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Recognition and mapping of DNA hypermodifications by type IV restriction enzymes


Project Description

Training Opportunity: This project will appeal to a motivated student who wants experience and training in both a UK academic setting, and in the world-class research labs of a leading US biotechnology company. In this joint academic-industrial proposal between Durham University and New England Biolabs, the student will gain skills in a range of techniques including molecular biology, microbiology, protein biochemistry, structural biology and next generation sequencing. The topic offers an exciting program of work in a rapidly developing field, with excellent job prospects. As this is a CASE studentship, the student will be based at Durham University, with a minimum of 9 months placement at New England Biolabs HQ, in Massachusetts, USA.

Project Background: Bacteriophages (phages) outnumber bacteria by ten to one. This huge selection pressure has led to the development of bacterial systems that protect from phage predation. Many of these phage-resistance systems have already proved invaluable to biochemists: the restriction-modification and CRISPR-cas systems underpin the recombinant DNA and genome editing revolutions. Unlike the more commonly used type II restriction enzymes, type IV restriction enzymes cleave modified DNA substrates. We have isolated and characterised a new type IV restriction enzyme, BrxU, which is able to recognise diverse DNA hypermodifications, including 5-methyl-, 5-hydroxymethyl- and 5-glucosyl-hydroxymethyl- cytosine, and then cleave the target DNA. The last few years has seen increased interest in mammalian cytosine modifications, with up to 4% of the human genome containing cytosine modifications that have roles in developmental processes, pluripotency of stem cells, neurodegenerative diseases and tumourigenesis. BrxU has the potential for use as a tool with which to map DNA hypermodifications in combination with next generation sequencing technologies. This will provide a platform to better understand the role of these epigenetic markers in developmental and disease processes.

Aims: The student will (1) examine the biochemistry of the BrxU protein, namely (i) sequence specificity, (ii) DNA-hypermodification substrate specificity and (iii) nucleotide substrate specificity (cleavage activity is NTP-dependent). Next, the student will focus on (2) structural studies to understand each of the identified preferences and the role of nucleotide binding and hydrolysis in cleavage activity. Finally, the student will (3) apply BrxU to map DNA modifications as part of a high-throughput next generation sequencing platform.

Supervisory Team: New England Biolabs are a global company with a proven track record in supplying tools and reagents for biotechnological and biomedical research. This industrial expertise will be complemented by the newly upgraded structural biology capabilities at Durham University.

HOW TO APPLY
Applications should be made by emailing with a CV (including contact details of at least two academic (or other relevant) referees), and a covering letter – clearly stating your first choice project, and optionally 2nd and 3rd ranked projects, as well as including whatever additional information you feel is pertinent to your application; you may wish to indicate, for example, why you are particularly interested in the selected project(s) and at the selected University. Applications not meeting these criteria will be rejected.
In addition to the CV and covering letter, please email a completed copy of the Additional Details Form (Word document) to . A blank copy of this form can be found at: https://www.nld-dtp.org.uk/how-apply.

Informal enquiries may be made to

Funding Notes

This is a 4 year BBSRC CASE studentship under the Newcastle-Liverpool-Durham DTP. The successful applicant will receive research costs, tuition fees and stipend (£15,009 for 2019-20). The PhD will start in October 2020. Applicants should have, or be expecting to receive, a 2.1 Hons degree (or equivalent) in a relevant subject. EU candidates must have been resident in the UK for 3 years in order to receive full support. Please note, there are 2 stages to the application process.

References

A complex suite of loci and elements in eukaryotic type II topoisomerases determine selective sensitivity to distinct poisoning agents (2019) Nucleic Acids Research 47(15): 8163-8179.

Identification and biosynthesis of thymidine hypermodifications in the genomic DNA of widespread bacterial viruses (2018) Proceedings of the National Academy of Sciences of the United States of America 115(14): E3116-3125.

Evolution of Pectobacterium bacteriophage ΦM1 to escape two bifunctional Type III toxin-antitoxin and abortive infection systems through mutations in a single viral gene (2017) Applied and Environmental Microbiology 83(8): e03229-16.

Novel m4C modification in type I restriction-modification systems (2016) Nucleic Acids Research 44(19):9413-9425.

Structure of type IIL restriction-modification enzyme MmeI in complex with DNA has implications for engineering new specificities (2016) PLoS Biology 14(4): e1002442.

Crystal structure and stability of gyrase–fluoroquinolone cleaved complexes from Mycobacterium tuberculosis (2016) Proceedings of the National Academy of Sciences of the United States of America 113(7): 1706-1713.

Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase (2016) Proceedings of the National Academy of Sciences of the United States of America 113(7): E839-E846.

Co-evolution of quaternary organization and novel RNA tertiary interactions revealed in the crystal structure of a bacterial protein–RNA toxin–antitoxin system (2015) Nucleic Acids Research 43(19): 9529-9540.

Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot (2013) Proceedings of the National Academy of Sciences of the United States of America 110(3): E241-9.

A processed non-coding RNA regulates an altruistic bacterial antiviral system (2011) Nature Structural and Molecular Biology 18(2): 185-190.

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