Biology subjects

Ashton, N. W.

Publications and source records attributed to Ashton, N. W..

9 recordsLinked to original sources

bioRxiv2026

A RAD18 SAP domain PIP motif enables PCNA mono-ubiquitination and USP1-BRCA1 synthetic lethality

The proliferating cell nuclear antigen (PCNA) sliding clamp is mono-ubiquitinated by RAD6-RAD18 in response to DNA damage, initiating the DNA damage tolerance pathway of translesion synthesis. The molecular basis by which RAD18 engages PCNA has, however, remained incompletely defined. Mono-ubiquitinated PCNA is subsequently poly-ubiquitinated with K48-linked chains that target PCNA for degradation. Ubiquitin-specific protease 1 (USP1) reverses PCNA mono- and poly-ubiquitination; accordingly, inhibiting USP1 causes the accumulation of mono-ubiquitinated PCNA at replication forks and a reduction in total PCNA levels. USP1 inhibitors promote the accumulation of ssDNA gaps (ssGAPs) in newly replicated DNA and are synthetic lethality in BRCA1-deficient cells. Here, we combine computational and structural approaches to identify and characterize a PCNA-interacting peptide (PIP) motif in RAD18. This PIP motif is required for RAD18-dependent DNA damage-induced PCNA ubiquitination and PCNA turnover. Mutation of the RAD18-PCNA interface reduces ssGAP accumulation and USP1 inhibitor sensitivity in BRCA1-deficient cells. Furthermore, cells adapted to prolonged USP1 inhibition exhibit reduced RAD18 levels, suggesting that deregulation of RAD18 contributes to a biologically relevant drug resistance mechanism. This resistance could be overcome by inhibiting the Ataxia telangiectasia and Rad3-related (ATR) kinase. Together, these findings define a molecular interface required for RAD18-dependent PCNA mono-ubiquitination and identify it as a key determinant of USP1-BRCA1 synthetic lethality.

biochemistry↗

bioRxiv2025

Disruption of Microhomology-mediated End-joining in Ewing Sarcoma

Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of the oncoprotein, EWS-FLI1, in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POL{Theta}, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POL{Theta} expression, impaired MMEJ, and cellular sensitivity to inhibitors of the Fanconi Anemia (FA), NHEJ, or HR pathways, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POL0 mitotic foci and increases MMEJ activity. Inhibitors of the FA, NHEJ, or HR therefore may provide a targeted therapy for patients with EwS. Highlights- Ewing sarcoma tumors have a deficiency in POL{theta} expression and a corresponding loss of MMEJ activity - EWSR1 is a splicing factor that interacts with other splicing factors such as FUBP1 and KHSRP/FUBP2 to accurately splice the POLQ mRNA. - The EWS-FLI1 fusion oncoprotein, or loss of EWSR1, causes a splicing defect, leading to exon25 skipping of the POLQ pre-mRNA and loss of POL{theta} expression - The MMEJ deficiency of EwS cells results in cellular sensitivity to inhibitors of Fanconi Anemia, Homologous Recombination or Non-Homologous End-Joining - Exon 25 skipping of POLQ mRNA is a predictive biomarker for HR inhibitors in human cancers

cancer biology↗

bioRxiv2024

Effects of an auxin antagonist, PEO-IAA, and an auxin transport inhibitor, NPA, on gametophore development in Physcomitrium patens (Hedw.) Mitt.

