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(WIS) The application of novel Top down Mass Spectrometry to uncover targeted mutagenesis of DNA repair proteins for exposomics


Project Description

Next generation sequencing of human tumour DNA consistently shows large numbers of mutations scattered throughout the genome (e.g. 1). The source of these mutations is a matter of much speculation but there is evidence that DNA is continuously undergoing damage by a broad range of exogenous and endogenous genotoxic agents, among which are the alkylating agents. These powerful mutagens can react with the nitrogen and oxygen atoms in DNA bases, but also with the phosphodiester backbone, resulting in phosphotriesters (PTE). While the biological effects of PTE in DNA are poorly understood (2), it has recently been shown that, at least in E.coli, methyl PTE are mutagenic (3). In E.coli, one of the two PTE stereoisomers is processed by a suicide enzyme, a PTE methyltransferase (PTMT), that transfers the alkyl group to a cysteine reside in its active site (4). In contrast, in mammalian cells PTE are highly persistent, and as they constitute a substantial fraction of total DNA damage by alkylating agents, this makes them suitable candidates as cancer risk markers and potentially informing strategies for prevention. Such studies are limited by the paucity of methods for undertaking either quantitation or gene-specific mapping of these lesion in the human genome. Hence the ultimate goal of this project is to quantitate the PTEs lesions in human DNA and to map them within the genome in order to test the hypothesis that these lesions are important in the genotoxic effect of alkylating agents. To facilitate this, site directed mutagenesis (5) will be used to generate versions of the E.coli PTMT protein that can quantitate PTE at the genomic level and determine their distribution at the DNA sequence level. Variant proteins will be generated, purified and characterised biochemically (e.g by the repair of PTE in substrate DNA ) and physically (e.g. X-ray crystallography, surface plasmon resonance). It will then be used to detect and quantify PTE in DNA in cultured human cells following exposure to alkylating agents and ultimately in human tissue DNA. The studentship will involve, molecular biology, biochemistry, x-ray crystallography and proteomics mass spectroscopy and will benefit from direct interactions with an ongoing CR-UK funded project primarily investigating alkylation damage to DNA bases.

Entry Requirements
Applicants must be from the UK/EU and have obtained (or be about to obtain) a minimum 2:1 Bachelors honours degree or equivalent in a relevant subject area.

Funding Notes

This project is available to UK/EU candidates. Funding covers fees (UK/EU rate) and stipend for four years. Overseas candidates can apply providing they can pay the difference in fees and are from an eligible country. Candidates will be required to split their time between Manchester and Weizmann Institute of Science, as outlined on View Website .

As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.

References

1. Nik-Zainal S, Kucab JE, Morganella S, Glodzik D, Alexandrov LB, Arlt VM, Weninger A, et al. The genome as a record of environmental exposure. Mutagenesis. 2015;30:763-70.
2. Jones GD, Le Pla RC, Farmer PB. Phosphotriester adducts (PTE): DNA's overlooked lesion. Mutagenesis. 2010;25:3-16.
3. Wu J, Wang P, Wang Y. Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells. Nucleic Acids Res. 2018;46:4013-21.
4. Sedgwick B, Robins P, Totty N, Lindahl T. Functional domains and methyl acceptor sites of the Escherichia coli ada protein. J Biol Chem. 1988;263:4430-3.
5. He C, Wei H, Verdine GL. Converting the sacrificial DNA repair protein N-ada into a catalytic methyl phosphotriester repair enzyme. J Am Chem Soc. 2003;125:1450-1.

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