PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication

DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) lightdamaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol h-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells

Investigations into the cellular and molecular biology of a cytoplasmic dynein mutation which leads to neurodegeneration

The aim of this project was to investigate into the effect of the Loa mutation in the function of the cytoplasmic dynein complex. Protein-protein interaction assays were performed on NIH3T3, NSC34 and HEK293T cells transfected with a 2 kb fragment of the wild type or mutant dynein heavy chain gene 1 bearing the Loa mutation and seven different intermediate chain isoforms. The results indicated a 2.5 fold increase in the binding affinity of the mutant dynein heavy chain 1 727 amino acid fragment for the dynein intermediate chain gene 2 isoform A, compared to the wild type.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Intermediate chain subunit as a probe for cytoplasmic dynein function: biochemical analyses and live cell imaging in PC12 cells.

Cytoplasmic dynein 1 is a multi-subunit motor protein responsible for microtubule minus end-directed transport in axons. The cytoplasmic dynein intermediate chain subunit has a scaffold-like role in the dynein complex; it directly binds to four of the other five subunits, the heavy chain and the three light chains. The intermediate chain also binds the p150 subunit of dynactin, a protein that is essential for many dynein functions. We reexamined the generation of rat cytoplasmic dynein intermediate chain isoforms by the alternative splicing of the two genes that encode this subunit and identified an additional splicing site in intermediate chain gene 1. We reinvestigated the expression of the intermediate chain 1 isoforms in cultured cells and tissues. The Loa mouse, which is homozygote lethal, contains a missense mutation in the region of the cytoplasmic dynein heavy chain gene that binds the intermediate chain. Protein binding studies showed that all six intermediate chains were able to bind to the mutated heavy chain. GFP-tagged intermediate chains were constructed and PC12 cell lines with stable expression of the fusion proteins were established. Live cell imaging and comparative immunocytochemical analyses show that dynein is enriched in the actin rich region of growth cones

DYNC1H1 mutation alters transport kinetics and ERK1/2-cFos signalling in a mouse model of distal spinal muscular atrophy

Mutations in the gene encoding the heavy chain subunit (DYNC1H1) of cytoplasmic dynein cause spinal muscular atrophy with lower extremity predominance, Charcot-Marie-Tooth disease and intellectual disability. We used the legs at odd angles (Loa) (DYNC1H1(F580Y)) mouse model for spinal muscular atrophy with lower extremity predominance and a combination of live-cell imaging and biochemical assays to show that the velocity of dynein-dependent microtubule minus-end (towards the nucleus) movement of EGF and BDNF induced signalling endosomes is significantly reduced in Loa embryonic fibroblasts and motor neurons. At the same time, the number of the plus-end (towards the cell periphery) moving endosomes is increased in the mutant cells. As a result, the extracellular signal-regulated kinases (ERK) 1/2 activation and c-Fos expression are altered in both mutant cell types, but the motor neurons exhibit a strikingly abnormal ERK1/2 and c-Fos response to serum-starvation induced stress. These data highlight the cell-type specific ERK1/2 response as a possible contributory factor in the neuropathological nature of Dync1h1 mutations, despite generic aberrant kinetics in both cell types, providing an explanation for how mutations in the ubiquitously expressed DYNC1H1 cause neuron-specific disease

Visualization of co-localization in Aβ42-administered neuroblastoma cells reveals lysosome damage and autophagosome accumulation related to cell death

Aβ42 [amyloid-β peptide-(1–42)] plays a central role in Alzheimer's disease and is known to have a detrimental effect on neuronal cell function and survival when assembled into an oligomeric form. In the present study we show that administration of freshly prepared Aβ42 oligomers to a neuroblastoma (SH-SY5Y) cell line results in a reduction in survival, and that Aβ42 enters the cells prior to cell death. Immunoconfocal and immunogold electron microscopy reveal the path of the Aβ42 with time through the endosomal system and shows that it accumulates in lysosomes. A 24 h incubation with Aβ results in cells that have damaged lysosomes showing signs of enzyme leakage, accumulate autophagic vacuoles and exhibit severely disrupted nuclei. Endogenous Aβ is evident in the cells and the results of the present study suggest that the addition of Aβ oligomers disrupts a crucial balance in Aβ conformation and concentration inside neuronal cells, resulting in catastrophic effects on cellular function and, ultimately, in cell death