Auxin sensing and polar auxin transport (PAT) are required for normal growth and development of stem internodes and leaves of gametophores of Physcomitrium (formerly Physcomitrella) patens. Auxin is required for the formation of leaf apical cells, and NPA-driven elevation of the intracellular auxin/cytokinin ratio stimulates bifurcation at the leaf margin. Auxin is implicated in the differentiation of the leaf midrib. Auxin sensing and PAT appear not to be needed for the transition of gametophore buds into leafy shoots.

plant biology↗

bioRxiv2024

The polyketide pathway in sporopollenin biosynthesis is specific to land plants (Embryophyta)

Background and AimsSporopollenin (SP) is a complex biopolymer in the outer wall of spores and pollen and provides protection from environmental stresses. Its extraordinary chemical resistance, especially to acetolysis, was widely used to identify SP in biological specimens. This broad definition of SP led to claims for its widespread occurrence among diverse embryophyte and non-embryophyte taxa. We previously proposed a biochemical definition that can be used to distinguish genuine SP from other chemically resistant cell wall materials. The definition was centred on ASCL (Anther-Specific Chalcone synthase-Like), an embryophyte-specific enzyme of the polyketide pathway that provides precursors for SP biosynthesis. Herein, we examine the evolution and distribution of all five enzymes (CYP703A, CYP704B, ACOS, ASCL and TKPR) of the polyketide pathway and propose a new, more comprehensive definition of SP. MethodsWe performed BLASTp searches, phylogenetic tree construction, protein modeling and sequence analysis to determine the presence or absence of ACOS and TKPR in embryophytes and streptophytic algae. Key ResultsWe found evidence that all five enzymes of the polyketide pathway evolved from ancestral enzymes of primary metabolism and ACOS, ASCL and TKPR were co-selected during evolution. The dosage of all five genes has been subjected to strict evolutionary control and, in some taxa, synteny has provided a selective advantage. All five enzymes are present in embryophytes but absent in green algae, indicating that the polyketide pathway and therefore SP is embryophyte-specific. ConclusionsThe addition of the polyketide pathway in the definition of genuine SP will allow separation of SP from algaenans and other chemically resistant SP-like algal spore wall substances. This study further signifies SP as an evolutionary innovation unique to the embryophyte lineage and encourages research on possible evolutionary relationship between algal spore wall SP-like materials and embryophyte SP.

plant biology↗

bioRxiv2024

DNA polymerase η is regulated by mutually exclusive mono-ubiquitination and mono-NEDDylation

DNA polymerase eta (Pol {eta}) is a Y-family translesion polymerase responsible for synthesizing new DNA across UV-damaged templates. It is recruited to replication forks following mono-ubiquitination of the PCNA DNA clamp. This interaction is mediated by PCNA-interacting protein (PIP) motifs within Pol {eta}, as well as by its C-terminal ubiquitin-binding zinc finger (UBZ) domain. Previous work has suggested that Pol {eta} itself is mono-ubiquitinated at four C-terminal lysine residues, which is dependent on prior ubiquitin-binding by its UBZ domain. Here, we show that Pol {eta} can be modified at the same lysine residues by the ubiquitin-like protein, NEDD8. Like ubiquitination, this modification is driven by non-covalent interactions between NEDD8 and the UBZ domain. While only a small proportion of Pol {eta} is mono-NEDDylated under normal conditions, these levels rapidly increase by inhibiting the COP9 signalosome, suggesting that mono-NEDDylation is maintained under strong negative regulation. Finally, we provide data to support that mono-ubiquitination is important for Pol {eta} foci formation and suggest that NEDDylation disrupts this process. These results reveal a new mechanism of Pol {eta} regulation by ubiquitin-like proteins.

biochemistry↗

bioRxiv2024

Early morphogenetic patterns of protonemata and gametophores of Physcomitrium patens (Hedw.) Mitt.

