Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and repair. It forms a homotrimeric ring that embraces the DNA and slides along it, anchoring DNA polymerases and other DNA editing enzymes. It also interacts with regulatory proteins through a sequence motif known as PCNA Interacting Protein box (PIP-box). We here review the latest contributions to knowledge regarding the structure-function relationships in human PCNA, particularly the mechanism of sliding, and of the molecular recognition of canonical and non-canonical PIP motifs. The unique binding mode of the oncogene p15 is described in detail, and the implications of the recently discovered structure of PCNA bound to polymerase δ are discussed. The study of the post-translational modifications of PCNA and its partners may yield therapeutic opportunities in cancer treatment, in addition to illuminating the way PCNA coordinates the dynamic exchange of its many partners in DNA replication and repair.

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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.

The type VI secretion system (T6SS) of Pseudomonas aeruginosa injects effector proteins into neighbouring competitors and host cells, providing a fitness advantage that allows this opportunistic nosocomial pathogen to persist and prevail during the onset of infections. However, despite the high clinical relevance of P. aeruginosa, the identity and mode of action of most P. aeruginosa T6SS-dependent effectors remain to be discovered. Here, we report the molecular mechanism of Tse5-CT, the toxic auto-proteolytic product of the P. aeruginosa T6SS exported effector Tse5. Our results demonstrate that Tse5-CT is a pore-forming toxin that can transport ions across the membrane, causing membrane depolarisation and bacterial death. The membrane potential regulates a wide range of essential cellular functions; therefore, membrane depolarisation is an efficient strategy to compete with other microorganisms in polymicrobial environments.