A lack of premature termination codon read-through efficacy of PTC124 (Ataluren) in a diverse array of reporter assays.

The drug molecule PTC124 (Ataluren) has been described as a read-through agent, capable of suppressing premature termination codons (PTCs) and restoring functional protein production from genes disrupted by nonsense mutations. Following the discovery of PTC124 there was some controversy regarding its mechanism of action with two reports attributing its activity to an off-target effect on the Firefly luciferase (FLuc) reporter used in the development of the molecule. Despite questions remaining as to its mechanism of action, development of PTC124 continued into the clinic and it is being actively pursued as a potential nonsense mutation therapy. To thoroughly test the ability of PTC124 to read through nonsense mutations, we conducted a detailed assessment comparing the efficacy of PTC124 with the classical aminoglycoside antibiotic read-through agent geneticin (G418) across a diverse range of in vitro reporter assays. We can confirm the off-target FLuc activity of PTC124 but found that, while G418 exhibits varying activity in every read-through assay, there is no evidence of activity for PTC124.

An in vivo analysis of the localisation and interactions of human p66 DNA polymerase delta subunit.

BACKGROUND: DNA polymerase delta is essential for eukaryotic DNA replication and also plays a role in DNA repair. The processivity of this polymerase complex is dependent upon its interaction with the sliding clamp PCNA and the polymerase-PCNA interaction is largely mediated through the p66 polymerase subunit. We have analysed the interactions of the human p66 DNA polymerase delta subunit with PCNA and with components of the DNA polymerase delta complex in vivo. RESULTS: Using the two-hybrid system, we have mapped the interaction domains for binding to the p50 polymerase delta subunit and with PCNA to the N-terminus and the C-terminus of p66, respectively. Co-immunoprecipitation experiments confirm that these interaction domains are functional in vivo. Expression of EGFP-p66 shows that it is a nuclear protein which co-localises with PCNA throughout the cell cycle. p66 is localised to sites of DNA replication during S phase and to repair foci following DNA damage. We have identified a functional nuclear localisation sequence and shown that localisation to replication foci is not dependent upon active nuclear import. Sub-domains of p66 act as dominant negative suppressors of colony formation, suggesting that p66 forms an essential structural link between the p50 subunit and PCNA. Analysis of the C-terminal PCNA binding motif shows that deletion of the QVSITGFF core motif results in a reduced affinity for PCNA, while deletion of a further 20 amino acids completely abolishes the interaction. A reduced affinity for PCNA correlates with reduced targeting to replication foci. We have confirmed the p66-PCNA interaction in vivo using fluorescence resonance energy transfer (FRET) techniques. CONCLUSION: We have defined the regions of p66 required for its interaction with PCNA and the p50 polymerase subunit. We demonstrate a functional link between PCNA interaction and localisation to replication foci and show that there is a direct interaction between p66 and PCNA in living cells during DNA replication. The dominant negative effect upon growth resulting from expression of p66 sub-domains confirms that the p66-PCNA interaction is essential in vivo.

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.

Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase delta subunit Cdc27.

BACKGROUND: DNA polymerases alpha and delta play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase alpha (Pol alpha)-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase delta (Pol delta) is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase epsilon (Pol epsilon). RESULTS: Here it is shown that the catalytic subunit of Pol alpha, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol delta, both in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM), in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function. CONCLUSIONS: A short protein sequence motif (DPIM) has been identified as mediating Pol alpha-Pol delta interactions in fission yeast. Despite being conserved across species, mutational analysis indicates the DPIM does not play an essential role in vivo, suggesting that interaction between the two polymerases is also non-essential.

Peptide aptamers define distinct EB1- and EB3-binding motifs and interfere with microtubule dynamics.

EB1 is a conserved protein that plays a central role in regulating microtubule dynamics and organization. It binds directly to microtubule plus ends and recruits other plus end-localizing proteins. Most EB1-binding proteins contain a Ser-any residue-Ile-Pro (SxIP) motif. Here we describe the isolation of peptide aptamers with optimized versions of this motif by screening for interaction with the Drosophila EB1 protein. The use of small peptide aptamers to competitively inhibit protein interaction and function is becoming increasingly recognized as a powerful technique. We show that SxIP aptamers can bind microtubule plus ends in cells and functionally act to displace interacting proteins by competitive binding. Their expression in developing flies can interfere with microtubules, altering their dynamics. We also identify aptamers binding to human EB1 and EB3, which have sequence requirements similar to but distinct from each other and from Drosophila EB1. This suggests that EB1 paralogues within one species may interact with overlapping but distinct sets of proteins in cells.

Anti-c-Met antibodies recognising a temperature sensitive epitope, inhibit cell growth.

c-Met is a tyrosine receptor kinase which is activated by its ligand, the hepatocyte growth factor. Activation of c-Met leads to a wide spectrum of biological activities such as motility, angiogenesis, morphogenesis, cell survival and cell regeneration. c-Met is abnormally activated in many tumour types. Aberrant c-Met activation was found to induce tumour development, tumour cell migration and invasion, and the worst and final step in cancer progression, metastasis. In addition, c-Met activation in cells was also shown to confer resistance to apoptosis induced by UV damage or chemotherapeutic drugs. This study describes the development of monoclonal antibodies against c-Met as therapeutic molecules in cancer treatment/diagnostics. A panel of c-Met monoclonal antibodies was developed and characterised by epitope mapping, Western blotting, immunoprecipitation, agonist/antagonist effect in cell scatter assays and for their ability to recognise native c-Met by flow cytometry. We refer to these antibodies as Specifically Engaging Extracellular c-Met (seeMet). seeMet 2 and 13 bound strongly to native c-Met in flow cytometry and reduced SNU-5 cell growth. Interestingly, seeMet 2 binding was strongly reduced at 4oC when compared to 37oC. Detail mapping of the seeMet 2 epitope indicated a cryptic binding site hidden within the c-Met α-chain.

Uncoupling the replication machinery: replication fork progression in the absence of processive DNA synthesis.

The precise coordination of the different steps of DNA replication is critical for the maintenance of genome stability. We have probed the mechanisms coupling various components of the replication machinery and their response to polymerase stalling by inhibition of the DNA polymerases in living mammalian cells with aphidicolin. We observed little change in the behaviour of proteins involved in the initiation of DNA replication. In contrast, we detected a marked accumulation of the single stranded DNA binding factor RPA34 at sites of DNA replication. Finally, we demonstrate that proteins involved in the elongation step of DNA synthesis dissociate from replication foci in the presence of aphidicolin. Taken together, these data indicate that inhibition of processive DNA polymerases uncouples the initiation of DNA replication from subsequent elongation steps. We, therefore, propose that the replication machinery is made up of distinct functional sub-modules that allow a flexible and dynamic response to challenges during DNA replication.

The p66 and p12 subunits of DNA polymerase delta are modified by ubiquitin and ubiquitin-like proteins.

Modification by ubiquitin-like proteins is now known to be important for the functions of many proteins involved in DNA replication and repair. We have investigated the modification of human DNA polymerase delta by ubiquitin and SUMO proteins. We find that while the p125 and p50 subunits were not modified, the p12 subunit is ubiquitinated and the p66 subunit can be modified by ubiquitin and SUMO3. We show that levels of p12 are regulated by the proteasome, either directly or indirectly, through a mechanism that is not dependent upon p12 ubiquitination. We have mapped two sites of SUMO3-specific modification on the p66 subunit. SUMOylation by SUMO3 but not SUMO2 is unusual: their level of homology is so high that they are normally classified as variants of the same protein. However, our findings show that these two proteins can be distinguished in vivo and may have specific functions.