In axenic culture, protonemata of Physcomitrium (formerly Physcomitrella) patens (Hedw.) Mitt. are comprised of chloronemata and caulonemata that can be distinguished morpho-structurally. On solid nutrient agar medium, a protonemal inoculum proliferates, initially generating primary chloronemata, and expands radially across the surface of the substratum forming approximately round colonies. The expansion of the colonies derives mainly from centrifugal elongation of newly formed caulonemata, which arise by differentiation of primary chloronemal apical cells in the central region. Most commonly, oblique cross walls separate cells of the uniseriate caulonemal filaments. Each oblique wall organises a smaller and a larger angle with the outer wall of a tubular caulonemal cell. The smaller angle may lie in four different positions, vertically upward or downward, and laterally, to the right or left, parallel to the surface of the solid medium. Chloronemal filaments, which contain cells separated by transverse cross walls, develop from side branch initials (SBIs) at or near the smaller angle at the distal end of caulonemal cells. Like the smaller angle of the oblique cross walls in caulonemata, these secondary chloronemata also show four different orientations, vertically, upward or downward, and laterally, right or left. A strong inverse correlation exists between the placement of SBIs and secondary chloronemata and the direction of caulonemal tip growth. If the caulonemal tips curve downward into the medium, chloronemata are situated vertically on the upper side of caulonemata; similarly, if they turn upward, chloronemata are situated on the lower side. A similar correlation exists when caulonemal tips curve to the right or left on the surface of solid medium, in which case laterally directed chloronemata grow in the opposite direction, i.e. on the outside of the curve. The procumbent caulonemata with their associated protruding secondary chloronemal branches are reminiscent of the heterotrichous branching habit of filaments of some green algae. Gametophore buds develop from a minority of caulonemal SBIs and also occasionally from chloronemata; a stalk cell, which resembles a chloronemal cell, supports each bud. Buds develop into leafy gametophores that are arranged in a fairy ring. We discuss the relative merits of several models that might explain the temporally and spatially regulated clustering of buds resulting in the formation of a fairy ring.

plant biology↗

bioRxiv2024

Takakia possesses a key marker of embryophyte sporopollenin

The enigmatic moss, Takakia lepidozioides, possesses a particular type III polyketide synthase, ASCL (Anther-Specific Chalcone synthase-Like), that is an identifying marker for genuine sporopollenin in the walls of embryophyte spores and pollen grains. By contrast, a survey of all algae with sequenced genomes confirms that they do not possess ASCL and, therefore, their spore walls are not composed of sporopollenin.

evolutionary biology↗

bioRxiv2023

The curvature of Physcomitrium patens caulonemal filaments in relation to their capacity forphototropic and thigmotropic responses and nutation

Light-grown, whole gametophytic colonies of Physcomitrium (formerly Physcomitrella) patens (Hedw.) Mitt. exhibit a spiral morphology resulting from the strongly coordinated curvature of the population of peripheral caulonemal filaments. The direction of curvature is predominantly clockwise when cultures are illuminated from above and anticlockwise when illuminated from below. In P. patens, side branch initials (SBIs) emerge from caulonemal subapical cells on the outside of the curve. By contrast, the curvature of caulonemata of Funaria hygrometrica is predominantly anticlockwise when colonies are illuminated from above and clockwise when illuminated from below. In F. hygrometrica, SBIs emerge from caulonemal subapical cells on the inside of the curve. We have discounted a role for gravity in these phenomena and discuss several other possible mechanistic explanations. We also document for the first time thigmotropism of protonemata of P. patens. O_TBL View this table: org.highwire.dtl.DTLVardef@9b2d80org.highwire.dtl.DTLVardef@1c83f39org.highwire.dtl.DTLVardef@1aecda3org.highwire.dtl.DTLVardef@13ebd83org.highwire.dtl.DTLVardef@be625_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOBox 1.C_FLOATNO O_TABLECAPTIONGlossary of Terminology C_TABLECAPTION C_TBL

plant biology↗

bioRxiv2023

D-serine- and p-fluorophenylalanine-resistant mutants of Physcomitrium patens are defective in amino acid uptake

Two amino acid analogue-resistant lines of Physcomitrium (formerly Physcomitrella) patens, DSR8 and PFR4, are resistant to a range of D-amino acids and an inhibitory concentration of L-lysine. Both are defective in the uptake of [35S]-L-methionine. Uptake by the wild-type line is pH-dependent (decreasing with raised external proton concentrations) and is depressed by dinitrophenol and by ammonium ions. We discuss the possible involvement of an active proton-general amino acid antiport pump in the plasma membrane.

plant biology↗