Biology subjects

Williamson, A. J.

Publications and source records attributed to Williamson, A. J..

4 recordsLinked to original sources

bioRxiv2022

Characterisation and sequence mapping of large RNA and mRNA therapeutics using mass spectrometry

Large RNA including messenger RNA (mRNA) has emerged as an important new class of therapeutic. Recently this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study we have developed an automated, high throughput workflow for the rapid characterisation and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilised on magnetic particles was performed in conjunction with high resolution liquid chromatography mass spectrometry analysis. Sequence mapping was performed using automated oligoribonucleotide annotation and identifications based on MS/MS spectra. Using this approach >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS Co-V2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation can be completed within 90 minutes. The ability to rapidly identify, characterise and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation and impurity analysis.

biochemistry↗

bioRxiv2021

Sulfate adenylyl transferase kinetics and mechanisms of metabolic inhibitors of microbial sulfate respiration

Sulfate analog oxyanions that function as selective metabolic inhibitors of dissimilatory sulfate reducing microorganisms (SRM) are widely used in ecological studies and industrial applications. As such, it is important to understand the mode of action and mechanisms of tolerance or adaptation to these compounds. Different oxyanions vary widely in their inhibitory potency and mechanism of inhibition, but current evidence suggests that the sulfate adenylyl transferase/ATP sulfurylase (Sat) enzyme is an important target. We heterologously expressed and purified the Sat from the model SRM, Desulfovibrio alaskensis G20. With this enzyme we determined the turnover kinetics (kcat, KM) for alternative substrates (molybdate, selenate, arsenate, monofluorophosphate, and chromate) and inhibition constants (KI) for competitive inhibitors (perchlorate, chlorate, and nitrate). These measurements enable the first quantitative comparisons of these compounds as substrates or inhibitors of a purified Sat from a respiratory sulfate reducer. We compare predicted half-maximal inhibitory concentrations (IC50) based on Sat kinetics with measured IC50 values against D. alaskensis G20 growth and discuss our results in light of known mechanisms of sensitivity or resistance to oxyanions. This analysis helps with the interpretation of recent adaptive laboratory evolution studies and illustrates the value of interpreting gene-microbe-environment interactions through the lens of enzyme kinetics.

microbiology↗

bioRxiv2021

Selective metal extraction by biologically produced siderophores during bioleaching from low-grade primary and secondary mineral resources

Siderophores are a class of biogenic macromolecules that have high affinities for metals in the environment, thus could be exploited for alternate sustainable metal recovery technologies. Here, we assess the role of siderophores in the extraction and complexation of metals from an iron oxide-rich metallurgical processing residue and a low-grade primary Ni ore. Evaluation of the biological siderophore bioproduction by three pseudomonads, P. fluorescens, P. azotoformans and P. putida identified that P. putida could generate the highest siderophore yield, which was characterized as a hydroxamate and catecholate mixed-type pyoverdine PyoPpC-3B. Key physiochemical parameters involved in raw siderophore mediated metal extraction were identified using a fractional factorial design of experiments (DOE) and subsequently employed in purified PyoPpC-3B leaching experiments. Further targeted experiments with hydroxamate and catecholate functional analogues of PyoPpC-3B confirmed their marked ability to competitively or selectively leach and chelate hard metal ions, including Al(OH)4-, Mn2+ and Zn2+. Interestingly, complexation of Mn and Zn ions exceeded the natural affinity of pyoverdine for Fe3+, thus despite the low metal recoveries from the materials tested in this study, this work provides important new insights in siderophore-metal interactions.

biochemistry↗

bioRxiv2019

Chd1 regulates repair of promoter-proximal DNA breaks to sustain hypertranscription in embryonic stem cells

Stem and progenitor cells undergo a global elevation of nascent transcription, or hypertranscription, during key developmental transitions involving rapid cell proliferation. The chromatin remodeler Chd1 binds to genes transcribed by RNA Polymerase (Pol) I and II and is required for hypertranscription in embryonic stem (ES) cells in vitro and the early post-implantation epiblast in vivo. Biochemically, Chd1 has been shown to facilitate transcription at least in part by removing nucleosomal barriers to elongation, but its mechanism of action in stem cells remains poorly understood. Here we report a novel role for Chd1 in the repair of promoter-proximal endogenous double-stranded DNA breaks (DSBs) in ES cells. An unbiased proteomics approach revealed that Chd1 interacts with several DNA repair factors including Atm, Parp1, Kap1 and Topoisomerase 2{beta}. We show that wild-type ES cells display high levels of phosphorylated H2A.X and Kap1 at chromatin, notably at rDNA in the nucleolus, in a Chd1-dependent manner. Loss of Chd1 leads to an extensive accumulation of DSBs at Chd1-bound Pol II-transcribed genes and rDNA. Genes prone to DNA breaks in Chd1 KO ES cells tend to be longer genes with GC-rich promoters, a more labile nucleosomal structure and roles in chromatin regulation, transcription and signaling. These results reveal a vulnerability of hypertranscribing stem cells to endogenous DNA breaks, with important implications for developmental and cancer biology.

molecular biology↗