked DNA and PCNA. Consistently, cryo-EM data show that when Lig1 and FEN1 are simultaneously bound to one PCNA ring, the nicked DNA is trapped within the ligase active site and poised for the end-joining reaction. Thus, PCNA functions as a bivalent platform for the synchronous coordination of Lig1 and FEN1 in Okazaki fragment sealing.
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