Biology subjects

Wigley, D. B.

Publications and source records attributed to Wigley, D. B..

3 recordsLinked to original sources

bioRxiv2022

Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.

biochemistry↗

bioRxiv2020

The type 2 diabetes gene product STARD10 is a phosphoinositide binding protein that controls insulin secretory granule biogenesis

ObjectiveRisk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the {beta}-cell, impaired glucose-induced insulin secretion and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids, and thus the pathways through which STARD10 regulates {beta}-cell function, are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the {beta}-cell and its effect on proinsulin processing and insulin granule biogenesis and maturation. MethodsWe used isolated islets from mice deleted selectively in the {beta}-cell for Stard10 ({beta}StarD10KO) and performed electron microscopy, pulse-chase, RNA sequencing and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay were performed on purified STARD10 protein. Results{beta}StarD10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased. Amongst the differentially expressed genes in {beta}StarD10KO islets, expression of the phosphoinositide binding proteins Pirt and Synaptotagmin 1 were decreased while lipidomic analysis demonstrated changes in phosphatidyl inositol levels. The inositol lipid kinase PIP4K2C was also identified as a STARD10 binding partner. STARD10 bound to inositides phosphorylated at the 3 position and solution of the crystal structure of STARD10 to 2.3 [A] resolution revealed a binding pocket capable of accommodating polyphosphoinositides. ConclusionOur data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis and insulin processing.

cell biology↗

bioRxiv2018

Human CtIP forms a tetrameric dumbbell-shaped particle which binds and bridges complex DNA end structures for double-strand break repair

CtIP is involved in the resection of double-stranded DNA breaks during the S and G2 phases of the cell cycle for repair by homologous recombination. Acting in concert with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with more complex structures including forked ssDNA overhangs and model nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

biochemistry↗